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+{"text": "When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis.Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches ,2 may leThe results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results. However, there are many sources of errors, both when purifying RNA, performing the RT reaction and during the PCR setup ,4. Q-PCRcriteria . In recocriteria ,6-9. We Lepeophtheirus salmonis) starvation experiment using the 2-\u0394\u0394CT method [-\u0394\u0394CT method, our results show that LsTryp1 transcript levels decrease following starvation and return to normal adult level when the louse subsequently gets access to food  were reared as earlier described [Salmo salar) and 3 lice were stored in RNA later (Ambion). The remaining 12 lice were starved in incubators with flowing seawater for 14 days. After starvation, 3 lice were sampled and stored as described above, and the remaining 9 lice were put in a tank with uninfected salmon where they could settle on their salmon hosts and resume feeding. After 15 days on their new hosts 3 lice were sampled and stored as described above. The experimental procedures were carried out in accordance with national regulations for use of animals in scientific research.Salmon lice  or 0.4 (CT = 32) from the most similar parallel at the same dilution. At least 4 dilutions were required for each stage. The resulting data were calibrated to unstarved lice and analysed as described by Kvamme et al.[\u03b1 = 0.05) from the other dilutions. The signal corresponding to the initial template concentration (R0) was derived using the average PCR efficiency for LsTryp1 and eEF1\u03b1 when the PCR efficiencies were not significantly different . When the PCR efficiency differed significantly, R0 was calculated using individual gene specific mean efficiencies. The mean R0 for each dilution of LsTryp1 was normalised to corresponding eEF1\u03b1 values. The normalised R0 values were calibrated to the values for unstarved lice. 95% confidence intervals (CI) were derived from normalised R0 values.The transcript levels of LsTryp1  and the 1 [eEF1\u03b1  in 1 sel1 [eEF1\u03b1 . The RNA1 [eEF1\u03b1  and cDNAescribed  and a meescribed . The latescribed . When usme et al.. When usRS and PF conceived the study and designed the Q-PCR assay. RS carried out the assay and analyses, and wrote the first draft of the communication. PF contributed to development of the communication. FN provided expert input for the writing and supervised the study."}
+{"text": "Saccharomyces cerevisiae has been associated with human life for millennia in the brewery and bakery. Recently it has been recognized as an emerging opportunistic pathogen. To study the evolutionary history of S. cerevisiae, the origin of clinical isolates and the importance of a virulence-associated trait, population genetics and phenotypic assays have been applied to an ecologically diverse set of 103 strains isolated from clinics, breweries, vineyards, fruits, soil, commercial supplements and insect guts.S. cerevisiae strains that are associated with ecology, geography and virulence. Shared alleles suggest that the clinical isolates contain genetic contribution from the fruit isolates. Clinical and fruit isolates exhibit high levels of recombination, unlike the genetically homogenous soil isolates in which no recombination was detected. However, clinical and soil isolates were more resistant to oxidative stress than any other population, suggesting a correlation between survival in oxidative stress and yeast pathogenicity.DNA sequence data from five nuclear DNA loci were analyzed for population structure and haplotype distribution. Additionally, all strains were tested for survival of oxidative stress, a trait associated with microbial pathogenicity. DNA sequence analyses identified three genetic subgroups within the recombining S. cerevisiae delineated three distinct groups, comprising primarily the (i) human-associated brewery and vineyard strains, (ii) clinical and fruit isolates (iii) and wild soil isolates from eastern U.S. The interactions between S. cerevisiae and humans potentiate yeast evolution and the development of genetically, ecologically and geographically divergent groups.Population genetic analyses of  S. cerevisiae has long been associated with humans as the fermentative agent in the production of bread, beer, and wine. Archeological evidence for the production of fermented beverages in China dates to 7,000 BCE, and molecular evidence demonstrating S. cerevisiae was the fermentative agent has been found in wine jars from ancient Egypt dating to 3,150 BCE S. cerevisiae embraces a wider range than domesticated strains found in the vineyard and the brewery. Wild strains have been isolated from mushroom fruiting bodies as well as oak tree-associated soils and fluxes S. cerevisiae are furthermore a major cause of spoilage of mango fruit and peach puree, and it has recently been identified in surveys of the fungal diversity in beetle guts S. cerevisiae ecological diversity, encompassing domesticated and wild isolates, has spurred interest in the life history and population genetics of this species. The population biology of an organism so tightly associated with humans is challenging to study due to sampling bias, limited sampling sizes, human influence, and non-random sampling S. cerevisiae have mainly been addressed using strains from grape berries, vineyards and other industrial applications. Fay and Benavides analyzed approximately 7 kb of coding and non-coding DNA sequences in 81 strains from vineyards, fermentation of sake, palm wine, ragi and cider, fruit sources, including lychee, fig, and mushrooms, oak tree and surrounding soil from New Jersey, and patients in the U.S. and Europe. This extensive analysis resulted in the recognition of domesticated, i.e. human-associated, and wild populations in S. cerevisiaeThe breadth of S. cerevisiae's has been reported as an emerging opportunistic pathogen S. cerevisiae causing infections S. cerevisiae and its commercially available preparations known as S. boulardii, that are used to treat antibiotic-related diarrhea, have been shown to cause a wide variety of infections, ranging from cutaneous infections and vaginitis to systemic infections of the bloodstream and vital organs in immunocompromised and immunocompetent individuals Candida albicans, the most common human fungal pathogen C. albicans and its status as genetics model system, S. cerevisiae, the benevolent baker's yeast, has acquired enhanced scientific value as a model pathogen in the study of virulence-related traits In addition to investigations into the origin and consequences of domestication, S. cerevisiae include growth at high temperature S. cerevisiae isolates from spoiled peach pure and grape juice were heat resistant Microbial virulence-related traits promote host invasion, colonization and virulence. One of these is the ability to survive oxidative stress, exerted by radical oxygen species (ROS), an integral component of mammalian host defenses, is associated with virulence in various bacteria S. cerevisiae, little is known about the emergence of this versatile yeast as a pathogen and the role of selection and/or adaptation in this evolutionary process. This study aims to (i) test for genetic differentiation between strains of a broad range of origins, (ii) investigate the evolutionary origin of clinical isolates, and (iii) identify an association between a virulence-related trait and pathogenicity. The targeted virulence trait is survival of oxidative stress. To investigate population structure and evolution of pathogenicity, an ecologically diverse sample of 103 S. cerevisiae isolates, comprising seven populations, was analyzed. Clinical isolates with confirmed virulence were obtained from the California Institute for Medical Research in Stanford and Duke University Medical Center in Durham, NC S. boulardii strains from France, Italy and Germany, complete the sampling Despite progress in understanding the population structure of domesticated and wild S. cerevisiae, expanding the previous findings. (ii) Clinical isolates are genetically similar to the isolates from fruit, which supports the hypothesis that monoculture or fermenting fruits may serve as a natural reservoir for the evolution of clinical isolates. (iii) Strains from the clinic and Pennsylvania soil tolerate oxidative stress better than any other group. However, the clinical isolates are genetically diverse while the soil isolates are identical at all loci. This strongly suggests that resistance to ROS is an adaptive property of the clinical strains.The results confirm previous findings and generated several novel conclusions. (i) At least three divergent groups representing different evolutionary trajectories were detected in S. cerevisiae, as confirmed by ITS sequencing and all strains were determined by FACS analysis to be diploid (data not shown). To ensure correct estimates of population genetic parameters haplotype phase within each locus was determined. For the majority of isolates the correct locus haplotype phase could be determined by visual analysis of sequence alignments. For 22 strains the correct phase of one ore more loci was identified using PHASE 2.1.1. All isolates in this study belong to the species S. boulardii form group C. Note that two fruit isolates in group C stem from grapes, two fruit isolates from Holly and a papaya. Interestingly, all group C vineyard isolates have been collected from Vitis vinifera in Australia or North Carolina. The two identical NC vineyard isolates that are outside the major clusters were isolated from V. rotundifolia grapes, the Muscadine grapes that are native to the southeastern United States. Seven of the nine isolates collected in Adelaide (AU) are identical. As measure of genetic differentiation pairwise Fst values were calculated for groups A, B, and C. Fst values showed significant genetic differentiation between the three groups and analysis of molecular variance (AMOVA) indicated that 25% of the observed variance occurs within these groups and 62% between them. The index of association (IA), measuring the extent of linkage equilibrium, was significantly different from zero for group A but not for B and C.Principal component analysis (PCA), a transformation method that reduces multidimensional data sets to lower dimensions, was employed to detect putative structure among 87 isolates for which complete sequence data for five loci could be obtained . ConfideA), nucleotide diversity (\u03c0), and deviation from Hardy-Weinberg equilibrium (HWE) were calculated , recombination, linkage equilibrium  confirmed significant differences in oxidative stress between groups of N\u226510 (P<0.0001). Clinical isolates differ significantly (P<0.001) from all groups but PA soil  Low levels of recombination and genetic diversity among soil populations (group B) suggest a predominantly clonal life style that contributes to differentiation from clinical and domesticated groups and maintenance of a wild lineage that also exhibits geographic structure because all of its isolates were collected in the eastern United States. Interestingly, the soil isolates share four of five haplotypes with beetle gut isolates, suggesting that insects acquire S. boulardii were not as resistant to ROS as the clinical strains. Indeed, strains of S. boulardii were less virulent compared to a clinical (YJM145) and a laboratory (Y55) strain S. boulardii isolates used here were cultured from commercial products sold in France, Italy and Germany. Their close association with vineyard strains suggests that S. boulardii may have originated from vineyard strains.The clinical and PA soil populations exhibited the highest mean resistance to ROS. Therefore ROS resistance in the clinical isolates arose either independently or was contributed by the soil isolates. The genetic diversity among the clinical isolates suggests multiple origins of the genetic material required for ROS resistance in these strains. Regardless, fungal resistance to ROS offers protection from oxidative host defenses and is undoubtedly an advantageous pathobiological property. Consistent with this conclusion, the probiotic strains of Most recently, a study conducted by Kvitek, Will and Gasch combined investigations into genetic diversity with stress response and identified a distinct sake fermentation clade but no discrete clades for clinical or oak-associated isolates S. cerevisiae beyond its prosaic service as a scientific tool in the laboratory or an agent of fermentation. As a species, S. cerevisiae entered two new life history trajectories while continuing its life in the soil and on decomposing fruit. Some strains of S. cerevisiae became domesticated in fermentation and brewing, and while others became pathogenic. With the accumulation of ecological and population genetic data, this versatile microbe becomes an invaluable model for evolutionary biology and population genetics. The current report offers a new paradigm for studying pathogenesis by identifying correlation(s) among the virulence traits of isolates and the ecology of their ancestral reservoirs. These correlations will identify the genotypes associated with pathogenic strains or the potential for pathogenicity and elucidate the evolution of pathogenicity.This investigation presents an exciting and provocative demonstration of the complex life history of S. cerevisiae strains were isolated from S. cerevisiae infected patients (N\u200a=\u200a15), soil in Pennsylvania , soil in North Carolina , vineyards in NC (N\u200a=\u200a10) and Australia , various fruits (N\u200a=\u200a18), brewery (N\u200a=\u200a16), commercial S. boulardii preparations (N\u200a=\u200a4), and the insect gut (N\u200a=\u200a2) . DNA was extracted using the CTAB buffer method  (N\u200a=\u200a2) . All str96Cleanup Kit (Millipore) or the ExoSAP protocol and their concentration determined electrophoretically Genomic DNAs were diluted 1\u2236100 and 2 \u00b5l added to a 25 \u00b5l reaction from TaKaRa Ex Taq kit with a final primer concentration of 0.6 \u00b5M . The PCRA were calculated in GenAlEx 6, DnaSP 4.50.3, and MultiLocus 1.3 A). Due to limited sampling size S. boulardii and beetle gut isolates were not included in HWE, Fst, \u03c0 and IA calculations. An UPGMA tree based on a pair-wise genetic distances between isolates included in PCA was build in PHYLIP 3.68 MLS1, ACT1, ADP1, PHD1 and RPB1 in PAUP* 4.0b10 applying the parsimony criterion, conducting heuristic searches, and using tree-bisection-reconnection (TBR) as branch swapping algorithm. These five loci were chosen for analysis based on their prior employment in phylogenetic studies of the Saccharomycetales, population genetic analysis of S. cerevisiae and C. albicans and their differential expression patterns upon phagocytosis by macrophages 2SO4, 1.7 g/l yeast nitrogen base (YNB) without amino acids and ammonium sulfate), incubated at 30\u00b0C while shaking (250 rpm), transferred once, and grown over night. In order to reduce variability in the cell suspension, the cells were washed twice with 0.9% NaCl solution and dissolved in 1\u00d7 phosphate buffered saline. After adjusting the cell number to 2\u00d7103 cells/ml, 20 mM tert-Butyl hydroperoxide  were added and treated and control samples incubated for one hour at 30\u00b0C, while shaking. 100 \u00b5l cell suspensions from both samples were spread on YPD plates, incubated for 48 h at 30\u00b0C and colonies counted. Survival was calculated as the ratio of treated over untreated cells. Each strain was tested three times and results were clustered by origin and plotted in box-and-whisker plots. A one-way ANOVA with Bonferroni correction was carried out on experimental triplicates of each strain in every group to assess differences in variation for all groups with N\u226510.Two-day old cultures from YPD plates were inoculated into liquid synthetic defined media Click here for additional data file.Figure S1UPGMA tree. The tree was generated from a pair-wise genetic distance matrix based on haplotype data of the 87 strains that were included in PCA. The strains are color-coded by origin (legend) and groups recognized in PCA indicated at internodes in the tree. Strains marked with * denote isolates that are not included in PCA groups A, B or C confidence envelopes. The numbered bar below the tree indicates total genetic distance observed in the data set.(1.34 MB TIF)Click here for additional data file."}
+{"text": "Neuroscientists have observed that the human brain is comprised of neurons. We have observed that babies start speaking at an early age, yet no young animals, including pets, have so far been seen to speak, at least not in the articulated fashion of human babies. To understand this highly cognitive ability, many psycholinguistic data have been gathered, from behavioral, to neurolinguistic, to recent neuroimaging studies, each measuring macroscopic properties of the brain. Nevertheless, the challenging question remains unanswered of how such complicated behavior emerges from the microscopic (or mescoscopic) properties of individual neurons and of networks of neurons in the brain.We would like to tackle this question by developing and analyzing a Potts attractor neural network model, whose units hypothetically represent patches of the cortex. The network has the ability to spontaneously hop (or latch) across memory patterns , thus producing an infinite sequence of patterns, at least in some regimes . We woulBefore training the network on the corpus, the critical issues to be addressed, and the central ones here, are: how should the words be represented in a cognitively plausible manner in the network? how should the correlation between words, in terms of both meaning and statistical dependences, be reflected in their  representations? how should two main characteristics of a word, the meaning (semantic) and the syntactic properties, be represented in the network?We represent words in a distributed fashion on 900 units, 541 out of which express the semantic content and the rest, 359 units, are representative of the syntactic characteristics of a word. The distinction between the semantic and syntactic characteristics of a word has been loosely inspired by a vast number of neuropsychological studies [The preliminary analysis of the produced patterns indicates the resemblance between the statistics of the representation of words and the patterns that can generate the latching behavior of the network. This is a promising step towards building a neural network that can spontaneously generate a sequence of words (sentences) with desired syntactic and semantic relationships between words in sentences."}
+{"text": "Boronic acid mol\u00adecules form inversion-related hydrogen-bonded dimers in an R               2               2(8) motif. The structure is consolidated by inter\u00admolecular C\u2014H\u22efO bonds and C\u2014H\u22ef\u03c0 inter\u00adactions.In the crystal structure of the title compound, C \u00c5                           b = 12.339 (4) \u00c5                           c = 16.983 (5) \u00c5                           \u03b2 = 95.651 (5)\u00b0V = 2548.0 (14) \u00c53                                                   Z = 4                           K\u03b1 radiationMo \u22121                        \u03bc = 0.13 mmT = 297 K                           0.52 \u00d7 0.45 \u00d7 0.42 mm                                 Bruker SMART CCD area-detector diffractometerSADABS; Bruker, 2000T                           min = 0.934, T                           max = 0.947Absorption correction: multi-scan (23787 measured reflections4486 independent reflectionsI > 2\u03c3(I)3727 reflections with R                           int = 0.052                                                            R[F                           2 > 2\u03c3(F                           2)] = 0.083                           wR(F                           2) = 0.186                           S = 1.19                           4486 reflections294 parametersH-atom parameters constrainedmax = 0.40 e \u00c5\u22123                        \u0394\u03c1min = \u22120.32 e \u00c5\u22123                        \u0394\u03c1                                 SMART  used to solve structure: SHELXS97  I, global. DOI: 10.1107/S1600536811051609/pk2368Isup2.hkl            Structure factors: contains datablock(s) I. DOI: 10.1107/S1600536811051609/pk2368Isup3.cml            Supplementary material file. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:"}
+{"text": "Arginine, an \u03b1-amino acid, has been reported to exert beneficial effects that ameliorate health problems and prevent excessive fat deposition. In this study, we investigated whether the activation of cell signaling by arginine can induce osteogenic differentiation and modulate excessive adipogenic differentiation in human mesenchymal stem cells (MSCs). Arginine potently induced the expression of type I\u03b11 collagen, osteocalcin, and ALP in a dose-dependent manner without causing cytotoxicity. Arginine significantly increased the mRNA expression of the osteogenic transcription factors runt-related transcription factor 2 (Runx2), DIx5, and osterix. Furthermore, arginine demonstrated its antiadipogenicity by decreasing adipocyte formation and triglyceride (TG) content in MSCs and inhibiting the mRNA expression of the adipogenic transcription factors peroxisome proliferator-activated receptor \u03b3 (PPAR\u03b3), CCAAT/enhancer-binding protein \u03b1 (C/EBP\u03b1), and fatty acid binding protein 4 (Fabp4). This effect was associated with increased expression of Wnt5a, and nuclear factor of activated T-cells (NFATc), and was abrogated by antagonists of Wnt and NFATc, which indicated a role of Wnt and NFATc signaling in the switch from adipogenesis to osteoblastogenesis induced by arginine. In conclusion, this is the first report of the dual action of arginine in promoting osteogenesis and inhibiting adipocyte formation through involving Wnt5a and NFATc signaling pathway. Human bone marrow mesenchymal stem cells (MSCs) are pleiotropic cells that differentiate into either adipocytes or osteoblasts ,2. ChangOsteogenesis and adipogenesis, which share a reciprocal relationship in the bone marrow, are complex processes that include the proliferation of precursor cells and their commitment to a specific lineage and then terminal differentiation ,7. In MSN-terminal kinase (JNK) pathway and the Wnt/calcium (Ca2+) pathway [2+ pathway ultimately leads to the binding of the nuclear factor of activated T-cells (NFATc) to specific DNA-binding sites [Wnt signaling is a key pathway that controls bone formation and adipogenesis by regulating critical events such as cell-fate determination and cell proliferation and differentiation . Wnt sig pathway ,17. The ng sites ,18,19. TAstragalus membranaceus Bunge, and it is one of the 20 most common natural amino acids [Arginine is a major compound produced by no acids . Oral adno acids , and it no acids . Argininno acids . Argininno acids . HoweverIn this study, we investigated whether arginine enhances osteogenic differentiation and inhibits adipocyte formation in MSCs by modulating osteogenic and adipogenic transcription factors and the Wnt signaling pathway.To examine how arginine affects cell proliferation, we treated MSCs with 0, 0.1, 1, and 10 \u00b5M arginine for 1, 3, 5, 7, and 10 days. Arginine dose-dependently enhanced cell proliferation after treatment for 48 h and increased the proliferation of cells in a statistically significant manner, by nearly 36%, at a concentration of 10 \u00b5M A. HoweveTo determine whether arginine can stimulate osteogenic differentiation, we measured the effect of arginine on the levels of the bone-formation markers type I\u03b11 collagen, osteocalcin, and alkaline phosphatase (ALP). Our results showed that the treatment of MSCs with 1 \u03bcM arginine for 3, 7, 14, and 21 days increased the mRNA expression of type I\u03b11 collagen, osteocalcin, and ALP in a statistically significant manner, but did not enhance the expression of type II\u03b11 collagen relative to the control level at each time point A. The exIn vivo studies have demonstrated that in mice in which the PPAR\u03b3 gene was deleted, the levels of TGs, free fatty acids, and cholesterol and the accumulation of hepatic TG were lower than those in control mice [To determine the effect of arginine on adipogenic differentiation in MSCs, 1 \u03bcM arginine was added to cells cultured in an adipogenic medium, and then adipocyte-specific gene expression was examined using quantitative real-time reverse-transcription PCR (qRT-PCR) during adipogenic induction. During the 14 days of differentiation, the induced adipocytes were stained with Oil red O. The staining-positive cells displayed substantial lipid accumulation at 3 days when compared with the uninduced cells A. The inrol mice . C/EBP\u03b1,rol mice . Lipid arol mice . Fabp4 brol mice . Our resTo investigate the roles of arginine in Wnt/\u03b2-catenin signaling during the osteoblastic and adipocytic differentiation of MSCs, cells cultured in an adipogenic medium were treated with arginine for 48 h and then Western-blotting analysis and RT-PCR were performed. Doses of arginine ranging from 0.1\u201310 \u03bcM did not affect the expression levels of phospho-\u03b2-catenin and \u03b2-catenin in MSCs A. SubseqNFATc1 represents a downstream of target of Wnt signaling and is a key regulator of adipogenic and osteogenic differentiation ,35. We tTaken together, our results suggest that arginine regulates Wnt/\u03b2-catenin independent signaling and also the mechanisms involving NFATc that control MSC fate and differentiation, and contributes to the increase in osteoblastogenesis and decrease in adipogenesis.l-Arginine, ALP activity assay kit, Alizarin red S staining kit, Oil red O staining kit, ascorbic acid, glycerophosphate, dexamethasone (DEX), 3-isobutyl-1-methylxanthine (IBMX), dimethyl sulfoxide (DMSO), and insulin (INS) were purchased from Sigma-Aldrich . Fetal bovine serum (FBS), fetal calf serum (FCS), antibiotics, Dulbecco\u2019s modified Eagle\u2019s medium (DMEM), Trizol R reagent, and SDS-polyacrylamide gels were purchased from Gibco-BRL . The RT-PCR system kit was purchased from TaKaRa Biotechnology . The real-time SYBR Green RT-PCR system kit was purchased from Bio-Rad . Hybond-C nitrocellulose membrane was purchased from Amersham Biosciences .2 flasks and incubated at 37 \u00b0C/5% CO2. After 48 h, non-adherent cells were removed from the flasks by changing the medium. Thereafter, the medium was changed once every 3 days. Typically, cultures reached 90% confluence by 14 days, at which point the cells were trypsinized using 0.25% trypsin-0.53 mM EDTA, counted, and then plated again. Cells from passages number of 4~6 were used in all experiments. To determine whether changes in gene expression depended on canonical or noncanonical Wnt signaling, MSCs were treated with arginine in the presence of DKK1 (50 ng/mL) or sFRP1 (250 ng/mL) (Sigma) and then gene expression was measured as described below . To investigate the role of NFATc in mediating the effect of arginine, MSCs were treated with arginine in the presence of CSA (100 ng/mL) or FK506 (10 ng/mL), and then gene expression was measured.Human bone-marrow MSCs were obtained from Lonza . Cells were seeded into 25-cm4 cells/well in 96-well plates and allowed to attach for 12 h in DMEM containing 10% FBS. To assess the effect of arginine on cell proliferation, MSCs were treated with culture medium containing various concentrations of arginine  for 48 h or with medium containing 0, 0.1, 1, and 10 \u03bcM arginine for 3, 5, 7, and 10 days. At each time point, the arginine containing medium was removed and the cells were incubated with fresh serum-free medium containing the Cell Counting Kit-8 (CCK-8) reagent WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitro-phenyl)-5--2H-tetrazolium) ; cells were incubated for 2 h at 37 \u00b0C in a CO2 incubator and then the amount of formazan dye generated, which is proportional to the number of living cells, was determined by measuring the absorbance at 570 nm using a multi-well plate reader . The percentage of proliferating cells was calculated by defining the cell viability measured in the absence of treatment as 100%.MSCs were seeded at a density of 2 \u00d7 104 cells/cm2 in 6-well plates. After 2 days, the medium was replaced with an osteogenic medium , and then fresh differentiation medium was added every other day and the cells were maintained at 37 \u00b0C in a humidified 5% CO2 atmosphere throughout the experiments until the cells were harvested. The differentiating cells were treated for 3, 7, 14, and 21 days with the osteogenic differentiation medium containing arginine at a final concentration of 1 \u03bcM, or the cells were treated with 0.1, 1, and 10 \u03bcM arginine. To determine the effects of arginine on osteogenesis in MSCs, the expression of osteogenesis-related biochemical markers such as collagen type 1\u03b1, osteocalcin, and ALP and transcription factors such as Runx2, DIx5, and osterix were measured using qRT-PCR and matrix calcium deposition was evaluated by performing Alizarin red S staining.To induce osteogenic differentiation, MSCs were plated at a density of 2 \u00d7 10After treatment with arginine for 21 days, mineralization was measured using Alizarin red S (Sigma) staining and phase-contrast microscopy. Cells were incubated with 2% Alizarin red at pH 4.2 for 10 min and then washed with distilled water; the sub-cultured cells were observed using phase-contrast microscopy to examine cell morphology and verify the presence of mineralized nodules. After staining, the cells were washed and photographed, and the dye retained by the cells was eluted by incubation with isopropanol and then quantified by measuring the absorbance at 510 nm.4 cells/cm2 in 6-well plates and grown to post-confluence for 3 days. The culture medium was subsequently replaced with the adipogenesis-induction medium  in order to commit the cells to the adipogenic lineage. This medium was changed every other day and maintained up to 14 days to ensure complete development of the adipocyte phenotype. To assess the effect of arginine, cells were treated for 3, 7, and 14 days with the adipocytic differentiation medium containing arginine at a final concentration of 1 \u03bcM, or cells were treated with 0.1, 1, and 10 \u03bcM arginine for 14 days, and then the cells were used for qRT-PCR and RT-PCR assays, Oil red O staining, and TG-content measurement. The expression of Wnt3a, Wnt5a, and \u03b2-catenin was also evaluated during the adipocytic differentiation of MSCs.MSCs were plated at a density of 3 \u00d7 10Intracellular lipid accumulation was measured using Oil red O staining; the staining of lipid droplets by this dye was used as an indicator of the degree of adipogenesis. Cells were carefully washed twice with phosphate-buffered saline (PBS) and then fixed and dried with 10% formalin for 20 min. Next, the 10% formalin was removed and 60% isopropanol was added to each well for 3 min and then the cells were washed thoroughly with PBS to remove unbound dye. Cells were incubated with the Oil red O solution for 20 min, and the staining of lipid droplets in differentiated MSCs was examined after rinsing the cells 3 times with distilled water. Cells were photographed, and then the dye retained by the cells was eluted by incubating the cells with isopropanol, and quantified by measuring the absorbance at 510 nm.The cellular content of TG was measured using a TG-determination kit . Briefly, the cells were rinsed 3 times with PBS and then scraped off the plate by using a rubber policeman. The cells were extracted using 1 mL of lysis buffer  and then sonicated for 30 s at 4 \u00b0C. Next, 20 \u03bcL of the cellular lysate was mixed with 3 mL of the enzyme solution supplied with the kit, and the mixture was incubated for 10 min at 37 \u00b0C. The absorbance at 550 nm was measured within 60 min. As an internal control, the DNA concentration in MSCs was determined using spectrophotometry and then the \u03bcg/\u03bcg value was calculated.Ct) method. The cycle of threshold (Ct) for each sample was averaged and normalized relative to that of GAPDH. The results were then analyzed using the comparative \u0394\u0394Ct method (2Ct)(\u2212\u0394\u0394) for the purpose of quantifying relative gene-expression levels. To perform RT-PCR, the diluted first-strand cDNA samples were amplified using TaqDNA polymerase (TaKaRa Biotechnology). The mRNA expression levels of Wnt5a, Wnt3a, and \u03b2-actin were evaluated by performing ethidium-bromide staining of PCR products. The signal intensity was quantified using Gel Doc EQ , and the relative expression of the mRNAs was normalized by dividing the signal of the target genes by that of the respective \u03b2-actin sample. The sequences of the primers used for qRT-PCR and RT-PCR assays are listed in Total cellular RNA was extracted from osteogenic cells or adipocytes by using the Trizol reagent and then centrifuged at 12,000 rpm for 10 min at 4 \u00b0C. Next, 1 \u03bcg of total RNA was reverse transcribed into cDNA for 60 min at 42 \u00b0C and then for 15 min at 72 \u00b0C by using an RT-PCR mixture (TaKaRa Biotechnology) that contained the RT buffer, oligo(dT) 12-mer, 10 mM dNTPs, 0.1 M dithiothreitol, reverse transcriptase, and RNase inhibitor. The qRT-PCR assay was performed in a 25-\u03bcL reaction mixture containing the SYBR Green PCR Master Mix (Roche Diagnostics). The template source was either 5 ng of cDNA or a purified DNA standard. Various primers were used for amplifying type II\u03b11 collagen, type I\u03b11 collagen, osteocalcin, ALP, Runx2, DIx5, osterix, PPAR\u03b3, C/EBP\u03b1, and Fabp4. To standardize mRNA levels, we amplified \u03b2-actin as an internal control. The relative expression of target genes in the examined samples was obtained using the difference in the comparative threshold , 5 min per wash, on a shaker. Next, the blots were incubated with goat anti-rabbit IgG-HRP  for 2 h at room temperature, and then washed 4 times in TTBS, 5 min each time. Immunoreactive proteins on the blots were visualized using ECL\u2122 Western Blotting detection reagents and the signals were detected using Image Station 4000R . Staining with an anti-\u03b2-actin antibody was used to verify that equal amounts of proteins were loaded in all lanes.Following incubation with various reagents, cells were lysed using the lysis buffer. Total proteins (20 \u03bcg/lane) were separated on 4%\u201312% SDS-polyacrylamide gels by preforming electrophoresis under reducing conditions, and then transferred to Hybond-C nitrocellulose membranes (Amersham Biosciences) at 300 mA for 90 min. After blocking with 5% non-fat skim milk for 2 h, the membranes were incubated with rabbit primary antibodies against phospho-\u03b2-catenin, anti-\u03b2-catenin and NFATc1  for 12 h at 4 \u00b0C. The membranes were washed 4 times in 1\u00d7 TTBS . In the case of two groups, Student\u2019s In conclusion, this study has presented the first experimental evidence of the pro-bone and anti-fat effects of arginine, which counteracts the age-related switch in MSC osteoblast-to-adipocyte differentiation through the Wnt and NFATc1 signaling pathways. A schematic diagram of these observed effects of arginine is shown in"}
+{"text": "Reconstructing the past variability of Arctic sea ice provides an essential context for recent multi-year sea ice decline, although few quantitative reconstructions cover the Holocene period prior to the earliest historical records 1,200 years ago. Photochemical recycling of bromine is observed over first-year, or seasonal, sea ice in so-called \u201cbromine explosions\u201d and we employ a 1-D chemistry transport model to quantify processes of bromine enrichment over first-year sea ice and depositional transport over multi-year sea ice and land ice. We report bromine enrichment in the Northwest Greenland Eemian NEEM ice core since the end of the Eemian interglacial 120,000 years ago, finding the maximum extension of first-year sea ice occurred approximately 9,000 years ago during the Holocene climate optimum, when Greenland temperatures were 2 to 3\u2009\u00b0C above present values. First-year sea ice extent was lowest during the glacial stadials suggesting complete coverage of the Arctic Ocean by multi-year sea ice. These findings demonstrate a clear relationship between temperature and first-year sea ice extent in the Arctic and suggest multi-year sea ice will continue to decline as polar amplification drives Arctic temperatures beyond the 2\u2009\u00b0C global average warming target of the recent COP21 Paris climate agreement. A seasonal cycle in polar atmospheric bromine was first discovered in the 1980s by Sturges and Barrie24611An understanding of polar tropospheric halogen chemistry has developed as ground- and ice-core based measurements have established clearer links between FYSI extent and halogen variability recorded in polar ice cores. Interactions between halogens and FYSI are monitored by satellite81525) derived from sea ice diatoms26Due to the poor geographical and temporal resolution of sea ice proxies, few quantitative reconstructions of Holocene Arctic total sea ice extent are available. The few reconstructions available are based on dinoflagellate cyst assemblagesThe drivers and feedbacks between polar climate and total sea ice remain a topic of active research. The ice- albedo feedback mechanism has been studied in detailHere we present bromine and sodium concentrations in the NEEM ice core to investigate Canadian Arctic sea ice variability since the last interglacial period. We define the Canadian Arctic as the area from which aerosols are entrained and transported to the NEEM site, comprising the Canadian archipelago, Baffin Bay and Hudson Bay regions see . Bromine1418Bromine and sodium concentrations have been determined in the North Greenland Eemian NEEM ice core34enr as Br/(Na*0.006) using the mass ratios of Br and Na and the average sea water Br/Na mass ratio (0.00618O temperature proxy (R2\u2009=\u20090.50), Brenr is positively correlated with \u03b418O (R2\u2009=\u20090.54) supporting the connection between warm climate conditions, greater presence of FYSI and enhanced bromine photochemistry.Bromine concentrations in the NEEM ice core record vary through the Holocene as well as during the rapid interstadial warming events . The lowtio 0.006. Althoug\u22121, a bromine-enriched air mass would travel more than 1,000\u2009km in 72\u2009hours, easily encompassing the distance from the Canadian Arctic to the NEEM site. In the absence of recycling, atmospheric bromine concentrations quickly decrease to levels an order of magnitude lower than those found in the FYSI scenarios. On the basis of the model results, we proceed with the interpretation that bromine enhancement at the NEEM site is quantitatively linked to the presence of FYSI and that bromine concentrations found at NEEM are reasonably independent of transport variability. This interpretation assumes explicitly that no bromine recycling occurs over MYSI or land ice, and that bromine enrichment may be employed as a quantitative indicator of bromine recycling over FYSI.To demonstrate the quantitative link between FYSI extent and bromine enrichment in the NEEM ice core, we have applied a 1-dimensional setup of a state-of-the-art chemistry transport model featuring interactive halogen photochemistry. The model is spun up with a constant atmospheric BrO concentration over open ocean and then transports the airmass over two types of surface: FYSI over which bromine is photochemically recycled; and a stable surface over which bromine is not recycled. This stable non-reactive surface is theorised to comprise a combination of MYSI and/or Greenland ice sheet surface (land ice). Three scenarios have been investigated, in which the airmass experiences bromine recycling for 48\u2009hours, 24\u2009hours, or not at all . This fienr 15.6\u2009\u00b1\u20093.2 (1\u03c3) 9.0\u201311.5 ky b2k), associated with the prevalence of FYSI, support existing evidence of a rapid loss of MYSI at the end of the glacial and a relatively ice-free Arctic Ocean. In comparison, bromine enrichment is lower during the last 3 ky (Brenr 10.6\u2009\u00b1\u20094.9 (1\u03c3) 0.03\u20133.03 ky b2k) and displays greater variability, suggesting greater extension of MYSI in the Canadian Arctic during the late Holocene. Expansion of MYSI in recent millennia is supported by reconstructions of Arctic sea ice determined from Arctic Ocean marine sedimentsBromine enrichment in the NEEM ice core is greatest during the early Holocene, indicating a substantial increase of FYSI and corresponding decrease of MYSI in the Arctic immediately after the glacial termination . Ice-cor13enr closely following changes in \u03b418O temperature proxy. Bromine enrichment during GI-1 is similar to late Holocene values, indicating a mixture of MYSI and FYSI in the Canadian Arctic. Between 11.8 and 12.8 ky b2k, consistently low Brenr values indicate a return to extensive MYSI cover in the Canadian Arctic.Over the last glacial period, bromine enrichment shows variability on the scales of Dansgaard-Oeschger (DO) events. DO events are rapid climate changes involving reorganisation of atmospheric and oceanic circulation on hemispheric and interhemispheric scales. Greenland interstadials (GI) 3 to 12  demonstrenr values found for glacial and late Holocene climates  to represent late glacial conditions (see 18O-log(Brenr) relationship  for the whole NEEM dataset presented here and therefore also use log(Brenr) for our linear model of FYSI response to Holocene temperature variability. The model . The residual standard deviation (RSD) was low for both halogens and ranged between 2\u201310%.Concentrations of Br and Na were determined by Inductively Coupled Plasma Sector Field Mass Spectrometry  equipped with a cyclonic Peltier-cooled spray chamber TraceSELECT\u00ae NH4OH, Sigma Aldrich), then 2% HNO3 acid , with 30\u2009s of MQ water between reagents. Between each analysis a single cleaning cycle was run to return the background to within 1% of the initial background level. Bromine was calibrated by external calibration using standards of 10 to 4,000\u2009pg g\u22121, prepared by diluting a 1,000\u2009mg L\u22121 standard solution  in MQ water. All calibration curves showed correlation coefficients greater than 0.99 .The analytical system was cleaned for 24\u2009h prior to each analysis session, consisting of alternating 180\u2009s washes of 5% ammonium solution (The Tropospheric HAlogen chemistry MOdel (THAMO)How to cite this article: Spolaor, A. et al. Canadian Arctic sea ice reconstructed from bromine in the Greenland NEEM ice core. Sci. Rep.6, 33925; doi: 10.1038/srep33925 (2016)."}
+{"text": "The cornea is the transparent outermost surface of the eye, consisting of a stratified epithelium, a collagenous stroma and an innermost single-cell layered endothelium and providing 2/3 of the refractive power of the eye. Multiple diseases of the cornea arise from genetic defects where the ultimate phenotype can be influenced by cross talk between the cell types and the extracellular matrix. Cell culture modeling of diseases can benefit from cornea organoids that include multiple corneal cell types and extracellular matrices. Here we present human iPS cell-derived organoids through sequential rounds of differentiation programs. These organoids share features of the developing cornea, harboring three distinct cell types with expression of key epithelial, stromal and endothelial cell markers. Cornea organoid cultures provide a powerful 3D model system for investigating corneal developmental processes and their disruptions in diseased conditions. The cornea is the outer protective layer of the eye that provides 2/3 of the refractive power required to focus light on the photosensitive retina2345in vitro modeling of the cornea has traditionally used individual constituent cell types of the epithelium, stroma or the endothelium7in vitro. In addition, studies of the ECM types they produce usually require long-duration cultures to elicit potential differences underlying disease. Therefore, we sought to develop three-dimensional (3D) corneal organoids comprising all the component cell types of the cornea in a stable system. This provides a unique opportunity to investigate cellular disease phenotypes for long periods of time, including developmental aspects, and incorporates influences of interactions between different cell types and the ECM.Despite its relative simplicity, in vitro12Recent efforts have made tremendous gains in developing self-organizing 3-dimensional organoids from embryonic stem cells, and subsequently later from induced pluripotent stem cells (iPSC)1112131415Methodological details of producing cornea organoids (C-ORG) from iPSCs are outlined in KRT)\u2019 KRT3 and KRT14 and these appear as bands of expected sizes in C-ORG samples  and the ECM marker Keratocan (KERA), as well as putative endothelial markers   on total RNA extracted from individual (not pooled) C-ORGs, donor corneas, as well as cultured corneal fibroblasts as references . The tra2122 samples . KRT 12  samples , and itsa sample . The PAX S100A4) 2627. TheCryo-sections of C-ORGs revealed a multi-layered architecture approximately 100\u2009\u03bcm thick. The sections were immunostained for epithelial, stromal and endothelial markers . The cen13032TEM of the organoids at low ,C and hi37Results presented here support the development of a corneal organoid structure from human iPSCs. These organoids express markers of the three cell types of the cornea - epithelium, stromal keratocytes and the endothelium. Moreover, key extracellular matrix collagens and proteoglycan core proteins that make up the stromal matrix were present in the organoids. In the central organoid collagen fibrils were present. The fibrils demonstrated the beginning of a hierarchal structure with fibrils becoming organized into small bundles (fibers) often with a ~90 degree displacement of adjacent structures.The emergence of the cornea organoids during the development of eye cups from iPSCs should be considered in the context of signals that regulate eye development. The vertebrate eye field develops under the regulation of the transcription factors TBX2/3/5As this study relies on cell mediated self-assembly and maturation of structures, the process of differentiation is orchestrated in a manner similar to that seen in development. In contrast, other tissue engineering approaches, such as culture of differentiated stromal fibroblasts in collagen matrix, hydrogels and other types of scaffolds attempt to recreate the local environment of the mature tissue. Alternatively, cell-based therapeutic approaches engage in transplanting limbal and mesenchymal stem cells and rely on their ability to populate and repair ocular surfaces. A recent study has published a 2-dimensional culture system whereby iPSCs demonstrated the ability to organize into a \u201cself-formed ectodermal autonomous multi-zone\u201d that could recreate different ocular epitheliaLUM, KERA) and COL1A1 and COLVA1. Our study on the other hand used iPSCs to develop cornea organoids, where the corneal status was more stringently tested at the transcript and protein level for the presence of epithelial, stromal and endothelial markers. The organoids show remarkably appropriate immunofluorescence staining for epithelial markers KRT3, KRT14 and p63. The outer layers also show some extracellular staining of perlecan and collagen type VIII. Perlecan, a heparan sulfate proteoglycan is a major component of the epithelial basement membrane, but it is also present in the Descemet\u2019s membrane of young corneasOne previous study has reported the development of cell spheres termed \u201ccornea orb\u201d from a human embryonic stem cell line and from a second cell line derived from a parthenogenetic, unfertilized eggThe organoid field in general is moving towards using induced pluripotent stem cells rather than embryonic stem cells to develop various organoids such as intestinal, kidney, liver and retinal organoidsAll experiments were carried out in accordance with the relevant guidelines and regulations set out by the Johns Hopkins Stem Cell Research and Oversight committee. Normal donor anterior stromal caps in Optisol \u2013GS  were obtained from endothelial keratoplasty from Tissue Banks International  and the Indiana Lions Eye and Tissue Bank  under established guidelines related to informed consent for research use of human donor corneas. These experiments are covered by the Johns Hopkins Medicine IRB approved protocol entitled \u201cPhenotypic and Genotypic Analysis of Keratoconus (NA_00006544).Stromal cells from donor corneas were extracted by serial extraction with collagenase as described before432, 10% CO2) cells were transitioned to a Neural induction medium: consisting of DMEM  with 1% B27 vitamin A (-)  and 38.8\u2009mg/L insulin , 128\u2009mg/L L-ascorbic acid , 28\u2009\u03bcg/l sodium selenium , 21.4\u2009mg/L transferrin , 38.8\u2009mg/L NaHCO3. Osmolarity was raised +30\u2009mOsm to ~330\u2013340\u2009mOsm by adding 0.88\u2009g/L NaCl. Aggregates were transferred at D10 to 15\u2009ml tubes, rinsed 3\u00d7 in HBSS, and treated with 100\u2009nM Smoothened agonist  from D10\u2013D18. Long term medium was a 3:1 mix of DMEM (Invitrogen #11965):F12 (#11765: Invitrogen) with 1% B27 (#17504044: Invitrogen), 10% heat inactivated FBS , 1\u2009mM pyruvate , 1xNEAA , 1X Glutamax  and 1\u2009mM taurine . Vesicles were excised from D10\u201312 (as late as D16) with tungsten needles and fed every 2\u20133 days in suspension in ultra-low binding T-75 flasks (Corning) or untreated 10\u2009cm polystyrene petri dishes. When 10\u2009cm dishes were used, sedentary aggregates were monitored to ensure that they didn\u2019t stick to the surface. Cells were maintained at 37\u2009\u00b0C in standard 20% O2/5% CO2. To increase survival and differentiation, 500\u2009nM all-trans retinoic acid  was added to the medium from D20 until D120  as per manufacturer\u2019s instructions. 1\u2009\u03bcg of RNA was reverse transcribed using the Superscript III RT kit . Quantitative PCR analysis was performed using the Bio-Rad CFX384 real time PCR machine, utilizing SYBR green chemistry (Applied Biosystems) 14\u2009\u03bcl reaction volumes. Briefly, 20\u2009nM of oligonucleotides were loaded with 3\u2009ng of cDNA per reaction. The primer list and PCR conditions are provided in The organoids were embedded in optimal cutting temperature compound (OCT) and flash frozen; then 10\u2009\u03bcm sections were taken on a cryostat. These were allowed to air dry for 30\u2009minutes before being fixed in 4% paraformaldehyde in PBS for 15\u2009minutes at room temperature. The sections were treated with 0.1% Triton X-100 in PBS for 5\u2009minutes at room temperature. The sections were then blocked in 3% BSA 2% normal goat serum for 1\u2009hour. Primary antibodies were then added at appropriate concentrations overnight at 4\u2009\u00b0C in a humidified chamber .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations."}
+{"text": "To assess the magnitude of difference in antibiotic use between clinical teams in the acute setting and assess evidence for any adverse consequences to patient safety or healthcare delivery.Prospective cohort study (1\u2005week) and analysis of linked electronic health records (3\u2005years).UK tertiary care centre.All patients admitted sequentially to the acute medical service under an infectious diseases acute physician (IDP) and other medical teams during 1\u2005week in 2013 (n=297), and 3\u2005years 2012\u20132014 (n=47\u2005585).Antibiotic use in days of therapy (DOT): raw group metrics and regression analysis adjusted for case mix.30-day all-cause mortality, treatment failure and length of stay.Antibiotic use was 173 vs 282 DOT/100 admissions in the IDP versus non-IDP group. Using case mix-adjusted zero-inflated Poisson regression, IDP patients were significantly less likely to receive an antibiotic (adjusted OR=0.25 (95% CI 0.07 to 0.84), p=0.03) and received shorter courses (adjusted rate ratio (RR)=0.71 (95% CI 0.54 to 0.93), p=0.01). Clinically stable IDP patients of uncertain diagnosis were more likely to have antibiotics held . There was no significant difference in treatment failure or mortality , but IDP patients were more likely to be admitted overnight (adjusted OR=3.53 (95% CI 1.24 to 10.03), p=0.03) and have longer length of stay (adjusted RR=1.19 (95% CI 1.05 to 1.36), p=0.007).The IDP-led group used 30% less antibiotic therapy with no adverse clinical outcome, suggesting antibiotic use can be reduced safely in the acute setting. This may be achieved in part by holding antibiotics and admitting the patient for observation rather than prescribing, which has implications for costs and hospital occupancy. More information is needed to indicate whether any such longer admission will increase or decrease risk of antibiotic-resistant infections. Clinical relevance of study population, conducted in \u2018real-life\u2019 hospital environment.Robust risk-adjusted multivariable analysis of prescribing to show differences in antibiotic use is not an artefact of case mix.Analysis of clinical outcome as well as prescribing outcome showing antibiotic reduction was achieved safely.Data collection by single physician unblinded to management team, possibility of recording bias, though objective criteria used where possible.Single-centre study; relevance to other institutions requires further exploration.Owing to the increasing profile of the threat of antimicrobial resistance, there has been an increased focus on measuring and reducing antibiotic use.Previous studies have shown that management input from an infectious diseases acute physician (IDP) can reduce antibiotic use in the acute hospital setting. Studies have demonstrated that IDP input for patients managed under other acute physicians is associated with shorter durations of treatment, with more patients having treatment stopped or shortened.11et alet al12A few studies have compared management of acute medical patients by infectious disease and non-infectious disease-trained hospitalists.Prescribing quality assessments in the acute medical setting have suggested that up to one-third of prescriptions may be inappropriate.The goal of our study was to identify whether general medical physicians could make significant, safe antibiotic reductions, and to assess the magnitude of this achievable reduction in an entire, mixed patient cohort from point of referral to the acute medical service, rather than in selected conditions.We also wished to investigate whether there was any evidence for adverse effects from lower antibiotic use on patient outcome. We were able to do this because in our institution, a tertiary centre, some acute medical unit teams are led by an IDP. Our primary objective was therefore to assess the difference in total amount of antibiotic used between IDP-led and non-IDP-led teams. Comparison at a consultant team level reflected working practices, and previous studies supporting the commonly held view among physicians that it is the \u2018senior doctor who ultimately decides what antimicrobial is prescribed\u2019.10.1136/bmjopen-2015-010969.supp2Supplementary figuresAntibiotic prescribing data were collected prospectively on all acute admissions to the medical service in a UK tertiary care centre  by the lead author, an active physician in the department, but not working during this period. Data were collected as a benchmarking audit of antimicrobial prescribing. In this service, patients are admitted from primary care and Emergency Department referrals, and managed under an on-call consultant acute physician and their team, who share an on-call team rota with three shifts per day . The team makes initial management decisions (including antibiotic prescribing) before or during a consultant ward round of all newly admitted patients. After initial care, a small proportion of patients are transferred for operational reasons to the nearest available bed under any team or to other non-acute specialities (<5%); the majority are managed under admitting team , readmission, escalation/restarting antibiotic treatment and disease-related complications  diagnosis codes, length of stay, mortality and readmissions. Patient-level prescribing and clinical response data were not available in this data set. During this period, the IDP performed clinical duties in the acute medicine service, and other departments together with periods of leave, thus admitting \u223c1% of patients in this data set.Antibiotic use was calculated using several metrics using previously published definitions,We hypothesised, based on small pilot data, that IDP management would lead to 30% less total antibiotic use, with no difference in clinical adverse composite outcome. In total, 367 patients provided at least 80% power to detect a reduction in overall antibiotic use from 300 to 210 DOT/100 admissions (30% reduction) based on 10\u2005000 simulations from a Poisson distribution, assuming that 20% patients were admitted under IDP during audit period, overall 30% patients received antibiotics and two-sided \u03b1=0.05. Acute medicine admits 300\u2013400 patients per week; 1\u2005week was therefore chosen as a representative audit sample including out-of-hours and weekend prescribing.2 or Fisher's exact tests for categorical factors. Antibiotic prescribing was modelled as a two-step process , representing clinical experience and national prescribing guidance,Baseline characteristics between IDP and non-IDP groups were compared using Wilcoxon Rank Sum tests for continuous and Pearson \u03c7The baseline antibiotic prescribing audit was approved by the Oxford University Hospitals Clinical Audit Service. Mortality and admission comparisons over 2012\u20132014 were an approved analysis within the Infections in Oxfordshire Research Database, which has Research Ethics Committee (14/SC/1069) and UK Health Research Authority (5-07(a)/2009) approval as an anonymised database without individual informed consent. The IDP gave consent to be identified in these data, and for the prescribing outcome analysis to be performed.During a 1-week period, the IDP-led team admitted 56 patients, and non-IDP Acute Physician teams admitted 241 patients. Teams admitted similar patients, except IDP patients had slightly lower white cell counts (WCC) and were more likely to be admitted with a frailty syndrome . Group mIn a patient-level risk-adjusted model , broad-spectrum antibiotic DOT (p=0.08), number of patients started on an intravenous antibiotic (p=0.12) or intravenous DOT (p=0.23). The reduction in total antibiotic use was not due to reduced use of antibiotic combinations; analysis by total length of therapy (which counts days on single or combination therapy as a single day) also showed a 39% unadjusted total reduction (129 vs 210 LOT/100 admissions) . AntibioThere was no difference in antibiotic prescription rates between IDP versus non-IDP groups in perceived high-risk patients , or in patients with low likelihood of infection. However, there was a difference in antibiotic use in cases of uncertain infective diagnosis without adverse features . In the There was no evidence that management under IDP was associated with risk-adjusted 30-day all-cause mortality or the composite \u2018treatment failure\u2019 outcome . Similar to our 1-week data set, this comprised patients admitted to the acute, unselected medical take, during which time the same consultant teams were operating with broadly the same antibiotic policy. Patient characteristics available in the 1\u2005week and 3-year data sets were broadly similar in terms of age, gender, Charlson score  and admission at a weekend  an attitude in the IDP team of \u2018you must justify why you gave antibiotics\u2019 compared with \u2018give antibiotics, just in case\u2019 (as previously described by others),Study strengths include the \u2018real life\u2019 clinical relevance of the study population and robust risk-adjusted multivariable analysis of patient-level data to study prescribing and clinical outcome. Limitations include the restricted audit size, which was also smaller than planned. While we were able to subsequently examine a larger administrative data set with similar patient characteristics to assess outcome, individual-level prescribing and clinical data were not available, requiring extrapolation that IDP and non-IDP antibiotic use would have been similar over the longer period of time. During the audit, data collection was by a single clinician unblinded to management team, with possibility of recording bias for clinical factors, although objective criteria were used wherever possible. Although acute physicians were made aware of the audit and the exact date were not announced, the IDP may have been more aware of the monitoring due to closer professional relationships with the lead author, and larger differences in prescribing found due to extra attention on antibiotic use in the IDP team. The study focused on one IDP; while IDPs with similar training and integration to acute medicine exist around the UK, replicating the findings with more IDPs at different locations is required before these findings can be generalised to IDPs as a group. The study was single centre; understanding generalisability to other centres remains important. Cost impact has not been assessed, though we note that as overnight stays currently cost over \u00a3250,Our findings confirm those of other studies of the potential for antibiotic use reduction in the acute mixed-case setting. A study of 129 adult patients admitted to a US tertiary hospital found 30% of total antimicrobial days were judged unnecessary by study investigators,29et al who found IDP management was associated with shorter lengths of stay.imilar to previous studies, we found IDP input associated with a reduction in antibiotic use.Our study adds to this body of work by demonstrating reduced antibiotic prescribing using patient-level data to adjust for case mix and conducting a robust analysis of clinical outcome data to assess patient safety, providing further evidence of the potential for safe antibiotic reduction. It also examines for the first time the effects of IDP management in patients at presentation, rather than after a period of assessment and provisional diagnosis of infection being made. It reports an association with reduced antibiotic use and increased admissions in the acute setting, contrary to previous studies.13In conclusion, our study provides further support that antibiotic reduction can be achieved safely in the acute setting, but at the expense of more and longer admissions. This could be done by replacing a perceived \u2018antibiotic safety net\u2019 (prescribe and discharge) with one based on observation and greater degree of diagnostic certainty (hold and observe). While the findings require replication in other healthcare settings, they suggest that attempts to reduce antibiotic use may come at the expense of increasing admissions and exposure to the hospital environment; this may increase costs to healthcare services and the burden on already-stretched acute services. Given that admission to hospital is a risk factor for resistant infection, it may also conversely increase the spread of resistance."}
+{"text": "Mitosis is a complex and dynamic process that is tightly regulated by a large number of mitotic proteins. Dysregulation of these proteins can generate daughter cells that exhibit genomic instability and aneuploidy, and such cells can transform into tumorigenic cells. Thus, it is important for faithful mitotic progression to regulate mitotic proteins at specific locations in the cells at a given time in each phase of mitosis. Ubiquitin-dependent modifications play critical roles in this process by regulating the degradation, translocation, or signal transduction of mitotic proteins. Here, we review how ubiquitination and deubiquitination regulate the progression of mitosis. In addition, we summarize the substrates and roles of some deubiquitinating enzymes (DUBs) crucial for mitosis and describe how they contribute error correction during mitosis and control the transition between the mitotic phases. In general, a tumor is caused by abnormal cells that undergo unrestricted divisions and proliferation. These events are the same as those that manifest upon the failure to control mitosis, and such a failure eventually results in cell death or tumorigenesis. Mitotic defects can occur at any phase of mitosis. Specifically, dysregulation of the spindle assembly checkpoint (SAC) leads to prolonged mitotic arrest and constitutes the major cause of several mitotic defects. Hence, the SAC is an important target for the development of antiproliferative chemotherapeutic strategies . Moreove(1)Prophase: Mitosis begins with the nuclear envelope breakdown (NEBD), an essential step for spindle assembly, followed by condensation of replicated DNA in the chromosome. During prophase, the two duplicated centrioles move to the opposite poles, where each pair forms a centrosome. The two centrosomes then nucleate the polymerization of microtubules from the opposite ends, forming the spindle.(2)Prometaphase: This phase is a dynamic part of mitotic progression. Microtubules rapidly assemble and disassemble by growing out from the duplicated centrosomes to find the accurate attachment site at the kinetochores of the chromosomes. The attached microtubules pull each chromosome from the opposite sites until all the chromosomes are bi-oriented and aligned.(3)Metaphase: The assembly of the mitotic spindle and its correct attachment to the kinetochore of sister chromatids are stabilized, completing the alignment of sister chromatids at the equator of the spindle for proper segregation of chromosomes toward the opposite poles of the spindle ,4,5. How(4)Anaphase: After the requirements of the SAC are satisfied, the cell enters anaphase. During this phase, microtubules attached to the duplicated chromosomes shorten from the opposite sites, separating the chromosome pairs and pulling each chromosome of a pair toward opposite spindle poles . Followi(5)Telophase: Once all the chromosomes reach their poles, the final phase of mitosis, termed telophase, begins. During telophase, the nuclear envelope reforms around the nuclei of daughter cells and chromosomes decondense .(6)Cytokinesis: This step refers to the division of the cytoplasm into two daughter cells. A cytokinetic furrow formed by the contraction of the actomyosin ring splits the cytoplasm into two domains. At this stage, two daughter cells remain connected by a narrow intracellular bridge containing antiparallel bundles of microtubules that overlap at the mid-body. The physical separation of the two daughter cells is finally accomplished by the fission of the plasma membrane via the process called abscission ,10.The cell cycle refers to a series of processes including DNA synthesis (S phase), cell growth (G1 phase), evaluation of the accuracy of the genomic materials (G2 phase), and cell division (M phase). Of these phases, mitosis (M phase), which occurs only in eukaryotic cells, is an important step in ensuring the stability of the entire genome by duplicating the genetic information and equally segregating it into two daughter cells . MitosisUbiquitination is a reversible PTM, which involves the covalent attachment of the small conserved protein ubiquitin (Ub) to a target protein, almost exclusively at a lysine residue . It requDeubiquitination is the reverse process of ubiquitination, that is, excise the conjugated monoubiquitin or polyubiquitin chains from the modified proteins . This prAlthough DUB paralogs are highly conserved in function, they can be divided into the following six families based on the architecture of their catalytic domains: ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), ovarian tumor proteases (OTUs), Machado-Josephin proteases (MJDs), JAB1/MPN/MOV34 (JAMMs), and MIU-containing novel DUB family (MINDY). The first four families are cysteine proteases, whereas JAMMs are metalloprotease . MINDY iSeveral E3 ligases participate in mitotic control at almost every phase. Of these, the anaphase-promoting complex/cyclosome (APC/C) E3 ligase plays an important part, as it controls the metaphase-to-anaphase transition by ensuring accurate chromosome segregation and regulates the mitotic exit by mediating the degradation of key mitotic cyclins ,20,21. AModulation of the localization of Aurora B affects its functions during mitosis. During metaphase, Aurora B participates in the destabilization of erroneous microtubule attachments at the kinetochores ,33. DuriSeveral DUBs have been reported involved in mitotic progression . For exaThe SAC plays an important role against chromosomal missegregation and generation of aneuploidy progeny, which is a remarkable common characteristic of tumorigenesis, by delaying sister chromatid separation until all chromosomes achieve bipolar kinetochore\u2013microtubule attachment. Once the last chromosome bi-orients on the mitotic spindle, APC/C activated by the binding of CDC20 ubiquitinates securin and cyclin B1 to induce their proteasomal degradation and then initiates chromosome segregation .\u2212/\u2212 mouse embryonic fibroblast (MEF) cells [\u2212/\u2212 MEF cells, abnormal spindle geometry and incomplete centrosome separation are increased, indicating that defects in these processes due to USP44 loss of function result in mitotic chromosome missegregation and aneuploidy. USP44 directly interacts with the centrosome component centrin and localizes to the centrosome during interphase. The DUB activity of USP44 and its ability to interact with centrin are critical for preventing chromosome lagging. Aneuploidy is very common in human lung adenocarcinoma, and reduced expression of USP44 is frequently observed in patients with lung adenocarcinoma [USP44 is a critical regulator of the SAC. Stegmeier et al. identified that USP44 is required for efficient SAC signaling and anaphase onset. They observed that the depletion of USP44 abolishes the checkpoint function of the SAC, but overexpression of a siRNA-resistant USP44 rescues the function of the SAC in USP44-depleted cells . In addiF) cells . An incrF) cells . SeveralF) cells . In USP4arcinoma . In otheUSP9X has been implicated in several disorders, including X-linked intellectual disability , ParkinsUSP9X has previously been reported to deubiquitinate and consequently stabilize the prosurvival BCL2 family member MCL1 by preventing its proteasomal degradation, thereby promoting cell survival and contributing to chemoresistance in B-cell lymphoma . HoweverRecently, Skowyra et al. showed that USP9X inhibits the degradation of SAC-controlled APC/C substrates, such as cyclin B1, cyclin A, and NIMA-related kinase 2A (NEK2A) during a mitotic arrest b. SpecifCYLD is a deubiquitinating enzyme that cleaves Lys63-linked polyubiquitin chains off its target proteins . OriginaUSP35 functions as a mitotic regulator by deubiquitinating Aurora B kinase and maintaining its stability during mitosis . Aurora While USP35 counteracts the ubiquitination effect of APC/C on Aurora B, Cezanne antagonizes the degradation of other APC/C substrates without the involvement of Aurora B. Cezanne, which is also called OTUD7B, belongs to the OTU family that has ubiquitin linkage specificity , and thiAs reviewed here, ubiquitination and deubiquitination catalyzed by E3 ubiquitin ligases and DUBs, respectively, regulate mitotic progression at all stages of mitosis. Ubiquitin-dependent modifications during mitosis have been shown to play important roles in the regulation of the key components of several events, including chromosome condensation, alignment, and segregation, for a faithful mitotic progression. Additionally, the misregulated expression of DUBs involved in the modifications supports aberrant mitotic progression, leading to the development of many types of cancers . As show"}
+{"text": "The feasibility of improving the extraction rate of common bean antioxidants by ultrasonic treatment was investigated. Scanning electron microscopy (SEM) and spectrum Fourier transform infrared spectrophotometer (FT-IR) analysis revealed that ultrasonic treatment substantially altered the cellular structure of common bean seed, resulting in increased surface area, eroded cell walls, and greater exposure of cellulose and hemicellulose. The highest antioxidant activity was obtained at optimal extraction conditions  which were optimized by response surface methodology. In terms of the extraction rate of common bean antioxidants, ultrasound-assisted extraction (UAE) exhibits about seven-fold higher extraction efficiency than conventional solvent extraction (CSE). In addition, 10 phenolic compounds in the common bean extracts were detected and quantified by high performance liquid chromatography (HPLC), including protocatechuic acid, catechin, chlorogenic acid, epicatechin, ferulic acid, coumarin, rutin, myricetin, cinnamic acid, and genistein. In summary, ultrasonic treatment is an ideal candidate methodology for improving the extraction rate of common bean antioxidants. Phaseolus vulgaris L.) are the second most important bean species in the world after soybean [In recognition of the great importance of pulses for sustainable agriculture and human nutrition, Food and Agriculture Organization of the United Nations (FAO) officially proclaimed 2016 as \u201cthe International Year of Pulses\u201d, aiming to draw attention to these important crops. Common beans  berries [Ultrasound-assisted extraction of antioxidants in common beans is influenced by several factors such as extraction solvent, extraction time, ultrasonic power, and solvent-to-solid ratio, which can be optimized to reduce process costs . Respons berries . In addiDried speckled kidney bean, a type of common bean  was purchased from Wal-Mart Stores in Kunming, Yunnan, China, and was produced in 2017. Prior to extraction, the dried beans were ground to fine powder using a laboratory-scale dry grinder . The powder was sifted through a 40-mesh sieve to obtain kidney bean powder of particle size less than 500 \u03bcm and was stored at 4 \u2103 and used within 1 month.g, 22 \u00b1 1 \u2103) and the supernatant was collected and stored at 4 \u2103, and analyzed within 24 h.The ultrasonic treatment of common beans was performed in a tunable ultrasonic bath. The sample (0.5 g) and certain volume of extraction solvent were added to a 50 mL tube. Ultrasonic power, extraction time, and extraction temperature were controlled by the equipment panel. Here, the extraction solvent consisted of water and acetone in different ratios, and the detailed ratios, extraction time, extraction temperature, and ultrasonic power were based on experiment design charts. After extraction, the sample was centrifuged  and the supernatant was collected and stored at 4 \u2103.In order to compare with UAE, CSE was carried out under the optimal extraction conditions obtained by response surface methodology. Samples (0.5 g) were extracted at 30 \u2103 for 68 min, using 18 mL 50% acetone as solvents in an electric hot water bath . After extraction, the sample was centrifuged , \u03bcmol Fe (II)/g dry weight (DW)) and Y2 , \u03bcmol Trolox/g DW), respectively. The Pareto Chart of Standardized Effects was used to illustrate the main effect or interactive effect, which is considered to be statistically significant when its magnitude is larger than the other contrast column effects [A two-level factorial (default generators) design was applied to build the distribution matrix, which was generated by Minitab 17 software . This design comprehensively examined the effects of ultrasonic time, extraction temperature, ultrasonic power, liquid-to-solid ratio, and acetone concentration on the common bean antioxidants and screened the key factors that have significant effects on the extraction process. The low and high levels were assigned as \u201c\u22121\u201d and \u201c+1\u201d, respectively,  and the  effects .1, 40-60-80 min), acetone concentration , and liquid to solid ratio  were selected as the major influential independent variables, and their related coded levels of independent variables were presented in 3 central composite design was used to evaluate the effects of the three independent variables on the extraction efficiency of antioxidants, which were reflected by two dependent responses Y1 and Y2.Based on the DOE results, the extraction time  was used for data regression analysis and estimation of regression equation coefficients. An empirical model was obtained by correlating the measured responses with independent variables using multiple regression analysis. The following equation was used to predict the second\u2212order response function for the experiments.The antioxidant activities of common bean extracts were evaluated by DPPH free radical scavenging activity (DPPH) assay and ferric-reducing antioxidant power (FRAP) assay according to our previously published work , and thev/v) formic acid in water) and solvent B (0.1% (v/v) formic acid in methanol). The analysis was performed by a gradient elution program: 0 min, 5% B (v/v); 3 min, 10% B (v/v); 13 min, 28% B (v/v); 23 min, 45% B (v/v); 38 min, 70% B (v/v); 43 min, 90% B (v/v); 48\u201363 min, 95% B (v/v). The sample injection volume was 20 \u03bcL, and the flow rate was set at 1.0 mL/min. Phenolic compounds were identified by comparison with the retention time and UV spectra of standards, and their contents were calculated based on the peak area under the maximum absorbance and calibration curve of the corresponding standard at 10\u2013100 \u03bcg/mL. The results were expressed as mg/100g DW.The supernatant obtained under optimal extraction conditions was collected and lyophilized and then dissolved in methanol for HPLC analysis. Major phenolic compounds in the common bean extracts were identified and quantified using Shimadzu LC-20AR HPLC system  equipped with a Shim-pack GIS C18 reversed phase column , a binary pump, and a diode array detector . The mobile phase consists of solvent A  was used to observe the surface structures of raw and treated samples. A spectrum Fourier transform infrared spectrophotometer  was applied to analyze the infrared signatures of the raw and treated common beans.p value less than 0.05 defined as statistical significance. The statistical software Design Expert 8.06 , IBM SPSS Statistics 19 , Minitab 17 software , and Origin 8.5  were used for statistical analysis.All the measurements were performed in triplicate, and the results were expressed as mean standard deviation (SD), with DOE technique offers a structured approach for generating a significance test of factor levels and providing a prediction model for response. Here, a two-level factorial (default generators) design was used to build the distribution matrix and the results are shown in p < 0.0001) for FRAP and DPPH values. The lack of fit for both models was not significant (p > 0.05), which indicated that the established models fully explain the relationship between independent variables and responses. The R2 and adjusted R2 (Adj. R2) were close to 1, indicating a high degree of correlation between the predicted values and experimental values. Moreover, the predicted R2 (Pred R2) of each model was in reasonable agreement with the Adj. R2, indicating that the model predicts the response value very well. The Adeq Precision of each model is larger than 4, indicating that the model can be navigated in the design space. Furthermore, the coefficient of variance (CV) of each model was rather low (CV < 3%), which further supported the good adaptability of the model, thereby assuring better reproducibility. The resulting response surface 3D graph corresponding to each response is shown in Experimental modeling results for antioxidant activity  show tha1, X2, X3), quadratic (X2X3), and interactive , acetone concentration (X2), and liquid-to-solid ratio (X3) showed a highly significant (p < 0.0001) positive effect on FRAP; moreover, their quadratic terms (p < 0.0001) negative effect on FRAP. The interaction between the acetone concentration and liquid-to-solid ratio (X2X3) revealed a significant (0.0024) negative effect on FRAP  and liquid-to-solid ratio (X3) showed a highly significant (p < 0.0001) positive effect on DPPH, and their quadratic terms (p < 0.0001) negative effect on DPPH; moreover, p = 0.0210) negative effect on DPPH. The interaction between the extraction time and acetone concentration (X1X2) had a significant (p = 0.0004) negative effect on DPPH. The interaction between the extraction time and liquid-to-solid ratio (X1X3) revealed a significant (p = 0.0421) positive effect on DPPH  were estimated by RSM, taking into account only the significant terms, which were shown in Equations (2) and (3):R2 value  fairly matched the adjusted R2 value   .2) and liquid-to-solid ratio (X3) revealed a positive effect for both responses. The interactive effects exhibited quite different patterns for the two responses (Y1 and Y2). Similar results were also obtained by others [In brief, acetone concentration /g DW and 50.9 \u03bcmol Trolox/g DW, which were well matched with the predicted values of 77.2 \u03bcmol Fe(\u2161)/g DW and 51.8 \u03bcmol Trolox/g DW with the coefficient of variance values of 2.68% and 1.84%, respectively.Two extraction methods (UAE and CSE) were compared with regard to their extraction efficiency. These two extraction methods were performed under the same optimum extraction conditions obtained by central composite design (CCD) .In terms of extraction efficiency, UAE gave FRAP and DPPH values of 75.2 \u03bcmol Fe(\u2161)/g DW and 50.9 \u03bcmol Trolox/g DW, respectively, while CSE offered 9.87 \u03bcmol Fe(\u2161)/g DW (FRAP) and 7.76 \u03bcmol Trolox/g DW (DPPH), indicating that the UAE method yielded about eight-fold higher FRAP and about seven-fold higher DPPH than CSE method. Producing higher antioxidant activities through UAE can be attributed to the ultrasonic wave facilitating solvent penetration into the sample matrix and increasing the rates of mass transfer of antioxidants to the extraction solvent . SimilarPhaseolus vulgaris L.) [Ten polyphenolic antioxidant compounds in the extracts, including protocatechuic acid, catechin, chlorogenic acid, epicatechin, ferulic acid, coumarin, rutin, myricetin, cinnamic acid, and genistein, were detected and quantified by HPLC with standard curves. The chromatography of standards is shown in aris L.) . In addiaris L.) . In addiaris L.) . Interesaris L.) . Flavonoaris L.) . Phenoliaris L.) . TherefoDifferences of the common bean slurries before and after ultrasonic treatment a show en2O molecules and produces hydroxyl and hydrogen radicals, which then excite a chain reaction. The chain reaction produces more free radicals or oxidizing species such as hydrogen peroxide, oxygen, and ozone [\u22121 was enhanced, and it represented the stretching vibration of O\u2013H and hydrogen bonds in phenolic and aliphatic structures [\u22121 represented the stretch vibrations of the C\u2013H bond in the methylene of cellulose [\u22121 change, which may be related to the demethylation of the syringyl structure of lignin [\u22121 is the C\u2013H signal in planar deformation of glucosyl group in lignin [\u22121, which is assigned to C\u2013O stretching and is likely the C\u2013O bond of cellulose and hemicellulose [In addition to the physical effects of ultrasound, the sonochemical effect is another important factor in the change of the physicochemical properties of common beans. The sonochemical effects originate from a series of complex free radical reactions in water. Initially, ultrasonication cleaves the O\u2013H bonds of Hnd ozone . The oxiructures . The enhellulose ,33. The f lignin . The enhn lignin . These rellulose . This inIn this study, ultrasound-assisted extraction of common bean antioxidants was optimized by RSM. The optimal extraction parameters were determined, and the predicted values were close to the experimental values. The optimal UAE conditions for common bean antioxidants included an extraction time of 68 min, extraction solvent concentration of 55%, liquid-to-solid ratio of 36 mL/g, extraction temperature of 30 \u2103, and ultrasonic power of 480 W. Under these optimal conditions, the maximum FRAP and DPPH values of common beans were 75.2 \u03bcmol Fe(\u2161)/g DW and 50.9 \u03bcmol Trolox/g DW, respectively.Compared to conventional extraction methods, ultrasonic treatment significantly increases the extraction rate of antioxidants, which were further analyzed using HPLC, and 10 phenolic compounds were detected, including protocatechuic acid, catechin, chlorogenic acid, epicatechin, ferulic acid, coumarin, rutin, myricetin, cinnamic acid, and genistein. In addition, the effects of ultrasound on common bean structures were analyzed using SEM and FT-IR, finding that ultrasonic treatment led to a loose structure and erosion of the surface of common beans, which could result in more rapid and greater penetration of the solvent into the plant material."}
+{"text": "Platycarya strobilacea is a rich source of ellagic acid (EA) which has shown antioxidant, anticancer and antimutagen properties. Response surface methodology (RSM) was used to optimize the conditions for ultrasonic extraction of EA from infructescence of P. strobilacea. A central composite design (CCD) was used for experimental design and analysis of the results to obtain the optimal processing parameters. The content of EA in the extracts was determined by HPLC with UV detection. Three independent variables such as ultrasonic extraction temperature (\u00b0C), liquid:solid ratio (mL/g), and ultrasonic extraction time (min) were investigated. The experimental data obtained were fitted to a quadratic equation using multiple regression analysis and also analyzed by appropriate statistical methods. The 3-D response surface and the contour plots derived from the mathematical models were applied to determine the optimal conditions. The optimum ultrasonic extraction conditions were as follows: ultrasonic extraction temperature 70 \u00b0C, liquid:solid ratio 22.5, and ultrasonic extraction time 40 min. Under these conditions, the experimental percentage value was 1.961%, which is in close agreement with the value predicted by the model.The infructescence of  Extracts from red raspberry leaves or seeds, pomegranates, or other sources are said to contain high levels of EA, and are available as dietary supplements in capsule, powder, or liquid forms. A recent profusion of pomegranate nutraceutical products, \u2018\u2018standardized to 40% EA\u201d has appeared in the marketplace [Platycarya strobilacea Sieb. et Zucc, EA is present in the heartwood, as reported by Tanaka et al. [P. strobilacea. Polyphenols are widely distributed in the plant kingdom and are important components of common foods, including tea, red wine, fruits, beverages and various medicinal plants. The importance of polyphenols arises from their effects on sensory properties, including astringency and colour, and possible health effects that they may have. One of the polyphenols is ellagic acid (EA), a dimeric derivative of gallic acid, which mainly exists in higher plants, including fruits and flowers, combined with its precursor, hexahydroxydiphenic acid or bound in the form of ellagitannins . EA was ketplace . EA was ketplace . Plants ketplace . In the a et al. , but theP. strobilacea can be affected by many factors including ultrasonic extraction temperature, liquid:solid ratio, and ultrasonic extraction time. In such situations, where multiple variables may influence the extraction yield, response surface methodology (RSM) is an effective technique for optimizing the process [The efficiency of the extraction of EA from the infructescence of  process ,11. The  process . As a po process .P. strobilacea using response surface methodology (RSM) employing a CCD (three factors and five levels) to study the effects of ultrasonic extraction temperature, liquid:solid ratio, and ultrasonic extraction time on the extraction yield of EA.The purpose of the present study was to optimize the process for production of EA from the infructescence of P. strobilacea, as shown by a typical HPLC chromatogram of the extracts ; x2, ultrasonic extraction time (min); x3, ultrasonic extraction temperature (\u00b0C). ANOVA was used to evaluate the significance of the coefficients of the models. The regression coefficient values of equation are listed in P-values were used as a tool to check the significance of each coefficient, which in turn may indicate the pattern of the interactions between the variables. For any of the terms in the model, a large regression coefficient and a small P-value would indicate a more significant effect on the respective response variables [P, the more significant was the corresponding coefficient. It can be seen from this table that the linear coefficients  and the quadratic term coefficients  were significant, with very small P-values (P < 0.05). The other term coefficients were not significant (P > 0.05). Response surface optimization is more advantageous than the traditional single parameter optimization in that it saves time, space and raw materials. A total of 20 runs were needed for optimizing the three individual parameters in the current CCD. ariables . Thus, tP > 0.05) are eliminated from the model through a \u201cbackward elimination\u201d process. The statistical parameters obtained from the ANOVA for the reduced models are given in P < 0.05 is obtained, implying that these models are significant. The adequate precision value is a measure of the \u201csignal (response) to noise (deviation) ratio\u201d. A ratio greater than four is desirable [To obtain a simple and yet realistic model, the insignificant terms . In the figures, the maximum predicted value indicated by the surface was confined to the smallest ellipse in the contour diagram. Elliptical contours are obtained when there is a perfect interaction between the independent variables ,17. The In P. strobilacea were ultrasonic extraction temperature 70 \u00b0C, liquid:solid ratio 22.5, and ultrasonic extraction time 40 min. Among the three extraction parameters studied, ultrasonic extraction temperature was the most significant factor affecting the EA extraction yield, followed by liquid:solid ratio, and ultrasonic extraction time according to the regression coefficients significance of the quadratic model  was collected from the ground in Liuan, Anhui Province, China. The infructescence was ground in a knife mill and the powdered sample was sieved to select particles smaller than 1 mm. EA was purchased from Sigma Chemical Co. . The solvents methanol and trifluoroacetic acid (TFA) were of analytical reagent (AR) purity grade. The CH3CN used for the analysis were of HPLC grade. Deionized water was used throughout.Dried infructescence of Y) was as follows:Y represents the response variable, a0 is a constant, ia, iia and ija are the linear, quadratic and interactive coefficients, respectively. iX and jX are the levels of the independent variables. The software uses this quadratic model to build the response surface. The adequacy of the model was determined by evaluating the lack of fit, coefficient of determination  and the Fisher test value  obtained from the analysis of variance (ANOVA) that was generated by the software. Statistical significance of the model and model parameters was determined at the 5% probability level (\u03b1 = 0.05). Three-dimensional surface response plots were generated by varying two variables within the experimental range and holding the other constant at the central point. RSM was applied to evaluate the effects of ultrasonic extraction temperature, liquid:solid ratio, and ultrasonic extraction time on the yields of EA. RSM was performed using the Design-Expert software program version 7.0. The coded and uncoded independent variables used in the RSM design are listed in et al. [The EA was extracted according to the method described by Bianco et al. . An amou3CN with the solvent programmed as follows: 0-8th minutes 7% B, 8th-25th minutes 7%-32% B, 25th-30th minutes 32%-35% B, 30th-35th minutes 35% B. EA peak was identified by comparing its retention time with those of standards and the concentration was calculated from the calibration curves. The analysis was carried out in triplicate.The polyphenol fraction was isolated by methanolic extraction and EA was determined in the extracts by HPLC with UV detection. The detector was operated at 357 nm wavelength which corresponded to the experimentally found maximum absorption of the EA standard. An aliquot of the extract were filtered through a 0.45 \u00b5m syringe filter prior to HPLC-UV analysis. EA was separated using an Inertex C18 column . The solvent flow rate was 1 mL/min and the mobile phase was composed of solvent (A) water  and solvent (B) CHExperimental data was analyzed by multiple regressions to fit the quadratic equation to all independent variables. Analysis of variance (ANOVA) was performed to evaluate significant differences between independent variables. To visualize the relationships between the responses and the independent variables, surface response and contour plots of the fitted quadratic regression equations were generated using Design-Expert software version 7.0.The extraction conditions have a significant effect on the EA extraction yield. The use of the contour and surface plots in RSM was effective for estimating the effect of three independent variables . The optimum set of the independent variables was obtained graphically in order to obtain the desired levels of EA extraction. An optimal experimental extraction yield of 1.961% was obtained when the optimum conditions of EA extraction  were used. Under these optimized conditions the experimental EA extraction yield agreed closely with the predicted yield of 2.028%."}
+{"text": "Annona squamosa L.) is a popular tropical fruit and its peel is a municipal waste. An ultrasound-assisted extraction method was developed for the recovery of natural antioxidants from sugar apple peel. Central composite design was used to optimize solvent concentration (13.2%\u201346.8%), ultrasonic time (33.2\u201366.8 min), and temperature (43.2\u201376.8 \u00b0C) for the recovery of natural antioxidants from sugar apple peel. The second-order polynomial models demonstrated a good fit of the quadratic models with the experimental results in respect to total phenolic content , FRAP , and TEAC  values. The optimal extraction conditions were 20:1 (mL/g) of solvent-to-solid ratio, 32.68% acetone, and 67.23 \u00b0C for 42.54 min under ultrasonic irradiation. Under these conditions, the maximal yield of total phenolic content was 26.81 (mg GA/g FW). The experimental results obtained under optimal conditions agreed well with the predicted results. The application of ultrasound markedly decreased extraction time and improved the extraction efficiency, compared with the conventional methods.Sugar apple ( Fruit wastes are one of the main sources of municipal wastes. Due to the high consumption and industrial processing of fruit edible parts, fruits residues  are generated in large quantities in large cities and become a severe environmental issue. However, if the phytochemicals of fruit residues were extracted effectively by applying efficient extraction technologies, their value could be added ,2. RecenAnnona squamosal L. (Annonaceae) is an important plant and widely distributed in tropical America and Asia [Annona squamosal bears edible fruits called sugar apple. It is a popular tropical fruit, typically globular or heart-shaped, 5\u201310 cm in diameter, with many round protuberances. The pulp is sweetly aromatic with a custard-like flavor, and is widely used to prepare juices, jellies, and compotes. The processing of the fruits will leave behind a substantial amount of non-edible parts. In previous studies, extremely high phenolic content and antioxidant capacities were found in sugar apple peel, indicating its potential to be utilized as a resource of natural antioxidants [and Asia . It has and Asia . Annona oxidants ,8. So faoxidants ,12,13. Ioxidants . Ultrasooxidants ,16.The extraction efficiency and antioxidant capacity of natural antioxidants could be markedly influenced by many factors, such as solvent type and concentration, solvent-to-solid, as well as extraction temperature and time . TherefoIn the preliminary experiment of our study, the effects of solvent type, solvent-to-solid ratio, acetone concentration, ultrasonic time, and temperature on the extraction of natural antioxidants from sugar apple peel were studied. Under the same extraction conditions , the responses followed a similar trend among all the solvents tested a.p < 0.05). At the solvent-to-solid ratio of 20:1, a plateau in the mass transfer was reached. There were, indeed, no significant differences for solvent-to-solid ratios between 30:1, 40:1, 50:1, and 20:1 (p > 0.05). In consideration of the solvent consumption and extraction efficiency, solvent-solid ratio of 20:1 (mL/g) was selected. The effects of extraction temperature and time were shown in In the literature, several frequently-used solvents were reported, such as ethanol, methanol, and acetone ,19,20,21The results of 20 runs using CCD design were shown in p < 0.0001) with F-values of 22.23, 42.05, and 27.36, respectively (R2) is a measure of degree of fit [R2 should be at least 0.80 [R2 values of the second-order polynomial models for TPC, FRAP, and TEAC were 0.9524, 0.9743, and 0.9610, respectively, which implied that 95.24%, 97.43%, and 96.10% of the variations could be explained by the fitted quadratic models, respectively. Furthermore, the absence of any lack of fit (p > 0.05) also strengthened the reliability of all models. The models were used for the construction of three dimensional response surface plots to predict the relationship between the responses and independent variables.The analysis of variance (ANOVA) of the quadratic regression models for TPC, FRAP, and TEAC showed that the models were significant  and temperature (X3), and the cross product effect between acetone concentration and temperature , were positive and significant at p < 0.01, while the quadratic effect of acetone concentration (X12) were negative and significant at p < 0.001. It was evident that the linear effects of time  and temperature (X3) showed significantly positive linear effects and negative quadratic effects, respectively (p < 0.001). Ultrasonic time showed significantly negative linear effect on the response (p < 0.05). The cross product effect between acetone concentration (X1) and time (X2) was positive and significant at p < 0.05, whereas the cross product effect between time (X2) and temperature (X3) was negative and significant at p < 0.01. The 3D response surfaces plot for the effects of independent variables  on FRAP values were given in In term of FRAP, acetone concentration (X2), cross product terms of time (X2) and temperature (X3), all the terms had significant effects on the antioxidant capacities (p < 0.01). The 3D response surfaces plot X1, X2, X3). Seen from In aspect of TEAC, as shown in The optimum extraction conditions obtained by RSM was used to validate the predictive model of extraction for TPC, FRAP, and TEAC values from sugar apple peel. Seen from The compounds 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ), 2,2\u2032-azinobis(3-ethylbenothiazoline-6-sulfonic acid) diammonium salt (ABTS), Folin\u2013Ciocalteu\u2019s phenol reagent and gallic acid were purchased from Sigma\u2013Aldrich . Acetone, ethanol, methanol, acetic acid, hydrochloric acid, potassium persulphate, iron(III) chloride hexahydrate, iron(II) sulfate heptahydrate, sodium acetate, and sodium carbonate were of analytical grade and obtained from Tianjin Chemical Factory . Deionized water was used throughout the experiment.Sugar apples were collected from markets in Guangzhou, China. Samples were cleaned with deionized water and the peel was separated. Immediately, the separated peel was ground into fine particles with a special grinder.i.e., acoustic power of 0.38 W/cm2), heating power of 800 W, equipped with a digital timer and a temperature controller. In RSM test, sample particles (0.5 g) were sonicated in the acetone (10 mL) for different time at set temperature. After sonication, the sample was centrifuged at 9600 g for 20 min, and then the supernatant was collected and diluted for analysis. The reaction process happened in a 15 mL centrifuge tube with screw cap and conical bottom, which was fixed in a plastic rack placing in the ultrasonic device.The ultrasound-assisted extraction process was performed in a ultrasonic device  with an electric power of 600 W  at 37 \u00b0C for 24 h.The FRAP assay was performed according to the procedure described by Benzie and Strain  with min\u2022+ stock solution was prepared from 7 mmol/L ABTS and 2.45 mmol/L potassium persulfate in a volume ratio of 1:1, and then incubated in the dark at room temperature for 16 h and used within two days. A 100 \u03bcL of the tested sample was mixed with 3.8 mL ABTS\u2022+ working solution and the absorbance was detected at 734 nm after incubation at room temperature for 6 min. The percent of inhibition of absorbance at 734 nm was calculated and the results were expressed as \u00b5mol Trolox/g FW of fruit peel.The TEAC assay was performed based on the protocol established previously  with minTotal phenolic contents were determined with Folin\u2013Ciocalteu method . BrieflyX1), ultrasonic time (X2), and ultrasonic temperature (X3) were chosen for independent processing variables. The range and center point values of three independent variables presented in Y is the predicted response, \u03b20 is an intercept, \u03b2i, \u03b2ii, and \u03b2ij are the coefficients of the linear, quadratic, and interaction terms, respectively. iX and jX are coded independent variables.Optimization of extraction conditions for natural antioxidants from sugar apple peel were carried out using response surface methodology (RSM). A three-variable, five level central composite design (CCD) was applied to determine the best combination of extraction variables. Acetone concentration (R2), and the Fisher test value  generated from the analysis of variance (ANOVA) by the software. The difference was considered significant at p < 0.05.The regression and graphical analysis of the experimental data were performed by Design Expert version 7.1.3 software . The suitability of the model was checked by evaluating the lack of fit, coefficient of determination  of solvent-to-solid ratio, 32.68% acetone and 67.23 \u00b0C for 42.54 min under ultrasonic irradiation. The application of ultrasound markedly decreased extraction time, and improved the extraction efficiency compared with the conventional methods.An ultrasound-assisted extraction method has been developed for the recovery of natural antioxidants from sugar apple peel. Response surface methodology was used to optimize the experimental variables such as solvent concentration, ultrasonic time, and temperature. The"}
+{"text": "Glycine max var) sprouts. Three influencing factors: liquid-solid ratio, period of ultrasonic assisted extraction and extraction temperature were investigated in the ultrasonic aqueous extraction. Then Response Surface Methodology (RSM) was applied to optimize the extraction process focused on DPPH radical-scavenging capacity of the antioxidants with respect to the above influencing factors. The best combination of each significant factor was determined by RSM design and optimum pretreatment conditions for maximum radical-scavenging capacity were established to be liquid-solid ratio of 29.19:1, extraction time of 32.13 min, and extraction temperature of 30 \u00b0C. Under these conditions, 67.60% of DPPH radical-scavenging capacity was observed experimentally, similar to the theoretical prediction of 66.36%.Response surface methodology (RSM) using a central composite design (CCD) was employed to optimize the conditions for extraction of antioxidants from black soybean ( Glycine max var) is the black seed of the soybean (Glycine max (L.) merr), also known as the black bean. It is mainly cultivated in the provinces of Shanxi and Hebei for both food and medicinal purposes. The Supplement to Compendium of Materia Medica states that black beans can be beneficial to sperm and bone marrow production, muscle strength, hair growth, and the immune system. Modern scientific research shows that black beans have hypolipidemic and antioxidant properties and can be used to beautify the skin [Black soybean  is a kind of optimization method which serves as an accurate, effective, and simple tool for optimizing the experimental process ,14, and The influences of extraction time, liquid-solid ratio and extraction temperature on antioxidant activities of extraction were shown in It can be seen from The effect of three independent variables, namely, extraction temperature (A), extraction time (B), liquid-solid ratio (C) on DPPH radical scavenging activity (Y) was investigated using a three-factor CCD-RSM experimental and the results are shown in 2 \u2212 0.01378B2 \u2212 0.03845C2. Analysis of variance for response surface quadratic model was shown in After data fitting to the experimental findings, one gets the following regression equation: Y = 13.3375 + 0.23845A + 1.06255B + 2.22142C \u2212 0.04950AB + 0.01833BC \u2212 0.09750AC \u2212 0.02269A2 are significant model terms. Values greater than 0.1000 indicate the model terms are not significant. There are many insignificant model terms (not counting those required to support hierarchy), so model reduction may improve the model.As shown in The analyses of the data were done using the Design-Expert 7.1 software. Response surfaces and contour plots were shown in The response surfaces and contour plots of 1 = (A\u221245)/15, X2 = (B\u221240)/10, X3 = (C\u221230)/10. The regression equation transfer into: Y = \u2212 405.5275 \u2212 100.62975X1 \u2212 17.1745X2 \u2212103.725X3 \u2212 5.10525X12 \u22121.378X22\u2212 3.845X32 \u2212 7.425X1X2 + 1.833X2X3 \u2212 14.625X1X3.To get the optimized value of the extraction conditions, the three independent variables, namely, extraction temperature (A), extraction time (B), liquid-solid ratio (C) were transformed as follows: XThe optimal conditions analyzed using Design Expert 7.1 were as follows: 30 \u00b0C of extraction temperature, 32.13 min of extraction time, and 29.19:1 of liquid-solid ratio. Under optimal conditions, the theoretical value of DPPH radical scavenging rate of black soybean sprout extract was 66.36%, and the measured actual scavenging rate was 67.60%, very close to the theoretical prediction. The results show that response surface methodology is a useful mathematical tool in finding the optimum extraction conditions for antioxidants from black soybean sprouts.Glycine max var) ; 1,1-diphenyl-2-picrylhydrazyl radical (DPPH\u2022), AR ; Vitamin C, AR .The following reagents and materials were obtained for this study: soybeans  was used in this investigation. Five grams of soybean seeds were soaked in distilled water (200 mL) for 16 h and then placed in a germinator . Germination occurred at 25 \u00b0C and 99% relative humidity in darkness with water sprayed every 60 min [Black soybean , peroxyl radical-trapping capacity (PRTC), Trolox equivalent antioxidant capacity (TEAC) and DPPH radical scavenging capacity (DPPH\u2022). DPPH\u2022 is a stable organic free radical which can be used to give a global measure of antioxidant activity by spectrophotometry; therefore, antioxidant activity of extracts were measured using DPPH\u2022 scavenging method . Ethanoli.e., extraction time, liquid-solid ratio and extraction temperature to be used in design of experiments. It involved extraction times of 20, 30, 40, 50, 60 min at fixed liquid-solid ratio of 30:1 (30 mL of deionized water to 1 g powder) and extraction temperature of 30 \u00b0C. Then, liquid-solid ratio was evaluated at different ratios from 10:1 to 50:1 at a fixed extraction time of 30 min and extraction temperature of 30 \u00b0C. Extraction temperature was evaluated from 20 to 75 \u00b0C at a fixed extraction time of 30 min and liquid-solid ratio of 30:1.A screening study was conducted to determine appropriate ranges of independent variables, Response surface methodology was employed to establish the optimum conditions for extracting antioxidants from black soybean sprout. The effect of three independent variables, namely, extraction time (20\u201340 min), liquid-solid ratio (20:1\u201340:1), extraction temperature 30\u201360 \u00b0C) on DPPH radical scavenging activity was investigated using a three-factor CCD-RSM experimental runs to determine the optimal parameters of the extraction process ,28,29,30\u201360 \u00b0C onThe analyses of the data were done using the Design-Expert 7.1. .In this study, RSM was used to investigate the main and interaction effects of important independent variables for extraction of antioxidants from black soybean sprouts on the basis of single-factor experiments. The optimum conditions for antioxidant extraction from black soybean sprouts were established. Research shows that the optimized experimental process increases the antioxidant activity of antioxidants from black soybean sprouts. The manufacturing process optimized using RSM needs lower energy, shorter time and offers simplified manipulation. The extract was green, healthly, safe and thus shows potential as an additive in cosmetic products. In addition, the successful extraction of antioxidants from black soybean sprout provides a basis for the development and utilization of black soybean resources."}
+{"text": "Cinnamomum camphora leaves. Response surface methodology of Box\u2013Behnken design was employed to optimize the HUCSE process, and the optimum operation conditions attained with an extraction yield of 7.95\u2009\u00b1\u20090.27\u2009mg/g were ethanol concentration 76%, homogenate/ultrasonic time 25\u2009min, solvent-to-solid ratio 22\u2009mL/g, and ultrasonic power 240\u2009W. A second-order kinetic mathematical methodology was performed to depict the behaviors of HUCSE and heat reflux extraction method. The results suggested that the developed HUCSE is an efficient and green method for the extraction of C. camphora flavonoids or other plant natural products, where the obvious higher parameters of extraction capacity at saturation, second-order extraction rate constant, and original extraction rate were obtained when compared to the heat reflux method. The antioxidant activity assays in vitro showed that the C. camphora flavonoids possessed strong antioxidant activity and are promising to be applied as a natural alternative antioxidant.A free-of-dust pollution extraction method combined-homogenate and ultrasonic cavitation system, namely, homogenate-combined ultrasonic cavitation synergistic extraction (HUCSE), was proposed for the efficient extraction of flavonoids from  Natural products have gained great interest as natural alternatives to substitute the traditional synthetic compounds for health purposes as their remarkable health-promoting activity and consumer preference for the cleaning labels , 2. SpecCinnamomum camphora is an evergreen tree, which is widely distributed in Southern China as a Chinese folk medicine for the cure of many diseases [C. camphora is even extensively cultivated in Jiangxi province as a landscape tree species and an economic crop for the production of essential oil and camphor [C. camphora are mainly on essential oils used for their antifungal and insecticidal activities [C. camphora leaves with remarkable antioxidant activity and antibacterial activity [C. camphora leaves at regular intervals facilitates C. camphora to grow vigorously [diseases . C. camp camphor . Many retivities \u201315, and activity \u201318. Moregorously , so it iC. camphora flavonoids is much necessary.Generally, plant materials are ground to powders using mechanical disintegrators prior to the subsequent extraction process for sufficiently obtaining the target compounds. Among the physical smashing ways, homogenate is an effective technique as it possesses many inherent merits , which is conducive to the dissolution of plant bioactive compounds into extraction solvent , 21. ExtC. camphora leaves. An RSM-based Box\u2013Behnken design (BBD) was applied to offer the optimal combination of ethanol concentration, homogenate/ultrasonic time, solvent-to-solid ratio, and ultrasonic power with which a maximum yield of C. camphora flavonoids can be attained by HUCSE and their antioxidant capability can be evaluated in vitro.The response surface method (RSM) is an efficient statistical optimization methodology which just needs a few numbers of experimental trials to investigate multiple variables and their interactive effects . RSM is C), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and 2,4,6-tripyridyl-s-triazine (TPTZ) were obtained from Aladdin Reagent Co. . Other analytically pure reagents were obtained from Beijing Chemical Reagents Co. . Deionized water used in the experiments was purified by a Milli-Q water system . C. camphor leaves were collected in November 2017 from Jiangxi Normal University campus  and authenticated by Prof. Ronggen Deng . Fresh leaves were dried at room temperature (20\u2009\u00b1\u20090.8\u00b0C) for 7\u2009days, stored in a nylon bag, and then placed in a dry and ventilated place before the subsequent experiments.Chromatographically pure rutin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), vitamin C  and 2\u2009mL of CH3COONa solution (1\u2009mol/L) were added, followed by adjustment to the scale with 60% ethanol aqueous solution. The mixtures were placed for 20\u2009min in atmospheric temperature and then detected at 400\u2009nm against the mixture without coloration as a reference by a UV-Vis spectrophotometer . The yields of the flavonoids were conveyed as milligram of rutin equivalents per gram of dry weight of C. camphora leaves based on the standard calibration curve. The calibration curve  ranged from 0 to 0.05\u2009mg/ml (R2\u2009=\u20090.9963). All experiments were conducted three times, and the results were conveyed as mean\u2009\u00b1\u2009SD.The quantification of g et al.  with sliC. camphora leaves were weighed and transferred into the HUCSE system accompanied with the addition of a definite volume of ethanol solution followed by treatment under the presetting conditions for flavonoids extraction. After each treatment, the suspension was centrifuged at 8,000\u2009\u00d7\u2009g for 20\u2009min to collect the crude extract solutions and then stored in a refrigerator prior to the analysis of flavonoids content by UV-Vis spectrophotometry. Each experiment trial was performed in triplicate for accuracy.Dried A, %), homogenate/ultrasonic time , solvent-to-solid ratio , and ultrasonic power . The scope and level of each factor as shown in BBD, a member of RSM, is characterized with a spherical and revolving design. It comprises a central point, and the intermediate points circumscribed on the sphere at the cube edges . Herein,\u03b20 is presented as the constant; \u03b2i, \u03b2ii, and \u03b2ij are defined as the coefficients of linear, quadratic, and cross-product, respectively. Xi and Xj are given as the independent factors at different levels.The Design-Expert  software was employed for performing the model establishment, experimental design, analysis of data, and graph plotting. A quadratic equation was fitted to investigate the correlation between the determined values and the four independent variables as follows:C. camphora leaves powders (60 meshes of particle size) was weighted and placed into a glass flask, and then a definite volume of ethanol aqueous solution was added before subjecting to extraction by HRE. The other extraction conditions applied in HRE were the optimal conditions based on the results of the optimizing process for HUCSE. After the extraction, the mixture was transferred to certification and then analyzed by UV\u2013Vis spectrophotometry as mentioned above.The conventional heat reflux method (HRE) was performed as the reference method to make a comparison with the proposed HUCSE . HRE wasC. camphora leaves by HUCSE and HRE were investigated on the basis of the second-order kinetic model. The model parameters h, k, Ys, and Yt are defined as the original extraction rate (mg/g\u00b7min), second-order extraction rate constant (mg/g\u00b7mim), extraction capacity (mg/g) at saturation, and the extract yield (mg/g) at any moment t (min), respectively. The dissolution rate of flavonoids contained in the solid transferring to solvent can be represented asThe second-order kinetic model offers an adequate 1 acceptable illustration with respect to the solid-liquid extraction procedures and has t\u2009=\u20090 to t and the corresponding Yt\u2009=\u20090 to Yt, the second-order model equation  can be denoted by equation  can be calculated experimentally by the intercept and slope of the t/Yt versus t plot.The three second-order kinetic model parameters /g extracts.According to the study of Benzie and Strain , the ant3Fe(CN)6 solution were mixed and incubated at 50\u00b0C for 20\u2009min and then 2.5\u2009mL 10% of trichloroacetic acid was added, followed by centrifugation at 10,000 \u00d7 g for 10\u2009min. Aliquots of 2.5\u2009mL of the upper layer were collected and then blended with 2.5\u2009mL of deionized water and 0.5\u2009mL 0.1% of FeCl3 and then were detected at 707\u2009nm. Positive control groups were carried out as mentioned above by replacing sample solution by VC, BHT, and BHA.The assay for the evaluation of reducing power was performed based on Oyaizu's study . 1\u2009mL ofANOVA was used to analyze the statistical significance of all data. BBD was performed by Design-Expert 8.0 software , and the actual responses were the average values of each experiment trial in triplicate. The other experiments were conducted in three replicates, and the results are presented as the mean\u2009\u00b1\u2009SD. OriginPro 2017 software was employed to fit the extraction kinetic curves for HUCSE and HRE according to the second-order kinetic model.C. camphora leaves (1\u2009g dry weight) were extracted by HUCSE at an ultrasonic power of 250\u2009W for 20\u2009min with various proportions of ethanol (0\u2013100%), and the solvent-to-solid ratio was 20\u2009mL/g. As shown in C. camphora flavonoids showed an increasing tendency. However, an upward trend was observed in the extraction yield when the ethanol concentration was over 75%. So, 75\u2013100% of ethanol concentration was applied for the following experiments as an apparent change of extraction yield was observed in this range.Ethanol is a generally used extractant owing to its remarkable penetration capacity into the plant materials, relatively low boiling temperature (easily recycled) and cost, safety, and nontoxicity . The proTo obtain the appropriate homogenate/ultrasonic time, a series of experiments were conducted at different HUCSE times, and the results are presented in The solvent-to-solid ratio is a crucial factor in the extraction procedure, excessive volumes of the solvent could give rise to the tedious extraction process and needless waste, and minute volumes of solvent may result in the incomplete separation. Hence, different solvent-to-solid ratios from 12 to 28\u2009mL/g with 4\u2009mL/g intervals were studied to analyze their effects. As presented in To analyze the effect of ultrasonic power on the extraction yield of flavonoids, tests were conducted at different ultrasonic powers  with 75% of ethanol concentration, 20\u2009min of homogenate/ultrasonic time, and 20\u2009mL/g of solvent-to-solid ratio, respectively. As shown in C. camphora flavonoids extraction and to determine optimal extraction conditions. The extraction yield was defined as the response. The design matrix, numbered, and actual forms at three levels  of four independent factors; the determined extraction yields by HUCSE process; and the predicted values generated from BBD are given in A), homogenate/ultrasonic time (B), solvent-to-solid ratio (C), and ultrasonic power (D) were studied as shown in BBD was used to build the combined influences of four factors on the HUCSE process for R2\u2009=\u20090.9903), the adjusted coefficient of determination (adjusted R2\u2009=\u20090.9754), and the coefficient of variation (C.V\u2009=\u20091.26%), which revealed that more than 99% of the obtained actual values can be interpreted by the selected model, and the model accuracy and generic availability are capable. The predicted R2 of 0.9516 was corresponded well with the adjusted R2. The adequacy precision estimated the signal-to-noise ratio, and a ratio higher than 4 is generally desirable. The adequacy precision of 38.78 revealed that the developed model could be employed to handle the design space.The model suitability was verified by the coefficient of determination  and a very low P-value (<0.0001) as shown in The significance of all the coefficients was examined by  P-value . The devC. camphora flavonoids suggested that the interactive influences of HUCSE process was extremely significant affected by the ethanol concentration, homogenate/ultrasonic time, and solvent-to-solid ratio and ultrasonic power, in both linear and quadratic manners. The interaction of solvent-to-solid ratio versus ultrasonic power, homogenate/ultrasonic time versus ultrasonic power, and ethanol concentration versus solvent-to-solid ratio or ultrasonic power showed extremely significant, highly significant, and significant influences on the extraction process, respectively. Nevertheless, the interaction effects between homogenate/ultrasonic time to ethanol concentration and solvent-to-solid ratio were statistically insignificant. By using multiple regression analysis on the determined values, the empirical relationship between the extraction yield and four factors in natural values was represented using a polynomial equation:Statistical analysis regarding the regression model for the extraction of The regression polynomial equation can be delineated by both the three-dimensional response surface and the two-dimensional contour graphs generated from BBD. These graphs offered a way to visualize the connection between response values and detailed experimental levels of four factors and the interactive effects between two test factors on the extraction yield in HUCSE process. The contour plot shapes can reflect the statistical significance of mutual interactions between the factors, with which a circular contour graph indicates that the interactive effect between the factors are insignificant, while an elliptical contour graph suggests that the interactive effect between the factors are significant . The conn=3), which was in good accordance with the predicted response by the model equation and further confirmed that the developed response model was capable for the HUCSE process optimization.As for the BBD optimization process, which needs less experiment trials to be performed for a three-level factorial than other experiment designs to illustrate the significant factors, the possible interactive effects between the variables studied on the extraction yield of target compounds to attain the optimal operation conditions and the satisfied response . By supeC. camphora flavonoids extraction. The variations of flavonoids extraction yield with changing the treatment time in the procedures of HUCSE and HRE were depicted as presented in Extraction kinetic modeling was employed to make a comparison of the inherent behaviors of concern on heating and mass transfer during HUCSE and HRE procedures for Ys (extraction capacity at saturation) and k (second-order extraction rate constant)  denoted that the second-order kinetic mathematical model was adequate for fitting the HUCSE processes of flavonoids. The parameter Ys was applied to determine the extraction efficiency of HUCSE compared to that of HRE; it was observed that HUCSE (Ys\u2009=\u200914.95\u2009mg/g) was more efficient for the extraction of flavonoids than traditional HRE (Ys\u2009=\u200913.05\u2009mg/g). Meanwhile, the proposed HUCSE possessed apparently higher original extraction rate (h) and the second-order extraction rate constant (k) for flavonoids separation in comparison to HRE. These results all declared that HUCSE is an efficient technique for the extraction of C. camphor flavonoids. It can be concluded that the synergistic effects of the homogenate and ultrasonic cavitation are the main reason that the proposed HUCSE method can dramatically shorten the extraction time and improve the extraction efficiency. A similar result was also observed in our previous study [The parameters of onstant)  were obtonstant)  of t/Ytus study .C. camphor flavonoids on scavenging DPPH radicals. Compared to the positive control sets of VC, BHT, and BHA, C. camphor flavonoids revealed relatively low DPPH radical-savaging activity at low concentrations, notably with a further increase of concentration, flavonoids showed approximately similar DPPH radical-savaging activity as Vc. FRAP test was performed in the present study to evaluate the antioxidant ability of C. camphor flavonoids. As presented in C. camphor flavonoids showed great potential as a natural antioxidant to substitute the traditional synthetic antioxidants based on the above results.Scavenging activity on DPPH-free radicals is an important indicator on the investigation of antioxidant activity of bioactive compounds .  FigureC. camphora flavonoids. BBD was employed to optimize the HUCSE process; the optimal operation conditions were attained and validated, and the results showed that the improved HUCSE method is adequate for the extraction of flavonoids. The second-order kinetic model was performed to investigate the extraction kinetics of HUCSE and HRE of flavonoids, and the kinetic parameters were obtained and analyzed. HUCSE, compared with traditional HRE, enjoyed the higher efficiency for flavonoids extraction and could be extended to isolate other bioactive compounds from plant materials. Additionally, C. camphora flavonoids had remarkable antioxidant ability compared to the commercial antioxidants.In this study, HUCSE technique was developed with the merit of no-dust pollution for the efficient extraction of"}
+{"text": "Catharanthus roseus leaves. The optimized method employed 60-mesh particles, 80% ethanol, a negative pressure of \u22120.075 MPa, a solid to liquid ratio of 1:20, 30 min of extraction and three extraction cycles. Under these optimized conditions, the extraction yields of VDL, CTR, VCR and VLB are 0.5783, 0.2843, 0.018 and 0.126 mg/g DW, respectively. These extraction yields are equivalent to those from the well-known ultrasonic extraction method and higher than the yields from maceration extraction and heating reflux extraction. Our results suggest that NPCE-RP-HPLC represents an excellent alternative for the extraction and quantification of vinca alkaloids for pilot- and industrial-scale applications. In the present study, an improved method termed negative-pressure cavitation extraction (NPCE) followed by reverse phase high-performance liquid chromatography (RP-HPLC) was developed for the extraction and quantification of vindoline (VDL), catharanthine (CTR), vincristine (VCR) and vinblastine (VLB) from  Catharanthus roseus (L.) G. Don (Madagascar periwinkle) is a terpenoid indole alkaloid (TIA)-producing plant that belongs to the Apocynaceae Family. This plant has historically been used to treat a wide assortment of diseases. Research shows that Catharanthus roseus (C. roseus) contains over 130 compounds, and many of which have cytotoxicity [C. roseus and it has become a model species for the study of secondary metabolism in plants. The interest in this species arises from its therapeutic role as the source of the anticancerous alkaloids vinblastine (VLB) and vincristine (VCR) [toxicity . Few spene (VCR) . VLB andne (VCR) . Vincrisne (VCR) . The antne (VCR) ,8.C. roseus have reported the isolation of vinca alkaloids by centrifugal partition chromatography, supercritical CO2 extraction, ultrasound-assisted extraction and microwave assisted extraction [Because they exist in trace amounts (0.01\u20130.1 mg/g DW) in the plant, the extraction of VLB and VCR and their precursors from the plant has been an important method on the industrial scale. In attempts to improve the production of valuable alkaloids such as VDL, CTR, VCR and VLB, several studies on traction ,11,12,13C. roseus leaves. This study evaluated the effects of particle size, solvent concentration, as well as NCPE operational variables including negative pressure, solid to liquid ratio, extraction time and cycles on the extraction yields of the four main vinca alkaloids. This work may provide valuable data for both pilot- and industrial-scale applications.In recent years, a new extraction method called negative-pressure cavitation extraction (NPCE) has increased in popularity; NPCE is more efficient at constant lower temperatures and intensities than classical extraction technologies such as maceration extraction, heat reflux extraction and ultrasonic assisted extraction. Since it was first developed in 2009, the NPCE technique has been widely used in the extraction of a number of bioactive compounds from Chinese herbs ,18,19,20The particle size of the plant material and solvent concentration has a large impact on extraction yields. This is because plant cell walls are broken, and the accessibility to the bioactive compounds is augmented for the extraction solvent. As shown in To increase the extraction efficiency and decrease the extraction time, the extraction solvent concentration was optimized. It is well known that increasing the ethanol concentration induces a reduction in the dielectric constant of the extraction solution and improves the solubility of bioactive compounds. A range of ethanol concentrations  were compared. As can be seen from In our experiment, the extraction pressure was adjusted by regulating the airflow. The effect of different extraction pressures on the extraction yields of vinca alkaloids are shown in The effect of the solid to liquid ratio on the extraction yields of the four main vinca alkaloids was determined by a series of experiments with different solid to liquid ratios . As is shown in The proper extraction time is necessary for optimal extraction efficiency and ensuring a distribution equilibrium of bioactive compounds between the plant material and the extraction solvent. In the present study, extraction was carried out for 25, 30, 35, 40 or 45 min and the results are shown in The effect of the number of extraction cycles on the extraction yields was investigated using 1, 2, 3, 4 or 5 cycles. Generally, an increase in the number of extraction cycles increases extraction yield by allowing greater exposure of the material to fresh solvent and favoring the material-solvent equilibrium. As shown in After optimization, NPCE was shown to be an efficient method with high extraction yields . We determined that the optimal parameters to be employed are: 60-mesh particle size, 80% ethanol, a solid to liquid ratio of 1:20 (w/v) and a 30 min extraction for three cycles. NPCE has received considerable attention for extracting several bioactive compounds from Chinese Herbs; to the best of our knowledge, NPCE of vinca alkaloids has not been reported. As a result, it is necessary to compare the extraction efficiency of NPCE with other extraction methods for the extraction of vinca alkaloids. C. roseus leaves.Different methods  were performed using the optimized conditions, and the results are summarized in C. roseus leaves (80% water content) were collected from the Greenhouse of Northeast Forestry University, Heilongjiang, China, and identified by Professor Shao-Quan Nie from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University, Harbin, China. The material was dried in the shade, powdered by a disintegrator  and then sieved through 20\u2013100 mesh sieves . The powder was kept at 4 \u00b0C prior to use.Reference standards of VDL, CTR, VCR and VLB were purchased from Shanghai Anticancer Phytochemistry Co., Ltd. . Methanol  and acetonitrile  used for HPLC were HPLC reagent grade. Deionized water was filtered using a Milli-Q Ultrapure water purification system from Millipore . Other reagents were all commercially available analytical grade chemicals.The apparatus (CN 2597047) used in the NPCE experiment was provided by the Engineering Research Centre of Forestry Bio-preparation, Ministry of Education, Northeast Forestry University, Harbin, China. The mode of action of NPCE is based on the continuous formation and collapse of tiny bubbles under the action of a vacuum, which was described in detail in our previous study ,22,23. C. roseus leaves were transferred into the extraction chamber with different concentrations of extraction solvent. Then, a vacuum pump was connected to the extraction pot through a condenser to generate negative pressure. The pressure was monitored by adjusting the airflow rate, which was supplied from the bottom of the device. The extraction process was performed under different conditions as described in the Results section. After each extraction, the extraction solutions were combined and dried on a rotary evaporator  at 45 \u00b0C. HPLC-grade methanol was added in a constant volume to prepare the samples for RP-HPLC analysis. The extraction efficiency of the compounds in a given sample was defined as follows:In the present study, 25 g of powdered C. roseus leaves were placed in a beaker with 500 mL of 80% aqueous ethanol solution. The beaker was placed at room temperature for 12 h, the filtered extraction solution was collected, and another 500 mL of 80% ethanol was added into the beaker for an additional 12 h to obtain optimal extraction efficiency. For maceration extraction (ME), as determined by the preliminary optimized experiments, 25 g of powdered of C. roseus leaves were added to a round-bottom flask with 500 mL of 80% ethanol. The round-bottom flask was then placed into a water bath and linked with a condenser at the joint of the flask. The extraction process was twice maintained at 80 \u00b0C for 3 h to obtain optimal extraction efficiency. For heat reflux extraction (HRE), after preliminary optimization experiments, 25 g of powdered C. roseus leaves were added to a triangular flask with 500 mL of 80% ethanol. The extraction was performed in ultrasonic bath at a frequency of 40 kHz with the maximum input power of 250 W at 45 \u00b0C for 30 min. Three replicates were performed to obtain the best extraction efficiency. Ultrasonic-assisted extraction (UAE) was run in an ultrasonic bath  using the conditions optimized for NPCE at normal pressure. In brief, 25 g of powdered of In this study, an RP-HPLC separation and analysis protocol is employed to detect the four alkaloids. UV absorbance is the method of choice for their detection, and 220 nm was the wavelength used. The criteria for the identification of the compounds were established based on comparisons of the retention time and spectrum of an unknown compound with HPLC library data of standards .18W column (4.6 mm \u00d7 250 mm). RP-HPLC analysis was carried out using a Jasco HPLC system , which included a PU-1580 pump, a 7725i manual injector and a UV-1575 detector connected to CkChrom chromatography data system software (Version 6.1). Separations were performed on a HiQ sil C3PO4). The mobile phase consisted of 620 mL solution A and 380 mL solution B. The flow rate was set at 2.0 mL\u00b7min\u22121 and the elution was monitored at 220 nm [The separation was carried out by an isocratic elution using solution A (methanol/acetonitrile = 4:1), and solution B .All experiments were conducted in triplicate\uff0cand the average values of extracted vinca alkaloids were used for statistical analysis. The one-way ANOVA test was used to calculate the significance of the differences in extraction efficiency of the target alkaloids. The results of RP-HPLC analysis are expressed as the means \u00b1 SD (C. roseus leaves and compared with other standard extraction methods. Under the optimal parameters, the extraction yields of VDL, CTR, VCR and VLB were 0.5783, 0.2843, 0.018 and 0.126 mg/g DW, respectively. These yields are comparable to those obtained by the well-known UAE method and significantly higher than those obtained by ME and HRE. The results indicated that NPCE offers a fast, environmentally friendly and straightforward route for vinca alkaloid production. Consequently, the NPCE-RP-HPLC described here represents an excellent alternative for the extraction and quantification of the four main C. roseus alkaloids in both pilot- and industrial-scale applications.NPCE was first applied to the extraction of VDL, CTR, VCR and VLB from"}
+{"text": "Alzheimer\u2019s disease is a complex disorder encompassing multiple pathological features with associated genetic and molecular culprits. However, target-based therapeutic strategies have so far proved ineffective. The aim of this study is to develop a methodology harnessing the transcriptional changes associated with Alzheimer\u2019s disease to develop a high content quantitative disease phenotype that can be used to repurpose existing drugs. Firstly, the Alzheimer\u2019s disease gene expression landscape covering severe disease stage, early pathology progression, cognitive decline and animal models of the disease has been defined and used to select a set of 153 drugs tending to oppose disease-associated changes in the context of immortalised human cancer cell lines. The selected compounds have then been assayed in the more biologically relevant setting of iPSC-derived cortical neuron cultures. It is shown that 51 of the drugs drive expression changes consistently opposite to those seen in Alzheimer\u2019s disease. It is hoped that the iPSC profiles will serve as a useful resource for drug repositioning within the context of neurodegenerative disease and potentially aid in generating novel multi-targeted therapeutic strategies. Further, biological state dynamics can be modelled through temporal patterns of expression6. In the therapeutic context, it has been established that disease-associated expression changes can distinguish between disease states and are consistent across independent data sets, thus facilitating the identification of robust biomarkers7. Disease-associated gene changes point to modulated pathways and affected cell types, thus providing valuable insights into mechanisms8. Interestingly, the quantitative nature of the transcriptional phenotype has allowed for a direct mapping of disease to potential therapeutic12. Here the obvious hypothesis is that drugs tending to reverse the expression changes seen in the disease state may act to reverse the biological changes associated with the disease itself. An important caveat here is that some expression changes associated with Alzheimer\u2019s disease (AD) may in fact be compensatory and beneficial. Drug repurposing or repositioning has resulted in successful initiatives across several maladies19. Further, and of more specific interest to the present project, drugs with profiles showing significant anti-correlation to AD gene changes have been shown to be conspicuous for their reported neuroprotective activities12. In a recent development, disease-associated gene expression changes have begun to be inferred from genomic risk variant data with the Genotype-Tissue Expression repository20 and harnessed to predict repurposing candidates for major psychiatric conditions21. Although there is some intriguing psychotherapeutic association of the candidate drugs in this approach, the predicted transcriptional perturbation does not have an overlap with that seen in diseased brain tissue . In the absence of further validation of the predicted gene changes, one must fall back on data from patient samples.Global gene expression profiling can be thought of as a high content quantitative phenotypic measure characterising tissue22. The main aim of transcription-based drug discovery is not target discovery, but rather the discovery of drugs that have a disease-modulating effect based on their global transcriptional activity. A particularly attractive aspect of the approach is that it naturally lends itself to repositioning existing drugs thereby bypassing the hurdles that novel entities must overcome on the road to the clinic. AD has been extensively studied in relation to the expression changes following pathological and cognitive decline26. The wealth of data points to consistent and characteristic changes associated with the disease and thereby makes a repositioning strategy particularly attractive.There are no established disease-modifying drugs for the treatment of AD, there have been no new symptomatic treatments licensed for AD for >20 years and the pipeline of emerging therapies is very limited. Target-based drug research in AD has led to many insights into the disease and provided the research community with useful tool compounds. However, the promising results seen in the laboratory have so far failed to be carried over to the clinic and this has led to researchers casting around for novel, non-target-based approaches11. A more extensive drug set has been profiled on a variety of induced pluripotent stem cell (iPSC)-derived cells, including neural stem cells and differentiated cortical neurons. However, this data constituting the LINCS project27 is based on profiling a set of 1000 landmark genes and then using an optimised linear mapping to generate full profiles. This motivated the present initiative to define the full expression profiles of the CMAP candidate drugs in the more AD relevant cell type of iPSC-derived cortical neurons. The new phenotypes can then be compared to the CMAP profiles and more pertinently scored against the disease profiles to see whether they preserve or enhance their anti-correlation with AD. In this context, iPSC-derived cortical neurons have now been established as a model system for the study of neurological diseases especially the tracing of the effects of disease-related genetic variants31. This model provides for an efficient moderate throughput platform to assess the transcriptional effects of the candidate drugs in a more neurological context. It must be remembered, however, that AD is a complex pathology also involving multiple cell types, such as microglia and astrocytes. In this context, assaying drug perturbations within isolated iPSC cultures facilitates an important but limited insight into the disease.The application of gene expression profiling to drug repositioning is limited at present by the fact that full drug profiles are available only on a restricted set of immortalised human cell lines. This data is provided by the Broad Institute connectivity map project (CMAP)The motivation for the work presented here is to generate a neuronal-specific transcriptional database of compounds with a view to drug repositioning in AD and other neurodegenerative conditions. The initial compound set was assembled based on CMAP profiles that showed a tendency to reverse AD-associated expression changes observed across a variety of independent studies. The drug candidates were then profiled for their transcriptional effects on iPSC-derived human cortical neurons. The results indicate that at the global level there is a degree of correspondence between the CMAP and iPSC profiles. Furthermore, 51 of the drugs have profiles that drive transcription changes counter to those in AD. The consistently regulated genes correspond to those implicated in AD. It is hoped that the transcriptional data for these drugs will be of use to the wider community of researchers interested in neurodegenerative conditions and facilitate further repositioning efforts.32 was queried for series containing samples derived from postmortem AD patient brains for various stages of the disease. Similarly, murine AD model brain samples were also collected based on relevant query key words: 5xFAD, 3xTG, Alzheimer\u2019s disease+mouse. Profiles were generated based on relative levels of non-disease and disease state sample averages, with the scaled fold level defined as c) and disease (d) samples. The statistical significance is measured by Student\u2019s t test and those folds falling below the 95% confidence interval were dropped as were those with folds of <20%. The human disease versus control AD set comprises 21 profiles derived from 13 series  showing intra-profile consistency based on the regression scores for significant (Student\u2019s t test p\u2009<\u20090.05) correlations, see Supplementary Table 43 represented by two profiles from two independent series and Clinical Dementia Rating (CDR)44 profiles from one series. Similarly, series corresponding to murine models of AD were gathered from 5xFAD and 3xTG mice resulting in seven profiles from three series  for the 5xFAD set and nine profiles from eight series  for the 3xTG set. Series corresponding to BRAAK stage progression  were generated with a linear mixed model analysis, by fitting the gene expression level across the samples in the series to a linear function of the BRAAK stage with categorical calls on cell type and gender as covariates. The resulting residual correlation Z score for gene expression against BRAAK stage constituted the BRAAK profile. Profiles corresponding to full BRAAK progression were not considered to be sufficiently different to the overt disease profiles derived from the same series, where disease assignment is also based on BRAAK staging. However, gene expression changes driving mild BRAAK pathology should capture early disease biology invisible in the overt profiles. In total, six profiles corresponding to mild BRAAK pathology, level 0 to level 3, formed the mild BRAAK AD set. Similar profiles were generated for psychiatric measures MMSE and CDR . In the case of the MMSE profile, the regression signs were reversed as MMSE scores decrease with disease progression, see Table The NCBI GEO databasementclass2pt{minimmentclass2pt{minimt test of p\u2009<\u20090.05. Owing to the categorical nature of the representative profiles, correlation with the iPSC profiles was based on an enrichment analysis. The enrichment score was generated based on a binomial probability sum with gene probabilities scaled according to their frequencies in SPIED53.Representative profiles for each set were based on genes showing consistent changes across the constituent profiles. In particular, the sense changes  for significantly regulated genes were summed over the profiles and only those genes retained that had an absolute regulation fraction of >20% and with a significant regulation statistic measured by Student\u2019s www.broadinstitute.org/connectivity-map-cmap) 11. This consisted of probe sets for each sample ranked according to expression level relative to batch control. The data consist of 6100 samples covering 1260 drugs and 4 cell types. The relative probe expression ranks, defined as R in the rank of a given gene\u2019s expression change (Rmax being the highest and Rmin being the lowest ranks), were averaged over replicates ignoring cell type and filtered based on significance using a one-sample Student\u2019s t test. For genes with multiple probes, the probe with the largest significant change was mapped to the gene. This resulted in a unique profile for each drug in CMAP. The compound data can be queried through SPIED53.CMAP data were downloaded from the Broad connectivity map site . Each drug treatment at a concentration of 10\u2009\u03bcM for 6\u2009h was performed on 3 independent HyCCN cultures (average density 300\u2009K/cm2) and RNA from each treated well extracted by direct cell lysis and recovery using the Absolutely RNA Microprep Kit . Each drug-treated plate also consisted of a vehicle-only control set of triplicate cultures. Integrity of total RNAs was determined using an Agilent Bioanalyser as per the manufacturer\u2019s instructions and only samples with RNA integrity number >7 were progressed to transcriptome analysis. Transcriptome changes driven by exposure to the candidate drugs were determined using the Nugen Ovation V2 labelling system (https://www.nugen.com/products) followed by Human U133 Plus 2 GeneChips as per the manufacturer\u2019s instructions .Human iPSC-derived cerebral cortical neurons  were cultured as per the manufacturer\u2019s guidelines . Rapamycin, which has a well-defined transcriptional signature, served as a positive control. The expression changes were either measured as scaled folds filtered for significance with Student\u2019s 54. Animal model data were included in this study because the expression changes seen in the model systems have established causes, i.e. the inserted mutations, 5xFAD or 3xTG in our case. Consequently, candidate drugs reversing these changes may have more focused mechanisms of action. Furthermore, the evidence for neuroprotection is to a large extent derived from experiments in animal models.To capture as much as possible of the transcriptional landscape of AD, different categories were defined based on overt disease versus healthy profiles, profiles following early pathological and cognitive measures, together with those from animal models, as described in \u2018Materials and methods\u2019. There is a good degree of overlap between the overt AD profiles and those following cognitive decline, see Table 19. The relatively small number of compounds that are put forward for rigorous bio-assaying to establish firmer evidence for a disease-modulating potential of course reflects the experimental resource required. The basis of the present project was to select candidates to populate a database of iPSC profiles for drugs biased towards their predicted anti-AD and wider neuroprotective activities. It was therefore reasoned that the thresholds for deeming a drug a repositioning candidate had to be relaxed to allow for over a hundred candidates to be taken forward. To this end, five AD-based profile sets that capture distinct aspects of the disease were separately queried against CMAP and three selection criteria were applied. In the first instance, data were gathered on the anti-correlation rank of each compound, with compounds showing a high rank in either of the profiles considered as candidates, see Supplementary Table In general, transcription-based repositioning results in tens of candidates out of a total of just over a thousand drugs constituting CMAP53, see \u2018Materials and methods\u2019. The top 1000 genes in the iPSC rank profile consists of 959 upregulated and 41 downregulated genes and this served as a query in the SPIED search. It is perhaps worth pointing out here that the level of gene expression unique to a given cell type will tend to be elevated relative to a background consisting of a variety of tissue types. An analogy would be in the context of division of labour one is characterised by what one does not by what one does not do. The top SPIED hits show a high correlation with human brain-derived samples, validating the cell\u2019s lineage, see Supplementary Table As a first step in establishing the phenotype of the model cell system, the overall iPSC transcriptional profile was queried against a database of publicly deposited gene expression profiles via SPIEDZ score threshold of |Z|\u2009>\u20093, see \u2018Materials and methods\u2019 for details, capture most of the compound-associated changes. The enrichment is that of the rank of a given iPSC compound score with itself in CMAP. The enrichment plot is shown in Fig. p\u2009=\u20095.1E\u22126.The extent to which an iPSC profile correlates with its CMAP equivalent can be assessed by querying the CMAP database with the iPSC profile and ranking the CMAP equivalent. The extensively studied perturbagen rapamycin served as a positive control and eight independent profiles were generated to assess the degree to which these profiles are consistent with each other and with the rapamycin profile in CMAP. The rapamycin profiles had consistently high overlaps among themselves, but less so with the CMAP profile, with only one returning rapamycin as a top hit, rank seven, in a CMAP query, see Supplementary Fig. Z scores are: 2.43 for ADC pairs and 0.77 for all other pairs. It is therefore of interest to see to what extent the ADC set regulate a common set of transcripts. In Fig. 56, with growing evidence that gene variation affecting mitochondrial function may play a role in AD58. The downregulated set appears to be less consistent. Nonetheless, the enrichment of immune-associated pathways points to a possible anti-inflammatory activity of the candidate drugs. Interestingly, the following drugs have been reported to have neuroprotective activity: fluocinonide59, kawain63, allantoin64, dipyridamole67, estriol68, levamisole69, mycophenolic acid70, neostigmine71, probenecid73, chlorpromazine74, and phenoxybenzamine75, and xamoterol has been reported to ameliorate neuroinflammation and pathology in 5xFAD mice76 and shown to enhance cognition in a Down syndrome mouse model77. The atypical antipsychotic risperidone prescribed to manage psychosis in AD has demonstrated neuroprotection in animal models of ischemia78. Furthermore, cholinesterase inhibition is a therapeutic strategy for AD79 and there are two such inhibitors in the candidate list with galantamine as an established AD therapeutic80, while neostigmine exhibits poor blood\u2013brain barrier penetrance and is therefore not in clinical use for AD. There does not appear to be any gene expression signature distinguishing compounds with reported neuroprotective activities from the other ADC compounds. This is to be expected as not all compounds have been assayed for neuroprotection and biological activity is not expected to be solely encoded in the transcriptome.Further to assessing the extent to which compounds orchestrate similar expression changes in the cancer cell lines and differentiated cortical neurons, it is critical to test whether the drugs also act in an anti-AD manner in the neuronal context. To this end, the drug profiles were scored against five representative AD reprofiles derived from the AD sets defined above, see \u2018Materials and methods\u2019 for details. Table www.norpd.no), showing that salbutamol use reduced PD risk81. A middle ground between target-based and epidemiological approaches is a methodology based on the disease phenotype gleaned from gene expression changes observed in pathological states. Underlying this approach is the observation that disease states can effectively be represented by characteristic expression changes, in the sense that these changes are consistent and can function as high content quantitative biomarkers. One avenue available to drug repositioning is to use these transcriptional phenotypes together with the hypothesis that an anti-correlation in phenotypes is indicative of the therapeutic potential of the compound. Whereas the transcriptional landscape of neurodegeneration and AD in particular has been well characterised, the corresponding data for compounds are either limited to full profiles defined on non-neuronal proliferating cells or partial profiles on iPSC-derived neuronal cells. The basis of the present study is to go some way to fill this gap in the compound-associated transcriptome with an emphasis on drugs with an anti-AD potential.Neurodegenerative diseases present a therapeutic challenge due to the difficulty in establishing a clear protein or mechanistic culprit for classic target-based intervention. Another hurdle is a consequence of the temporal extent of disease progression and the probable need to treat before overt symptom onset. This is a particular problem in designing clinical trials. With this in mind, alternatives to target-based approaches are increasingly being pursued. One recent report compared Parkinson\u2019s disease (PD) incidence and chronic therapeutic use data from the Norwegian Prescription Database (82. This approach would have the advantage of including non-neuronal factors contributing to AD pathology such as inflammation. However, practical considerations limit whole-animal approaches to smaller drug sets and will therefore form part of a subsequent endeavour based on a more limited set of drug candidates selected based on the iPSC data.In the context of defining the neurotherapeutic potential of candidate drugs, a further development would be to treat wild-type or mutant AD mice with the compounds and measure expression changes in the brain, along the lines of the DrugMatrix projectIn the present work, we have established an AD transcriptional profile landscape and shown this to have a high degree of internal consistency. This disease-associated transcriptional landscape served as the basis for selecting a series of candidate drugs from the CMAP database of cancer cell line profiles, which were then assayed for their transcriptional effect on iPSC-derived cortical neurons. The iPSC profiles show a degree of overlap with the corresponding CMAP profiles, with a highly significant overall comparison in terms of the ranks observed for iPSC queries of CMAP. Out of the 153 iPSC drug profiles, 51, termed the ADC set, showed a high degree of anti-correlation with transcriptional changes seen in AD. A pathway enrichment analysis performed on each of the ADC set showed that pathways related to mitochondrial function were commonly upregulated while commonly downregulated pathways represented immune-associated pathways. Interestingly, these pathological features are found in multiple neurodegenerative disorders, such as PD and Huntington\u2019s disease, and it would be of interest to investigate whether these compounds may have wider therapeutic potential. Notably, 18 of the ADC drugs already have established neuroprotective ability in published studies. Whereas we expect that initial CMAP filtering against AD profiles has led to increased likelihood of discovering compounds that tend to reverse AD-associated expression changes in the context of iPSC cultures, this can only be rigorously assessed by generating iPSC profiles for a series of compounds randomly selected from the CMAP database, which is outside the scope of the present study. In conclusion, approaches to identifying a broader range of candidate therapies for AD are urgently needed. It is therefore expected that the iPSC database will serve as a useful platform for drug repositioning across multiple neuropathological disorders as well as AD.Supplementary Figure LegendsSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4Supplementary Table 5Supplementary Figure 1Supplementary Figure 2Supplementary Table 6"}
+{"text": "Major biological processes rely on the spatial organization of cells in complex, highly orchestrated three-dimensional (3D)tissues. Until the recent decade, most of information on spatial neural representation primarily came from microscopic imagingof \u201c2D\u201d (5-50 \u03bcm) tissue using traditional immunohistochemical techniques. However, serially sectioned and imaged tissuesections for tissue visualization can lead to unique non-linear deformations, which dramatically hinders scientists\u2019 insight intothe structural organization of intact organs. An emerging technique known as CLARITY renders large-scale biological tissuestransparent for 3D phenotype mapping and thereby, greatly facilitates structure-function relationships analyses. Since then,numerous modifications and improvements have been reported to push the boundaries of knowledge on tissue clearingtechniques in research on assembled biological systems. This review aims to outline our current knowledge on next-generationprotocols of fast free-of-acrylamide clearing tissue (FACT) and passive CLARITY (PACT). The most important question is whatmethod we should select for tissue clearing, FACT or PACT. This review also highlights how FACT differs from PACT onspanning multiple dimensions of the workflow. We systematically compared a number of factors including hydrogel formation,clearing solution, and clearing temperatures between free-acrylamide and acrylamide-based passive sodium dodecyl sulfate(SDS) tissue clearing and discussed negative effects of polyacrylamide on clearing, staining, and imaging in detail. Suchinformation may help to gain a perspective for interrogating neural circuits spatial interactions between molecules and cellsand provide guidance for developing novel tissue clearing strategies to probe deeply into intact organ.  Biological systems are capable of forming complex neuronal network of feedforward, feedback and horizontal circuits , the mos2 transparent for deep microscopic imaging  are among the first to make fixed tissues as large as 2 cm imaging , compare imaging , dibenzy imaging , 3D imag imaging , 12, see imaging , ClearT imaging , clear u imaging , 16, sys imaging , and ul imaging . HoweverA cutting-edge technique (termed CLARITY) developed by Chung and Deisseroth , provideThree clear contributions have been made via these techniques: stabilizing tissue structures using hydrogel embedding , use of In this review, we summarize some important findings related to the FACT and the PACT for whole tissue imaging, with systematically comparing differences on recent published protocols between free-acrylamide method and PACT method for whole tissue imaging, including gel formation, clearing solution concentrations, clearing temperature, and negative effect of polyacrylamide. Before, it should be noted that this article discusses most of publishedresearch and review articles comparing or introducingacrylamide-based methods together or with other methodsof clearing. It means the newly developed method, the FACT, has not been compared with acrylamide-based clearing methods. Based on comprehensive comparisons between the two tissue clearing techniques, FACT and PACT, we propose that FACT method performs better in the whole clearing processing, which may give novelinsights into mapping the architecture of neural circuitsand developing new approaches for tissue visualization. CLARITY technique has gained considerable success owing to its potential for mapping detailed structural and molecular information of intact biological systems and is supported by extensive research data, the applications of hydrogel embedding and electrophoretic tissue clearing (ETC) are at the very heart of the CLARITY clearing procedures associated with tissue preservation and clearing efficiency, both of these two techniques have had broad impact on the rate of tissue clearing, and had been incorporated into the design, or redesign, continually. Although active transport organ-electrophoresis approach which capitalizes on the highly charged nature of ionic micelles, can accelerate the lipid extraction by orders of magnitude, the payoffs of ETC are inevitable tissue degradation during sample heating and complexity to implement. Extending their prior work, Chung et al.  developeNevertheless, the main limitation of the slow rate of clearing makes the protocol impractical for scaling up, not to mention intact biological body mapping. On the basis of the passive CLARITY method, the concept of the PACT was first scientifically described by Yang et al. , when thRecently, an exciting new wave of improvements avoids incomplete tissue hydrogel hybridization and fine has emerged from free-of-acrylamide SDS-based cytostructure destruction in this protocol. However, the tissue clearing (FASTClear), which greatly reduces most prominent disadvantages of FASTClear is being the whole clearing time. Most notably, the replacement limited by immunolabelling to the full thickness of tissue. of acrylamide hydrogel by formaldehyde largely Moreover, problems of tissue shrinkage and archival formalin-fixed tissues clearing still exist. To challenge the above imperfections in 3D tissue imaging, simultaneously with FASTClear, the FACT  was intrIncrease of our knowledge on gel formation that preserves proteins of cytoarchitecture but may conversely influence tissue expansion by clear solution, depth of antibody or light penetration in tissue, and speed of lipid removal is crucial. The hydrogel-embedding clearing protocols, including active CLARITY, passive CLARITY, PACT and so on, were developed based on polymerizing the fixed tissue into an acrylamide hydrogel prior to the process of lipid-removal. This hypothesis of hydrogel embedding provides a beautiful and simple account for how the use of hydrogel may provide physical framework for the cleared tissues and act as an impediment allowing for deep penetration of the antibodies into the tissue . It steInterestingly, a more careful examination of different polyacrylamide concentrations in modified CLARITY protocol, found that the retention of RNA and penetration of antibodies improved with reducing acrylamide hydrogel composition from 4 to 1% . FurtherSimultaneous with the FASTClear, the FACT was developed  for furtOne plausible explanation for PFA connection with peptides which is consistent with most of these data, is that aldehyde plays a crucial role in reaction with nitrogen or other atoms within proteins and peptides and therefore, creates a methylene bridge made of a -CH2- cross link . The maThe optimal methods for 3D tissue mapping dependon a set of processing factors that include selection ofappropriate ingredients, optimization of pH, suitable incubation temperatures, thickness of sample, and perhaps also effort-based manipulation proficiency. For lipid removal, the clear solution composition at certain pH and solution concentration, could contribute to thespeed of diffusion of detergent micelles and the rate oflipid solvation by detergent micelles in tissues . Each oEarly recordings revealed that the presence of free radicals which is associated with pH value of solution used in the tissue clearing process, closely affect the clearing speed , 57-59, Recent studies have also revealed that temperature is also one of the most sensitive factors during clearing incubation process to preserve the fluorescent signal. Exposure to high temperatures can accelerate delipidation of tissue; however, 50\u00b0C adopted in FASTClear method, resultiThe limitations of hydrogel crosslinking have substantial effects on the clearing process, including clearing speed, protein preservation, tissue character, which are thought to contribute to the index of clearing quality. The slow clearing speed using polyacrylamide has been brought under the spotlight. Theoretical basis of the FASTClear postulatPreservation of structural integrity is the most prominent factor of 3D tissue imaging, which is thought to provide an intuitive and detailed picture of how neural circuits and other connectomics work. Polyacrylamide was once considered to chemically incorporate native biomolecules into the hydrogel mesh, however, investigation of the necessity of hydrogel involvement has just started. Although, analysis based on daily measurement of tissue weight (served as an indicator of tissue and water absorption) and tissue size (served as an indicator of tissue size change) area revealed increased daily protein loss by removal of hydrogel from brain fixation comparing with the PACT and passive Clarity approaches. Intriguingly, the total protein losses showed no significant differences among hydrogel-based and FACT groups . This dFurthermore, the field has begun to appreciate that acrylamide-embedded tissues during SDS clearing undergo expansion and become more fragile because of loss of structural integrity , 36. ConTherefore, PFA-based tissue clearing protocols can achieve a rapid clearing without influencing total protein loss and tissue area under specific conditions, comparing with hydrogel-based protocols. However, given that some degree of protein loss is inevitable in all protocols, and given the potential of more or less deformation of samples during lipid removing process occur no matter it is in PACT or FACT approaches. More effort should be made to further preserve more tissue structure details upon tissue transparency.Avariety of unfavorable factors of polyacrylamide, such as specific antibody, non-specific staining, low antibody penetration, and being time consuming on staining, made it heavily implicated. According to hydrogel-based hypothesis, pores of polyacrylamide matrices assist lipid exchange and therefore enhance antibody penetration by changing hydrogel composition . This hyPolyacrylamide hydrogel application in imaging are also subjected to modifications such as alteration of depth of penetration and light scattering, to increase background during imaging. Tissues are opaque for conventional light microscopy, mainly owing to their lipids, induce light scattering , 19 whicAs mentioned above, hydrogel polymerization can be considered for the expansion and fragility of tissue during the imaging procedure. Deformation of tissue disrupts the evaluation of fine structures such as neuronal processes and microglia. In addition, lesions to tissue swelling is irreversible, a major adverse event for 3D tissue imaging quality, which RI cannot be corrected by using FocusClear. Abnormalities in tissue processing caused by polyacrylamide, heavily influence fluorescent signal intensity, antibody penetration depth as well as light scattering. In contrast, the absence of hydrogel in the FACT method circumvents these limitations and optimizes the preservation of antigenicity . MoreoveAided by merging and adjusting the FASTClear and PACT methods, Xu et al.  effectivWe have witnessed tremendous progress in refining clearing methods introduced for identifying complex cytoarchitecture and mapping neural circuitry, involved in simplified protocols, higher quality and speed of data generation, safety and economical materials and reagents application. Notably, the PACT and the FACT methods have recently attracted considerable attention as they could optimize the clearing time, temperature, and reagents and preserve the fluorochrome signal. Notably, the FACT technique which has been developed by merging and modifying the FASTClear and PACT methods, includingavoiding hydrogel perfusion and embedding, decreasingthe temperature to 37\u00b0C, adjusting the clear solution pH to 7.5, has been identified with reinforcing properties compared with PACT for mapping detailed structures suchas neuronal processes and branches. As FACT facilitates thewhole workflow of tissue clearing including clearing timesaving, fluorescent signals preservation, cytoarchitectural retention, confocal microscopy optimization, data collectionacceleration, the distribution of such a 3D tissue map provides a more intuitive brain tissue picture of how connections of large populations of cells and their networks and howphysiological and pathological conditions lead to changesin expression of corresponding proteins and molecules. The FACT protocol suggests optimized conditions of rapid high-resolution imaging for brain tissue, which has made a big step over the PACTprotocol. In the following, we highlight several frontiers which should be addressed by further investigations to deepen our understanding of the FACT.To test the validity and versatility of the FACT in 3D tissue imaging, a compelling set of experiments with tissue-type specificity and temporal precision recordings, remains warranted. Recently, a series of other tissue clearing methods has strongly established the visualization efficiency of various tissues including the spinal cord , whole eAlthough some articles have been recently done using FACT protocol , 69; butA potential problem of using scanning microscopes is that low scanning speeds make them impractical for imaging large fields of samples, resulting in light falloff, lens distortions, potential micro movements of the tissues inside the chamber . The molIt would be an effective and reliable tool tosimultaneously map subcellular molecular architecturebetween neighboring or distant cells of various organs, such as the brain and stomach, using multiple fluorophore signal in the same biological samples to examine how feedback loops and neuronal circuits works among central and peripheral system. Moreover, based on molecular-level and projection-based neural circuit tracing, it would be especially useful to comprehensively explorepathological and physiological conditions by comparing the functionality of protein and cell population. Theadvantages of the FACT protocol, such as maximal preservation of fluorophore signal hold promise for classifying and sub-classifying cytoarctitectures, and for molecular localization and projection in other projects."}
+{"text": "For illustration purposes, the previous travel time probability density (BTTPD) approach is extended to assess the 3-d deep groundwater susceptibility to non-point source contamination within a sequence stratigraphic framework observed in the Kings River fluvial fan (KRFF) aquifer. The BTTPD, which represents complete age distributions underlying a single groundwater sample in a regional-scale aquifer, is used as a quantitative, transient measure of aquifer susceptibility. The resultant 3-d imaging of susceptibility using the simulated BTTPDs in KRFF reveals the strong influence of regional-scale heterogeneity on susceptibility. The regional-scale incised-valley fill deposits increase the susceptibility of aquifers by enhancing rapid downward solute movement and displaying relatively narrow and young age distributions. In contrast, the regional-scale sequence-boundary paleosols within the open-fan deposits \u201cprotect\u201d deep aquifers by slowing downward solute movement and displaying a relatively broad and old age distribution. Further comparison of the simulated susceptibility index maps to known contaminant distributions shows that these maps are generally consistent with the high concentration and quick evolution of 1,2-dibromo-3-chloropropane (DBCP) in groundwater around the incised-valley fill since the 1970s\u2019. This application demonstrates that the BTTPDs can be used as quantitative and transient measures of deep aquifer susceptibility to non-point source contamination.Groundwater susceptibility to non-point source contamination is typically quantified by stable indexes, while groundwater quality evolution  can be a long-term process that may last for decades and exhibit strong temporal variations. This study proposes a three-dimensional (3- The ultimate goal in most groundwater protection studies is to ascertain groundwater vulnerability, which refers to the likelihood that an aquifer will become contaminated as a function of the contaminant type, source type and distribution, and the system hydrogeology ,2. CurreDepth to water table, Recharge, Aquifer, Soil, Topography, Impact of the vadose zone, and hydraulic Conductivity. The DRASTIC index motivated many other indicators to map aquifer vulnerability in the last three decades. For example, the SINTACS index modified the DRASTIC index by adding options for the weight rating of each parameter embedded in DRASTIC [Concentration, Overlying layers, and Precipitation [First, the subjective rating (or index) method assigns a numerical score or rating to each (subjective) attribute affecting groundwater vulnerability, without the computational burden of statistically analyzing or numerical modeling of contaminant transport dynamics . This ap DRASTIC . The aqu DRASTIC . The vul DRASTIC . The COPpitation ,23. The pitation .Second, the statistical method uses descriptive statistics or complex regression to analyze contaminant concentrations and spatial distribution, or link observed contamination and environmental factors ,26. For Third, the process-based method simulates the process of groundwater flow and contaminant transport at the study site, and hence the advantage of this method is the detailed information on the spatiotemporal dynamics of contaminant migration and comprehensive information of groundwater vulnerability. Examples of the process-based method can be found in the recent review of Katyal et al. .As a specific process-based method, the backward travel time probability density (BTTPD) approach was proposed by Fogg et al.  to addreAlthough tremendous efforts have been made to assess groundwater vulnerability, there are two historical challenges. The first challenge is that efforts are needed to assess deep aquifer vulnerability for the following two reasons. First, the indices produced by the rating and statistical methods may only reflect potential of contaminant loading to the water table and do not represent vulnerability of deeper aquifers, especially in highly heterogeneous settings . The worThe second challenge is that previous studies usually quantified the groundwater vulnerability as a static value, while groundwater quality evolution is a long-term process. A transient index may describe better and provide more information about the vulnerability of real-world aquifers.d), deep aquifers. To check the feasibility of the transient, 3-d index map, we will apply it to assess the vulnerability of Kings River fluvial fan (KRFF) aquifer, located southeast of Fresno, California, USA  [vulnerability, by adjusting the initial/boundary conditions in flow/transport models and/or adding reactive terms in transport dynamics. Clearly, susceptibility analysis, or the intrinsic capability for contaminants to intrude on an aquifer system, is a prerequisite for reliable vulnerability analysis.Different from previous vulnerability assessment studies, here we address the less restrictive ristics) . Thus, cFogg et al.  applied X at time t originates from location Y at time s (s is a time before t), \u0398 is the effective porosity, D is the local hydrodynamic dispersion tensor. Note that here the dispersion tensor has the same value but different meanings in the backward and forward ADEs. In the forward-in-time ADE, D accounts for the uncertainty in concentration caused by molecular diffusion and differential advections; whereas in the backward-in-time ADE, D accounts for the uncertainty in the initial location and travel time of the particle as it moves backward [The BTTPD obeys the Chapman-Kolmogorov backward differential equation , which hbackward . The abobackward ,37,41. Award ADE ,38,43, wd continuous imaging of aquifer susceptibility instead of Fogg et al.\u2019s [d susceptibility image can provide whole-system mapping of aquifer susceptibility. It may also help us to distinguish the responses of different subsurface materials, including aquifers and aquitards, to contaminations. Aquitard susceptibility is considered in this study since aquitard materials can form significant sources/sinks of contaminants found in aquifers. Second, the susceptibility of a single aquifer system is treated as a time-dependent transient variable, not the static index calculated by Fogg et al. [d susceptibility maps to evaluate the influence of subsurface heterogeneity on aquifer susceptibility. This evaluation has not been done by Fogg et al. [Three logic extensions of Fogg et al.\u2019s  approachet al.\u2019s  discreteg et al. . A transg et al.  due to td susceptibility map for a million-node model in a PC. We circumvent this problem by distinguishing particles based on their releasing locations during one simulation of particle tracking. In other words, particles with labels are simultaneously released from each node and are then back tracked to obtain the BTTPDs  during one simulation. Second, if the node is close to the model boundaries, many particles can exit the model through the lateral or bottom boundaries. The groundwater age refers to the elapsed time since the water enters into the groundwater system through the water table. Therefore, the age is unknown for particles exiting boundaries other than the top boundary  in backward particle tracking experiments. In this study, we adjust the ages for these particles based on the depths at which they exit the model as was also proposed by Weissmann et al. [The particle-tracking algorithm used by Fogg et al.  needed tn et al. . Note thThe backward-in-time method is much more computationally efficient than forward methods, i.e., see . The sold advective flow field and dispersion, each particle history produces a different travel time, therefore generating full distributions of groundwater age within the modeled groundwater \u201csample\u201d. As will be shown later in this paper, such estimates of complete age distributions are useful and necessary not only for estimating susceptibility, but also for analyzing long-term evolution of groundwater quality as well as the transient susceptibility. The entire age distribution within single water sample generated by the particle tracking method distinguishes this study from other age simulation methods, such as Goode [In addition, because each particle takes on a different path due to the 3-as Goode , where o2 area of the Kings River fluvial fan aquifer  . ChannelThe third structure type is a coarse-grained incised-valley fill (IVF) deposit identified across the study area ,57. ThisHydrofacies within each of the stratigraphic sequences were simulated using transition probability geostatistics, as described by Weissmann and Fogg  and WeisDefinition of sequence stratigraphy is important for fluvial fan characterizations . WeissmaK) distributions, we apply the U. S. Geological Survey software MODFLOW [d, steady-state flow field. The conductivities assigned to each hydrofacies were 1.0 \u00d7 10\u22122, 1.0 \u00d7 10\u22123, 1.0 \u00d7 10\u22125, 1.0 \u00d7 10\u22126, 1.3 \u00d7 10\u22127 m/s for gravel, sand, muddy sand, mud, and paleosol, respectively [K of overlying beds for each cell at the water table [Using the geostatistical realizations to generate stochastic hydraulic conductivity  and the effective porosity (0.33). The single effective porosity was used for all hydrofacies (assigned with different K), since the spatial variation of porosity is relatively smaller than that for K, and the impact of the spatial variation of porosity on solute transport is secondary compared with the impact of K on transport [X \u00d7 Y \u00d7 Z) m. This assumption is based on (1) the conclusion of LaBolle and Fogg [The modeled flow velocities were incorporated into the particle tracking algorithm to simulate the groundwater BTTPD. The same transport parameters in Weissmann et al.  were useransport . The totand Fogg , who fouand Fogg , who foud susceptibilities are shown by the following five slices across the model domains: three horizontal slices at 5, 20, and 40 m below and parallel to the water table, respectively; one vertical slice parallel to the general groundwater flow direction and through (inside and below) the IVF; and another vertical slice parallel to the general groundwater flow direction and outside the IVF (X = 7100 m), where the high-K IVF truncates most parts of the uppermost paleosol. The vertical slice outside the IVF (X = 3600 m) contains all of the three layers of paleosols, with the uppermost paleosol lying close to the water table.The simulated 3- the IVF a. The thK hydrofacies since young water can also be found in some low-K zones or aquitards. Most of the discrete regions representing old waters, however, generally correspond to the location of aquitards. At a few small, separate high-K zones surrounded by low-K materials we observe very old water. Second, the IVF appears to significantly influence groundwater age and susceptibility. Inside or underlying the IVF, the system is dominated by relatively young water along the whole valley, independent of the corresponding hydrofacies type. This is especially true for saturated zones 20 and 40 m below the water table, where the shape of valley can be seen clearly from the correspondent susceptibility maps. Outside of the valley, the areas with relatively young water are not well connected horizontally, but intermingled with areas representing very old water.The susceptibilities along the three horizons  provide The susceptibility along the two vertical slices basically shows different behaviors from each other . In the To further explore the influence of heterogeneity on aquifer susceptibility, we compared the complete BTTPD along two vertical columns inside of the two vertical slices  through the IVF, the simulated BTTPD has three main characteristics  outside the IVF , which is different from the classical index  method [Although the influence of local heterogeneity on solute transport has been well-known for decades ,63,64, t) method .The regional-scale IVF significantly increases the susceptibility of aquifers by enhancing rapid downward solute movement. This is because of the ~80% of sandy-materials deposited in this valley. These high permeable materials incise through the uppermost paleosol layer, and pull in higher recharge than other areas in the model domain ,65. TherFor areas inside and below the IVF, the mean groundwater age may somewhat reflect the aquifer susceptibility, because the mean is fairly close to the mode and also the age distributions are relatively narrow. In addition, the relatively uniform susceptibility within/below the IVF at a same depth is caused by relatively homogeneity of the incised valley fill deposits and lack of extensive aquitard units within it. For aquifers inside and below the IVF, groundwater age usually increases with depth gradually. Hence, the regional-scale IVF has a dominant effect on aquifer susceptibility, overwhelming the influence of local-scale heterogeneity discussed above.2 of DBCP contaminated groundwater in the California Central Valley in 1986 [The measured concentration of pesticides is used here to test the susceptibility maps, and speculate on the historic role of the IVF on contaminant transport in KRFF aquifer system. 1,2-dibromo-3-chloropropane (DBCP) was a widely used nematicide during the 1950s through the 1970s, with the most frequent use between 1960 and 1979 [ in 1986 ). It was in 1986 ,67, muchThe regional-scale boundary paleosols within the open-fan deposits affect the BTTPD in two ways: (1) the relatively low permeability of paleosols retards the vertical transport of particles; and (2) the paleosols change the age distribution at a well by bringing more particles from deep zones, where the water age is generally old. This indicates that the overall sequence stratigraphic framework including paleosols decreases the deep aquifer susceptibility to contamination by altering pathways and delaying the vertical transport of contaminants. Therefore, the sequence boundary paleosols have the almost opposite effect on aquifer susceptibility as the IVF does.The BTTPDs for aquifers affected by the boundary paleosols contain heavier late-time tails than those affected by the IVF, and thus we cannot apply the BTTPD the same way as discussed in the previous section. Especially, for the open fan deposits bounded by paleosols, the mean groundwater-age-based susceptibility estimate will be misleading because the mean groundwater age is significantly influenced by the long late-time tail, missing the peak and the main part of a broad age distribution.One could argue that the simulated BTTPDs discussed above based on the single stochastic realization rather than on multiple realizations may not represent the full range of outcomes for transport and susceptibility in this complex aquifer system. Different aquifer realizations may have different distribution properties for both high- and low-permeable materials, resulting in different early-time tail, peak, and late-time tail in BTTPDs. Therefore, the details of BTTPD discussed above, such as the number of peaks and the actual spread length, may vary apparently between multiple aquifer realizations.However, as indicated by the numerical experiments explored by Weissmann et al. , the genGroundwater vulnerability was usually evaluated by a static index without information on long-term water quality evolution and deep-aquifer response to non-point source contamination. To address this historical issue, this study develops three-dimensional transient maps built upon reliable physical models , by extending the backward travel time probability density approach proposed by Fogg et al.  and LaBoFirst, the BTTPD can be used to derive transient, multi-dimensional indexes of aquifer susceptibility to non-point source contamination, which provides much more useful information than the classical, static index. The BTTPD can, if interpreted appropriately, provide the complete information to decipher the long-term evolution of groundwater quality affected by multi-scale groundwater circulation driven by aquifer/aquitard heterogeneity. Particularly, the cumulative BTTPDs with target time intervals can determine likelihood that an aquifer will be contaminated as a function of space and time.Second, the regional-scale incised-valley fill deposits increase the susceptibility of aquifers by enhancing rapid downward solute movement and displaying relatively narrow and young age distributions, due to the coarse-grained nature of the IVF and the incision of the uppermost paleosol layer. The historical record of non-point source pollutants  also implied the relatively more rapid downward movement of pollutants, although the data is not adequate to show the detailed evolution of pesticides across the large area of the study site.Third, the sequence boundary paleosols postpone the contamination of aquifers at all depths by retarding the vertical movement of contaminants and changing the proportions of particle plumes. Therefore, the existence of paleosol boundary decreases the susceptibility of saturated zones to recent contaminants with the price of an increased and a longer-term susceptibility to old contamination."}
+{"text": "Choosing or altering the planned statistical analysis approach after examination of trial data (often referred to as \u2018p-hacking\u2019) can bias the results of randomised trials. However, the extent of this issue in practice is currently unclear. We conducted a review of published randomised trials to evaluate how often a pre-specified analysis approach is publicly available, and how often the planned analysis is changed.A review of randomised trials published between January and April 2018 in six leading general medical journals. For each trial, we established whether a pre-specified analysis approach was publicly available in a protocol or statistical analysis plan and compared this to the trial publication.n\u2009=\u200931, 35%) and analysis population , followed by the use of covariates  and the approach for handling missing data . Many protocols or statistical analysis plans were dated after the trial had begun, so earlier discrepancies may have been missed.Overall, 89 of 101 eligible trials (88%) had a publicly available pre-specified analysis approach. Only 22/89 trials (25%) had no unexplained discrepancies between the pre-specified and conducted analysis. Fifty-four trials (61%) had one or more unexplained discrepancies, and in 13 trials (15%), it was impossible to ascertain whether any unexplained discrepancies occurred due to incomplete reporting of the statistical methods. Unexplained discrepancies were most common for the analysis model (Unexplained discrepancies in the statistical methods of randomised trials are common. Increased transparency is required for proper evaluation of results. The results of a clinical trial depend upon the statistical methods used for analysis. For example, changing the analysis population or method of handling missing data can change the size of the estimated treatment effect or its standard error. In some instances, these differences can be large and may affect the interpretation of the trial \u20136. If inGuidelines such as ICH-E9  , The Lancet, New England Journal of Medicine (NEJM), and PLOS Medicine. We searched for articles in PubMed with a publication type of \u2018randomized controlled trial\u2019 or categorised with the MeSH term \u2018random allocation\u2019, or including the keyword \u2018random*\u2019 in the title or abstract, restricted to the aforementioned included journals and publication period. The full search strategy is shown in Appendix 1 in Additional\u00a0file\u00a0In this review, we examined randomised controlled trials published between January and April 2018 in six general high-impact medical journals: Articles were eligible for inclusion if they reported results from a phase 2\u20134 randomised trial in humans. Exclusion criteria were pilot or feasibility study, phase 1 trial, non-randomised study, secondary analysis of previously published trial, cost-effectiveness as the primary outcome, more than one trial reported in the article, results of an interim analysis, or if the protocol or SAP was not in English.One author screened the title and abstract of each paper for eligibility. The full texts of these articles were then assessed independently by two reviewers to confirm eligibility. For all eligible studies, one author searched the main text, supplementary material, and references to identify whether a protocol and/or SAP was available.Data was extracted onto a pre-piloted standardised data extraction form by two reviewers independently  if one outcome was listed as the primary, we used this; (b) if no outcomes or multiple outcomes were listed as being primary, we used the outcome that the sample size calculation was based on; and (c) if no sample size calculation was performed or sample size was calculated for multiple primary outcomes, we used the first clinical outcome listed in the objectives/outcomes section. We identified the primary analysis as follows: (a) if a single analysis strategy was used, or multiple strategies were used with one being identified as primary, we used this; (b) if multiple strategies were used without one being identified as primary, we used the first one presented in the results section.For each article, we extracted general trial characteristics, whether protocols or SAPs were available, including the given dates of these documents and, if available, the blinding status of trial statisticians. For published protocols or SAPs, we used the date of publication. For protocols or SAPs available as supplementary material with the results article or elsewhere, we used the given date on the documents. For articles with a protocol or SAP, we compared the method of analysis in the trial publication against the method specified in the earliest available protocol or SAP which included some information on the analysis of the primary outcome . We assessed the following four analysis elements: (i) analysis population , (ii) the statistical analysis model, (iii) use of baseline covariates in the analysis, and (iv) the method for handling missing data. We chose these elements as they are specified in the SPIRIT guidelines and have been used in previous reviews , 26.We evaluated two types of discrepancies for each analysis element. The first, termed a \u2018change\u2019, occurred when the analysis element in the trial publication was different to that specified in the original analysis plan. The following examples would constitute changes: (a) if an intention-to-treat analysis population was originally specified, but a per-protocol analysis was used; (b) if the functional form of the statistical analysis model was changed, such as from a mixed-effects regression model to generalised estimating equations (GEE); (c) if the original analysis plan specified the analysis would not adjust for baseline covariates but the trial publication adjusted for one or more patient characteristic; or (d) if a complete case analysis was originally specified, but multiple imputation was used.The second discrepancy, termed an \u2018addition\u2019, occurred when the original analysis plan gave the investigators flexibility to subjectively choose the final analysis method after seeing trial data. This could occur if the original analysis plan (i) contained insufficient information about the proposed analysis or (ii) allowed the investigators to subjectively choose between multiple different potential analyses. The following examples would constitute additions: if the original analysis plan stated that (a) both a per-protocol and intention-to-treat analysis population would be used, without specifying which was the primary analysis ; (b) either parametric or non-parametric methods would be used depending on distributional assumptions, but did not define an objective criteria for assessing distributional assumptions (as the investigators could then present whichever method gave the most favourable result); (c) the analysis would adjust for important baseline covariates, but did not define how these covariates would be chosen ; or (d) multiple imputation would be used, but did not define what the method of imputation would be, or what variables would be included in the imputation model .We classified each discrepancy as being \u2018explained\u2019 or \u2018unexplained\u2019. Discrepancies were classified as explained if they had been specified in a subsequent version of the protocol or SAP  or if the trial publication explained that an alteration to the pre-specified analysis approach had been made. Otherwise, discrepancies were classified as unexplained.The main outcome measures were (i) the number of trials with a publicly available pre-specified analysis approach for the primary outcome , (ii) the number of trials with no unexplained discrepancies from the publicly available pre-specified analysis approach, and (iii) the total number of analysis elements for each trial with an unexplained discrepancy.Secondary outcomes were, for each analysis element described earlier, (i) the number of trials with at least one unexplained discrepancy (either change or addition), (ii) the number of trials with at least one unexplained change, and (iii) the number of trials with at least one unexplained addition.Outcomes were summarised descriptively using frequencies and percentages. We performed two pre-specified subgroup analyses, where we summarised outcomes separately according to trial funding status  and type of intervention . Two post hoc subgroup analyses were performed. The first summarised outcomes according to whether a pre-specified analysis approach was made available prior to the publication of the results article . The second summarised outcomes between trials with a standalone SAP available versus those without (regardless of the availability of a protocol).All statistical analyses were performed using Stata version 15 .Our search identified 197 articles, of which 101 were eligible see Fig.\u00a0. GeneralProtocols were available for 90 trials (89%) . SAPs were available for 46 trials (46%) . Of the 90 trials with an available protocol, the earliest version available was dated before recruitment began for 45 (50%) trials, 19 (21%) were dated during recruitment, 8 (9%) were dated after recruitment ended, and 18 (20%) did not have a date. Of the 46 trials with an available SAP, the earliest version of the SAP was dated before recruitment began for 9 (20%) trials, 13 (28%) were dated during recruitment, 13 (28%) were dated after recruitment ended, and 11 (24%) did not have a date.Overall, only 11 trials (11%) stated in the trial publication, protocol, or SAP that the statistician was blinded until the SAP was signed off, and 10 (10%) stated the statistician was blinded until the database was locked.Overall, 89 of 101 trials (88%) had a publicly available pre-specified analysis approach for the primary outcome. Eleven trials did not have an available protocol or SAP, and one trial had a protocol with no information on the analysis and no SAP. The document containing the original analysis plan  was dated before the start of recruitment for 41 of 89 (46%) trials, during recruitment in 19 (21%) trials , and after the end of recruitment in 8 (9%) trials . In 21 trials (24%), no date was available.For 46 of 89 trials (52%), the document containing the original analysis plan was made publically available prior to the trial publication , whereas the document for the remaining 43 trials (48%) was only made publically available at the point the trial was published .n\u2009=\u20095, explained discrepancies only n\u2009=\u200917). A further 54 trials (61%) had one or more unexplained discrepancies .Of the 89 trials with an available pre-specified analysis approach, only 22 (25%) did not have any unexplained discrepancies  or two  unexplained discrepancies. Only 11 (12%) had three and 2 (2%) had four unexplained discrepancies. Unexplained discrepancies were most common for the statistical analysis model  and analysis population , followed by the use of covariates  and handling of missing data . Table\u00a0Most trials had one n\u2009=\u20095, 28% orOverall, 29 trials (33%) had at least one explained discrepancy. Most discrepancies were explained in a later version of the protocol or SAP; only 2 trials explained a discrepancy in the trial publication. Of the 29 trials with an explained discrepancy, only 6 (21%) stated that the statistician was blinded until the SAP was signed off, and 4 (14%) until the database was locked.A total of 43/61 (66%) trials that had no for-profit funding had at least one unexplained discrepancy, compared to 11/28 (45%) trials that had some for-profit funding. One or more unexplained discrepancy occurred for 25 of 47 (53%) pharmaceutical intervention trials, 10 of 12 surgical trials (83%), 9 of 9 (100%) psychological/behavioural/educational trials, 9 of 19 (47%) other trials, and 1 of 2 trials (50%) with multiple intervention types.Fewer trials with a SAP available had unexplained discrepancies than trials without an available SAP, though this figure was still high (SAP available 22/46 [48%] with \u2265\u20091 unexplained discrepancy vs. no SAP 32/43 [74%]). Trials with a SAP still had a relatively high number of additions to the analysis method, indicating that methods were not being adequately pre-specified within these SAPs .A total of 34/46 (74%) trials in which the original analysis plan was made available prior to the trial publication had at least one unexplained discrepancy, compared to 20/43 (47%) trials where the analysis approach was only made available at the time of trial publication.See Additional\u00a0file\u00a0In our review of 101 trials published in high-impact general medical journals, we found that most had a pre-specified analysis approach for the primary outcome available in either a protocol or SAP. This is essential to allow transparent assessment of whether inappropriate changes were made to the statistical methods. However, most pre-specified statistical analysis approaches were available in a document that was dated after the trial had begun, or had no date available. It is therefore possible that the analysis approach in these documents may have already been changed from the pre-trial version. Moreover, there was poor reporting around the blinding status of statisticians rendering it impossible to verify that the pre-specified analysis approach had not been written after seeing unblinded trial data. In this case, even if no alterations were made to the pre-specified approach, results still may be subject to bias.Only 25% of trials did not have any unexplained discrepancies between the trial publication and the pre-specified analysis approach, and only 6% had no discrepancies at all. Most trials had at least one unexplained discrepancy (61%), with 32% of trials having two or more. In 15% of trials, it was impossible to assess whether there were unexplained discrepancies due to poor reporting of the statistical methods used. Of note, 33% of trials had one or more explained discrepancies; however, less than a quarter of these trials reported that the statistician was blinded to treatment allocation until the analysis plan was finalised or the database was locked. These alterations may therefore have been made based on unblinded trial data, despite being explained. It was also surprising that only two trials explained a discrepancy in the trial publication, despite requirements by the CONSORT [Our results are broadly consistent with previous reviews. Spence et al.  evaluateThe key issues we identified in this study were (i) low availability of pre-trial protocols and analysis plans, (ii) poor pre-specification of statistical methods within protocols and analysis plans, (iii) frequent unexplained discrepancies in the final trial publication, (iv) poor reporting of the blinding status of statisticians in relation to modifications of analysis methods or access to trial data, and (v) poor descriptions of the actual analysis methods used in the final publication. Increased adherence to guidelines such as SPIRIT, CONSORT, and the guidelines for Statistical Analysis Plans , 27, 28 Our study had some limitations. We only included articles from six high-impact medical journals; it is likely that trials published in other journals may have lower availability of protocols and SAPs and higher rates of unexplained discrepancies. Protocols and SAPs were identified via searching the main text, supplementary material, and references of articles; it is possible that we might have missed a few articles, but we expect this number to be very small, as in our experience when protocols/SAPs are available they are referenced to within the paper. Comparisons were based on the first available protocol or SAP; however, many were dated after the trial had begun, so there may have been discrepancies before this that we missed. We also assumed the dates available on protocols and SAPs included as part of the supplementary material alongside trial publications were accurate. However, we had no way to verify this, and it is possible that some of these dates are incorrect, which could imply a higher proportion of analysis approaches were written later on in the life cycle of the trial than what we have reported here.In conclusion, unexplained discrepancies in the statistical methods of randomised trials are common. Increased transparency around the statistical methods used in randomised trials is required for proper evaluation of trial results.Additional file 1. Protocol and data extraction form \u2013 contains protocol and data extraction form used for this study.Additional file 2. Supplementary material \u2013 contains additional methods and results."}
+{"text": "Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome (ARDS) but it is unknown whether prone positioning improves outcomes in mechanically ventilated patients with moderate to severe ARDS due to COVID-19.A cohort study at a New York City hospital at the peak of the early pandemic in the United States, under crisis conditions. The aim was to determine the benefit of prone positioning in mechanically ventilated patients with ARDS due to COVID-19. The primary outcome was in-hospital death. Secondary outcomes included changes in physiologic parameters. Fine-Gray competing risks models with stabilized inverse probability treatment weighting (sIPTW) were used to determine the effect of prone positioning on outcomes. In addition, linear mixed effects models (LMM) were used to assess changes in physiology with prone positioning.P< 0.005). Using LMM to evaluate the impact of positioning maneuvers on physiological parameters, the oxygenation-saturation index was significantly improved during days 1\u20133 (P< 0.01) whereas oxygenation-saturation index (OSI), oxygenation-index (OI) and arterial oxygen partial pressure to fractional inspired oxygen (PaO2:FiO2) were significantly improved during days 4\u20137 .Out of 335 participants who were intubated and mechanically ventilated, 62 underwent prone positioning, 199 met prone positioning criteria and served as controls and 74 were excluded. The intervention and control groups were similar at baseline. In multivariate-adjusted competing risks models with sIPTW, prone positioning was significantly associated with reduced mortality (SHR 0.61, 95% CI 0.46\u20130.80, Prone positioning in patients with moderate to severe ARDS due to COVID-19 is associated with reduced mortality and improved physiologic parameters. One in-hospital death could be averted for every eight patients treated. Replicating results and scaling the intervention are important, but prone positioning may represented an additional therapeutic option in patients with ARDS due to COVID-19. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has had a profound impact on global public health. The ongoing COVID-19 pandemic has presented numerous clinical management challenges further compounded by overwhelmed health systems. The initial critical care experience in Hubei province, and more broadly in China, inadequately informed preparations for what has been seen in Europe and North America., 4Several interventions for ARDS have been evaluated over the last two decades. In particular, prone positioning is one of few therapeutic interventions for patients with severe ARDS that has demonstrated improved oxygenation and a survival benefit. Awake prThe South Bronx is a socioeconomically disadvantaged borough in New York City (NYC) that had the highest per capita COVID-19 case count in the United States at 2941 per 100,000 residents with very high hospitalization and death rates., 12 The We sought to determine if patients on mechanical ventilation with moderate to severe ARDS who underwent standardized prone positioning had lower mortality and improved within-person physiologic changes. As we rapidly evaluate drugs and interventions for COVID-19, it is crucial to understand if serial prone positioning could be a complementary therapeutic intervention for the most critically ill.A cohort design with participants from the peak of hospitalizations for COVID-19 in exposed (prone positioning) and non-exposed (non-prone-positioning) groups. During the COVID-19 pandemic, much of the hospital was converted into make-shift intensive care units and virtually all inpatients had confirmed COVID-19. During this time, a multidisciplinary prone team including personnel from the United States Air Force Medical and Nursing Corps, the United States Army, civilian contractors, and hospital occupational and physical therapy was assembled to offer positioning maneuvers which were otherwise rarely done due to crisis operations. Details of the prone positioning process, including peri-maneuver checklists, team size and roles, supplies and team schedule are included in a priori): arterial oxygen partial pressure to fractional inspired oxygen (PaO2:FiO2) <150 mm Hg, positive end-expiratory pressure (PEEP) >10 cm of water and FiO2 > 0.6. The ultimate decision for initiating and discontinuing positional movements was made by the primary team overseeing and coordinating care for each patient. Prone positioning was not mandatory, but was routinely available, 24 hours a day, seven days a week. The study received institutional review board approval (IRB # 20 \u2013 007).Participants were identified from a single level 1 trauma hospital in the South Bronx, New York City, and were included across all hospital services . All sequential adult patients (>17 years of age) were included if they were intubated, had not undergone prone positioning by others, met criteria for prone positioning, and had confirmed SARS-CoV-2 infection by real-time real-time reverse transcription-polymerase chain nasal swab  from March 25 through May 2, 2020. The prone team offered positional services for mechanically ventilated patients who met the following criteria , acute physiology and chronic health evaluation (APACHE-II) score and vasopressor use were the primary confounders by indication. In the mortality analysis, the APACHE-II score was evaluated at the time of intubation., oxygenation saturation index (OSI), PaOtubation. BMI and Characteristics of the cohort were summarized using descriptive statistics as appropriate. Fine-Gray models were used to assess the association between prone positioning and death, accounting for hospital discharge as a competing risk. ParticipLinear mixed models (LMMs) were used to assess the association of prone vs supine positioning with physiologic parameter levels among the exposed. Outcomes were natural log transformed to meet normality assumptions. The LMMs included nested random effects for participant and positioning episode, and allowed for autocorrelation of the residuals. In addition to estimating overall positional effects, we also estimated these effects in days 1\u20133 and 4\u20137 of each episode. Pearson correlation coefficients were also used to characterize degree of agreement for OI, OSI, PaO2:FiO2 and SpO2:FiO2, to support clinical utility in practice. Analyses were performed in Stata .During the study period, 335 individuals were intubated and placed on mechanical ventilation. Sixty-two underwent prone positioning while 199 who did not undergo positioning changes, but met criteria to do so, were selected as contemporary controls. Seventy-four individuals were excluded for failing to meeting prone positioning criteria or for having undergone prone positioning by providers outside of the standard protocol. A study flow diagram depicts the inclusion and exclusion of participants across groups in Compared to contemporary controls, the prone positioning group had fewer deaths and a longer time to death in those who expired, in spite of similar length of stay and ventilator-free days. Estimates of the association between prone positioning and mortality are summarized in aO2:FiO2, SpO2:FiO2 and PaO2. For days 4\u20137 of prone positioning, improvement was seen in the PaO2:FiO2, SpO2:FiO2 and PaO2. Only the OI failed to show improvement at any time and OSI did not show improvement for days 4\u20137. During crisis operations with enhanced infection control and use of transport ventilators for routine ventilation, it may be difficult to obtain PaO2 and mean airway pressure values and so proxy variables may be helpful. We therefore looked at Pearson correlation coefficients amongst ratios and indices. Overall, PaO2:FiO2 and SpO2:FiO2 are moderately correlated (p = \u22120.51), and OSI and OI, and OSI and SpO2:FiO2, are closely correlated . The correlations did not differ when split into days 1\u20133 and 4\u20137. Results are summarized in aO2:FiO2 . No clear evidence for interaction between positioning and time was found.In analyses using LMMs to estimate the association of positioning with physiological indices, 19 of 62 exposed participants contributed more than one episode. In these analyses, prone vs supine positioning was significantly associated with overall improvement in PaO2:FiO2 . In modeWe report results from a comprehensive cohort study assessing the potential benefits of prone positioning in COVID-19 patients with moderate to severe ARDS. We found a nearly 40% reduction in mortality with prone positioning, an effect that appears sustained on cumulative incidence curves. With respect to physiologic parameters, there were meaningful changes across all ratios and indices to suggest that prone positioning is associated with improvements in within-person physiology and that the benefit may persist beyond three days. Our findings across both analyses were robust to various adjustments, modifications, sensitivity analyses and nested comparative testing.Fundamentally, this study has three key findings. First, we demonstrated a mortality benefit with prone positioning with a number needed to treat of eight. The durability of the finding is important, and ensuring that it can be replicated in other settings will be essential to justify a recommendation, but we have no evidence to attribute the survival benefit in the intervention arm to bias. Second, it appears that there is a benefit to additional days of prone positioning beyond 3 days. The effect seen with 4\u20137 days of prone positioning may be heavily influenced by a smaller group that realized a differential benefit, but 34 of 89 positioning sequences resulted in at least four days of intervention, representing a relatively large proportion of individuals. Third, prone positioning resulted in significant changes in physiologic parameters which may support the underlying hypothesis that prone positioning improves ventilation-perfusion matching. AdditionOur results are consistent with recent multi-center data suggesting a mortality benefit of prone positioning in patients with ARDS whether intubated or not., 19, 20 Some limitations of this study should be noted. First, this is a single center retrospective cohort study in a resource constrained environment under crisis operations. As a result, although patients had critical care needs, they were frequently cared for in ad-hoc intensive care units by non-critical care personnel. The decision to initiate or discontinue the intervention under study was left to the treating primary team without defining endpoints. We attempted to address any residual confounding through IPTW and no differences in the results were noted. If the prone team was consulted and the patient had moderate to severe ARDS and met criteria for prone positioning, it was felt that they could benefit from the intervention in addition to lung protective ventilation. Although this was pragmatic for this setting, if prone positioning is implemented elsewhere, the prone teams could consider establishing an opt-out approach with tailored entry and exit criteria, normal cadence of evaluation for candidacy for prone positioning and a mechanism for real-time data capture and quality control assessments. Finally, the results may not be readily generalizable to all populations, in particular those with milder disease and those that don\u2019t reflect the ethnic diversity seen in the Bronx. The institutional mortality proportion was high (>75%) and therefore the impact of the intervention may be attenuated in the setting of advanced interventions .There are also some notable strengths of this work. We were able to collect detailed physiologic data in a structured manner to systematically evaluate the impact of the intervention. Also, our population has been gravely understudied in the COVID-19 pandemic and we\u2019ve been able to contribute significantly to both describing their clinical course as well as critical care interventions for socioeconomically marginalized minority populations. Regarding outcome, we were able to include all patients who would have been eligible for prone positioning as controls creating a sound counterfactual for a contemporaneous comparison of both exposed and unexposed. Finally, compared to existing literature for patients with COVID-19, this study provides results for a large intervention group.In summary, we present data supporting prone positioning as an intervention to prolong survival and improve physiologic parameters in patients on mechanical ventilation with moderate to severe ARDS due to COVID-19. The findings should be replicated across institutions, but prone positioning may be an important consideration for health systems, particularly in the setting of an evolving suite of complementary interventions in the care of such vulnerable patients."}
+{"text": "Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distresssyndrome (ARDS) but it is unknown whether prone positioning improvesoutcomes in mechanically ventilated patients with moderate to severe ARDSdue to COVID-19.A cohort study at a New York City hospital at the peak of the early pandemicin the United States, under crisis conditions. The aim was to determine thebenefit of prone positioning in mechanically ventilated patients with ARDSdue to COVID-19. The primary outcome was in-hospital death. Secondaryoutcomes included changes in physiologic parameters. Fine-Gray competingrisks models with stabilized inverse probability treatment weighting (sIPTW)were used to determine the effect of prone positioning on outcomes. Inaddition, linear mixed effects models (LMM) were used to assess changes inphysiology with prone positioning.P < 0.005). Using LMM toevaluate the impact of positioning maneuvers on physiological parameters,the oxygenation-saturation index was significantly improved during days 1-3(P < 0.01) whereas oxygenation-saturation index(OSI), oxygenation-index (OI) and arterial oxygen partial pressure tofractional inspired oxygen (PaO2: FiO2)were significantly improved during days 4-7 .Out of 335 participants who were intubated and mechanically ventilated, 62underwent prone positioning, 199 met prone positioning criteria and servedas controls and 74 were excluded. The intervention and control groups weresimilar at baseline. In multivariate-adjusted competing risks models withsIPTW, prone positioning was significantly associated with reduced mortality(SHR 0.61, 95% CI 0.46-0.80, Prone positioning in patients with moderate to severe ARDS due to COVID-19 isassociated with reduced mortality and improved physiologic parameters. Onein-hospital death could be averted for every 8 patients treated. Replicatingresults and scaling the intervention are important, but prone positioningmay represent an additional therapeutic option in patients with ARDS due toCOVID-19. In the setting of critical COVID-19 illness, SARS-CoV-2infection often results in severe pneumonia and hypoxemia with many patientsdeveloping acute respiratory distress syndrome (ARDS).2 Hypoxemic respiratory failure with ARDS has poor outcomes overall andCOVID-19 associated ARDS is no exception.4Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause ofcoronavirus disease 2019 (COVID-19), has had a profound impact on global publichealth. The ongoing COVID-19 pandemic has presented numerous clinical managementchallenges further compounded by overwhelmed health systems. The initial criticalcare experience in Hubei province, and more broadly in China, inadequately informedpreparations for what has been seen in Europe and North America.5 Awake prone positioning outside of the intensive care unit (ICU) is safe andmay decrease respiratory rate and improve oxygenation with early applicationpotentially delaying need for intubation in patients with COVID-19.68 In the ICU setting, prone positioning of patients receiving non-invasiveventilation or high-flow nasal canula, with or without sedation, may also be beneficial.8 Physiologically, prone positioning may improve matching of ventilation andperfusion, but studies have not linked physiologic changes to clinical outcomes,especially in COVID-19.10Several interventions for ARDS have been evaluated over the last 2 decades. Inparticular, prone positioning is one of few therapeutic interventions for patientswith severe ARDS that has demonstrated improved oxygenation and a survival benefit.12 The pressing challenge that COVID-19 brought to NYC necessitated externalsupport through the United States Departments of Defense and Homeland Security,re-distribution and up-training of local hospital staff, support from clinicalvolunteers, and augmentation through healthcare worker staffing agencies. Given thehigh volume of critically-ill patients admitted to the hospital, a multidisciplinaryteam was assembled to provide prone positioning given the support for the practicein other populations with ARDS.The South Bronx is a socioeconomically disadvantaged borough in New York City (NYC)that had the highest per capita COVID-19 case count in the United States at 2941 per100,000 residents with very high hospitalization and death rates.We sought to determine if patients on mechanical ventilation with moderate to severeARDS who underwent standardized prone positioning had lower mortality and improvedwithin-person physiologic changes. As we rapidly evaluate drugs and interventionsfor COVID-19, it is crucial to understand if serial prone positioning could be acomplementary therapeutic intervention for the most critically ill.A cohort design with participants from the peak of hospitalizations for COVID-19in exposed (prone positioning) and non-exposed (non-prone-positioning) groups.During the COVID-19 pandemic, much of the hospital was converted into make-shiftintensive care units and virtually all inpatients had confirmed COVID-19. Duringthis time, a multidisciplinary prone team including personnel from the UnitedStates Air Force Medical and Nursing Corps, the United States Army, civiliancontractors, and hospital occupational and physical therapy was assembled tooffer positioning maneuvers which were otherwise rarely done due to crisisoperations. Details of the prone positioning process, including peri-maneuverchecklists, team size and roles, supplies and team schedule are included in2:FiO2) < 150 mm Hg, positiveend-expiratory pressure (PEEP) \u226510 cm of water and FiO2\u2009\u2009\u2265\u2009\u20090.6.Patients with a do not resuscitate order were not explicitly excluded from thestudy. In addition, continuous venovenous hemodialysis (CVVHD), nitric oxide orextracorporeal membrane oxygen (ECMO) were not available in the facility andintermittent hemodialysis or paralytics were rarely used. The ultimate decisionfor initiating and discontinuing positional movements was made by the primaryteam overseeing and coordinating care for each patient. Prone positioning wasnot mandatory, but was routinely available, 24 hours a day, 7 days a week. Thestudy received institutional review board approval (IRB # 20-007). The pronepositioning service was advertised through critical care ,hospital medicine, and physical medicine and rehabilitation leadership. Theseservices had knowledge of and direct access to every patient who wasmechanically ventilated in the hospital, even when they were not the primaryteam.Participants were identified from a single level 1 trauma hospital in the SouthBronx, New York City, and were included across all hospital services . All sequential adult patients (>17 years of age)were included if they were intubated, had not undergone prone positioning byothers, met criteria for prone positioning, and had confirmed SARS-CoV-2infection by real-time reverse transcription-polymerase chain nasal swab from March 25 throughMay 2, 2020. The prone team offered positional services for mechanicallyventilated patients who met the following criteria (established a priori):arterial oxygen partial pressure to fractional inspired oxygen , acute physiology and chronic health evaluation (APACHE-II) score andvasopressor use were the primary confounders by indication. In the mortalityanalysis, the APACHE-II score was evaluated at the time of intubation.13 BMI and age were categorized for ease of interpretation and clinicalutility. The study team obtained the study data through manual electronicmedical record chart abstraction .The primary exposure was positional maneuvers, defined as regular alternationbetween prone and supine positioning. The primary outcomes of interest werein-hospital mortality and, among exposed patients, differences in physiologicalparameters in prone vs supine position. In the mortality analysis, every patienthad at least 30 days elapse following initiation of, or meeting criteria for,prone positioning. Follow-up of unexposed controls began when the participantfirst met prone positioning criteria during the 2 weeks after intubation. In theanalysis of positioning effects on physiologic parameters among exposedpatients, repeated measures of the oxygenation index (OI), oxygenationsaturation index (OSI), PaO14 Participants remaining in the hospital at the end of follow-up werecensored. The proportional sub-distribution hazards assumption was assessedvisually through cumulative incidence curves. To minimize confounding byindication, we used standard regression adjustment as well as a doubly robustapproach adding stabilized inverse probability treatment weights (IPTWs) to thefully adjusted model.1517 A sensitivity analysis was done to identify changes in results byexcluding controls that died within 48 hours of intubation. In addition, numberneeded to treat was calculated by the inverse of the averaged absolute riskdifferences at 30 days, for all participants at their actual and counterfactualvalues of prone positioning, and in combination with their observed covariate values.18Characteristics of the cohort were summarized using descriptive statistics asappropriate. Fine-Gray models were used to assess the association between pronepositioning and death, accounting for hospital discharge as a competing risk.Linear mixed models (LMMs) were used to assess the association of prone vs supinepositioning with physiologic parameter levels among the exposed. Outcomes werenatural log transformed to meet normality assumptions. The LMMs included nestedrandom effects for participant and positioning episode, and allowed forautocorrelation of the residuals. In addition to estimating overall positionaleffects, we also estimated these effects in days 1-3 and 4-7 of each episode.Pearson correlation coefficients were also used to characterize degree ofagreement for OI, OSI, PaO2: FiO2 and SpO2: FiO2, to support clinical utility inpractice. Analyses were performed in Stata .During the study period, 335 individuals were intubated and placed on mechanicalventilation. Sixty-two underwent prone positioning while 199 who did not undergopositioning changes, but met criteria to do so, were selected as contemporarycontrols. Seventy-four individuals were excluded for failing to meet pronepositioning criteria or for having undergone prone positioning by providers outsideof the standard protocol. A study flow diagram depicts the inclusion and exclusionof participants across groups in Compared to contemporary controls, the prone positioning group had fewer deathsand a longer time to death in those who expired, in spite of similar length ofstay and ventilator-free days. Estimates of the association between pronepositioning and mortality are summarized in aO2: FiO2,SpO2: FiO2 and PaO2.For days 4-7 of prone positioning, improvement was seen in thePaO2: FiO2, SpO2:FiO2 and PaO2. Only the OI failed to showimprovement at any time and OSI did not show improvement for days 4-7. Duringcrisis operations with enhanced infection control and use of transportventilators for routine ventilation, it may be difficult to obtainPaO2 and mean airway pressure values and so proxyvariables may be helpful. We therefore looked at Pearson correlationcoefficients among ratios and indices. Overall, PaO2:FiO2 and SpO2: FiO2 aremoderately correlated (p = \u22120.51), and OSI and OI, and OSI andSpO2: FiO2, are closely correlated.The correlations did not differ when split into days 1-3 and 4-7. Results aresummarized in aO2:FiO2. In models allowing positioning effects to differ in days 1-3 and4-7, prone positioning was associated with improved OSI during days 1-3 (p <0.01) as well as improved OSI, OI and PaO2:FiO2 during days 4-7 . No clear evidence for interaction between positioning and timewas found.In analyses using LMMs to estimate the association of positioning withphysiological indices, 19 of 62 exposed participants contributed more than 1episode. In these analyses, prone vs supine positioning was significantlyassociated with overall improvement in P2:FiO2. In modWe report results from a comprehensive cohort study assessing the potential benefitsof prone positioning in COVID-19 patients with moderate to severe ARDS. We found anearly 40% reduction in mortality with prone positioning, an effect that appearssustained on cumulative incidence curves. With respect to physiologic parameters,there were meaningful changes across all ratios and indices to suggest that pronepositioning is associated with improvements in within-person physiology and that thebenefit may persist beyond 3 days. Our findings across both analyses were robust tovarious adjustments, modifications, sensitivity analyses and nested comparativetesting.10 Additionally, we demonstrated the utility of relatively accessible clinicalinformation in the ICU as reasonable surrogates to monitor changes inphysiology.Fundamentally, this study has 3 key findings. First, we demonstrated a mortalitybenefit with prone positioning with a number needed to treat of 8. The durability ofthe finding is important, including for a longer time period, and ensuring that itcan be replicated in other settings will be essential to justify a recommendation,but we have no evidence to attribute the survival benefit in the intervention arm tobias. Second, it appears that there is a benefit to additional days of pronepositioning beyond 3 days. The effect seen with 4-7 days of prone positioning may beheavily influenced by a smaller group that realized a differential benefit, but 34of 89 positioning sequences resulted in at least 4 days of intervention,representing a relatively large proportion of individuals. Third, prone positioningresulted in significant changes in physiologic parameters which may support theunderlying hypothesis that prone positioning improves ventilation-perfusion matching.620 There are recommendations for prolonged prone positioning of 12-16 hoursdaily for mechanically ventilated adult patients with COVID-19 and refractoryhypoxemic respiratory failure,21 but the optimal duration of the intervention, its impact on physiologicparameters and details regarding how to organize and structure an intervention teamduring a crisis have not been completely evaluated. We acknowledge that pronepositioning in mechanically ventilated patients is a resource-intensiveintervention, particularly in overwhelmed healthcare systems during pandemicconditions. Before adopting prone positioning techniques, staff education andcommitment is paramount. If justified by hospitalized patient volume, we recommendidentifying personnel and assigning them to a dedicated prone team and tailoringreadily available checklists to institutional needs and constraints  and therefore theimpact of the intervention may be attenuated in the setting of advancedinterventions  or the added attention andcare of a multidisciplinary team could in and of itself change patient\u2019s outcometrajectories, even though they were not involved in care decisions and did notintervene beyond prone positioning. Finally, there may be channeling bias due todisease severity or survivor bias, resulting in lower or higher probability ofexposure, respectively. The net result of this could be a bias toward or away fromthe null. We attempted to address this by ensuring that the populations werecomparable at baseline and by systematically including all possible patients. Atintubation, there was little difference in the quick SOFA, Charlson Comorbidityindex and the APACHE-II scores suggesting that the groups were similar, at leastwith respect to severity of illness.There are also some notable strengths of this work. We were able to collect detailedphysiologic data in a structured manner to systematically evaluate the impact of theintervention. Also, our population has been gravely understudied in the COVID-19pandemic and we\u2019ve been able to contribute significantly to both describing theirclinical course as well as critical care interventions for socioeconomicallymarginalized minority populations. Regarding outcome, we were able to include allpatients who would have been eligible for prone positioning as controls creating asound counterfactual for a contemporaneous comparison of both exposed and unexposed.Finally, compared to existing literature for patients with COVID-19, this studyprovides results for a large intervention group.In summary, we present data supporting prone positioning as an intervention toprolong survival and improve physiologic parameters in patients on mechanicalventilation with moderate to severe ARDS due to COVID-19. The findings should bereplicated across institutions, but prone positioning may be an importantconsideration for health systems, particularly in the setting of an evolving suiteof complementary interventions in the care of such vulnerable patients."}
+{"text": "Small intestinal hemangiomas are uncommon tumors that frequently present with gastrointestinal bleeding (GIB). Diagnosis, detection, and treatment can be challenging and may require surgical intervention. An 81-year-old female presented with melena. Video capsule endoscopy revealed active bleeding in the proximal jejunum and push enteroscopy identified a polypoid nodule with central umbilication. The patient underwent laparoscopic resection and jejunal submucosal hemangioma was detected. Submucosal hemangiomas are a rare cause of GIB. As the most common site of submucosal hemangiomas is the mid-jejunum, they are not easy to detect. Surgical intervention is usually required for a definitive diagnosis and definitive treatment. Intestinal hemangiomas are uncommon tumors, comprising only 0.05% of all intestinal neoplasms\u00a0. These bThe diagnosis of intestinal hemangioma can be challenging due to their location, and often no preoperative diagnosis is identified. Endoscopic resection is feasible for small lesions. However, surgical resection is the preferred treatment modality for large lesions or small lesions not amenable to endoscopic treatment. A case of an 81-year-old female with a jejunal hemangioma that was identified by capsule endoscopy and push enteroscopy before undergoing surgical resection is presented herein.An 81-year-old female presented with a one-week history of melena. The development of melena was associated with fatigue, pallor, palpitations, and shortness of breath on exertion. Hematemesis, hematochezia, nonsteroidal anti-inflammatory drug (NSAID) use, use of anticoagulants or antiplatelet agents were denied. Presenting blood pressure was 130/70 mmHg and the heart rate was 106 beats/minute. Her physical examination was unremarkable except for pallor and lower limb edema.\u00a0Admission hemoglobin was 4.7 g/dL, which was lower than her baseline of 8 g/dL. Transfusions were administered to maintain hemoglobin above 7 g/dL, and a proton pump inhibitor infusion was initiated. Esophagogastroduodenoscopy (EGD) was performed; benign-appearing and non-bleeding polyps were detected in the gastric fundus and body Figure\u00a0.The patient\u2019s hemoglobin continued to trend downward, and melena continued. Subsequently, colonoscopy did not detect significant abnormalities other than moderate diverticulosis, internal hemorrhoids, and old melenic liquid throughout the colon and ileum but no active bleeding Figure\u00a0.\u00a0Due to ongoing transfusion requirements, a video capsule endoscopy was performed, and active bleeding was identified shortly after the capsule passed the duodenum. Push enteroscopy was then performed, and a polypoid nodule with central umbilication and red spot was detected in the proximal jejunum Figure\u00a0.\u00a0The lesion was biopsied and post-biopsy bleeding developed Figure\u00a0.Hemostasis was achieved by injection of epinephrine (1:10000 concentration) and application of three hemostatic clips. After endoscopic intervention, the patient\u2019s hemoglobin remained stable, and further transfusions were not required.Due to concerns for malignancy and further bleeding a laparoscopic segmental jejunal resection was performed. Histological examination of the resected specimen confirmed a jejunal submucosal hemangioma with surrounding hemorrhage, an organizing hematoma, and no evidence of dysplasia or malignancy. The hemoglobin remained stable after surgery, and the patient was discharged home. On follow up, her hemoglobin normalized, her fatigue improved, and her melena resolved.\u00a0Intestinal hemangiomas are rare tumors and they are therefore an uncommon cause of GI bleeding. Unlike arteriovenous malformations, hemangiomas are neoplastic lesions secondary to endothelial hyperplasia\u00a0. They maThe presentation of intestinal hemangioma is variable, with the most common presenting symptom being GI bleeding and anemia\u00a0-8. BleedThe diagnosis of intestinal hemangiomas is challenging as no imaging study can provide a definitive diagnosis. Due to their location in the small intestine, they are often not detected during upper endoscopy or colonoscopy. The advent of newer endoscopic techniques such as capsule endoscopy, antegrade and retrograde single and double-balloon enteroscopy, and push enteroscopy have allowed for enhanced detection of these lesions\u00a0. While tPush enteroscopy, unlike capsule endoscopy, also plays a role in the management of intestinal hemangioma, offering an opportunity for biopsy, hemostatic intervention, and definitive treatment\u00a0-13. As oThis case was presented as a poster at the American College of Gastroenterology annual meeting\u00a0.Submucosal hemangiomas are a rare cause of GIB. They can present with melena and severe anemia. As the most common site is mid-jejunum, it is difficult to detect submucosal hemangiomas. In this case, the lesion was detected by video capsule endoscopy and was reached by push enteroscopy. Surgery is usually required for a definitive diagnosis and for definitive treatment."}
+{"text": "Results showed that low N concentrations (5\u2010 and 20\u2010mM N) promoted mean final germination proportion of all eight species by 4.4% and 6.4%, but high concentrations (40\u00a0mM\u00a0N) had no effect. The mean germination rate was decreased 2.1% and 5.1% by higher N concentration (20\u2010 and 40\u2010mM N) levels, but germination start time showed the opposite trend, delayed by 0.7, 0.9, and 1.8 d for the 10, 20, and 40\u00a0mM\u00a0N treatments. Final germination proportion, mean germination rate, and germination start time were significantly different among species in response to N concentration treatments. The final germination proportion of Allium tenuissimum and Chenopodium glaucum were suppressed by increased N concentration, whereas it increased for Potentilla bifurca, Plantago asiatica, and Setaria viridis. Our findings provide novel insights into N deposition\u2010induced species loss based on seed germination factors in semi\u2010arid grassland communities.Seed germination plays an important role in mediating plant species composition of grassland communities under nitrogen (N) enrichment. Shifts of plant community structure with N\u2010enhanced deposition in terrestrial ecosystems have occurred globally. Despite numerous studies about the effects of enhanced N deposition on mature plant communities, few studies have focused on seed germination. Using a laboratory experiment, we report the effects of five N concentrations, including 0, 5, 10, 20, and 40\u00a0mM N (NH Low N concentrations promoted mean final germination proportion of all species. The responses of eight species to N addition were differences. Mean germination rate was lower at higher N concentration levels. The long\u2010term mean annual precipitation is 385.5\u00a0mm, with\u00a0~\u00a090% distributed May\u2013October. The mean annual temperature is 2.1\u00b0C, with a range from \u221217.5\u00b0C in January to 18.9\u00b0C in July. According to FAO classification, the soil type is Haplic Calcisols, with an average of 16.95% clay, 20.30% silt, and 62.75% sand. Four perennial forbs (2.24NO3). Each N treatment level had 3 replicates of 50 seeds. NH4NO3 is a common source of N in grassland experiments. The emergence of the radicle was the criterion for germination , germination rate (GR), and germination start (GS).The FGP is the proportion of sown seeds that germinated  and n is the number of seeds used in an experiment  between seed sowing and the start of germination  was used to determine the relationship between pairs of FGP, GR, and GS. All statistical analyses were performed using the R statistical software environment .Two\u2010way analysis of variance (ANOVA) was used to determine the statistical significance of N treatment, species, and their interactions on FGP, GR, and GS. The Duncan's post hoc tests were used for multiple comparisons when a significant effect was detected. One\u2010way ANOVA was used for multi\u2010comparisons of the effect of N concentration on FGP, GR, and GS of each species, followed by Duncan's post hoc tests. A linear model (3A. frigida had the highest GP (95.3) and GR (28.1). C. glaucum had the earliest GT. P. bifurca had the lowest FGP (16.1) and GR (1.9), and the latest GT (5.3).Two\u2010way ANOVA showed that both N addition and species had a significant effect on final germination (FGP), germination rate (GR), and starting time (GT). Moreover, there was an interaction effect of N and species on FGP, GR, and GT Table\u00a0. A. frig3.1p\u00a0<\u00a0.05, Figure\u00a0p\u00a0<\u00a0.05; Figure\u00a0A. tenuissimum, P. bifurca, P. asiatica, and C. glaucum. Compared to the control, FGP of A. tenuissimum was suppressed by 28.7% under the N4 treatment, and FGP of P. bifurca was enhanced by 17.3%, 19.3%, and 14.6% under the N1, N2, and N3 treatments, respectively. FGP of P. asiatica was enhanced by 26.0%, 16.7%, 26.0%, and 26.0% under the N1, N2, N3, and N4 treatments, respectively, and FGP of C. glaucum was suppressed by 22.0% under the N4 treatment , and annuals and biennials (AB) functional groups  and two AB (P. asiatica and S. viridis). Moreover, higher nitrogen significantly delayed the mean GS of all species, as well as for the PF and AB functional groups. Water uptake is a fundamental requirement for the initiation and completion of seed germination . Mengzhou Liu: Formal analysis ; Methodology . Xudong Huanag: Formal analysis ; Funding acquisition (lead). Wei Hu: Funding acquisition (supporting). Ning Qiao: Investigation (supporting). Hongquan Song: Investigation . Bing Zhang: Conceptualization ; Data curation ; Formal analysis ; Investigation . Rui Zhang: Funding acquisition (supporting). Zhongling Yang: Data curation ; Funding acquisition (supporting). Yinzhan Liu: Conceptualization ; Investigation . Yuan Miao: Data curation . Shijie Han: Formal analysis . Dong Wang: Conceptualization (lead); Data curation (supporting); Formal analysis (supporting); Funding acquisition (supporting); Investigation (supporting); Methodology (supporting); Project administration (supporting)."}
+{"text": "Efficacious HIV-1 vaccination requires elicitation of long-lived antibody responses. However, our understanding of how different vaccine types elicit durable antibody responses is lacking. To assess the impact of vaccine type on antibody responses, we measured IgG isotypes against four consensus HIV antigens from 2 weeks to 10 years post HIV-1 vaccination and used mixed effects models to estimate half-life of responses in four human clinical trials. Compared to protein-boosted regimens, half-lives of gp120-specific antibodies were longer but peak magnitudes were lower in Modified Vaccinia Ankara (MVA)-boosted regimens. Furthermore, gp120-specific B cell transcriptomics from MVA-boosted and protein-boosted vaccines revealed a distinct signature at a peak (2 weeks after last vaccination) including CD19, CD40, and FCRL2-5 activation along with increased B cell receptor signaling. Additional analysis revealed contributions of RIG-I-like receptor pathway and genes such as SMAD5 and IL-32 to antibody durability. Thus, this study provides novel insights into vaccine induced antibody durability and B-cell receptor signaling. In the case of experimental HIV vaccines, efficacy is correlated with Envelope (Env)-specific antibody responses4. In particular, Env IgG3 titers that mediate antibody-dependent cellular phagocytosis (ADCP) and antibody dependent cellullar cytotoxicity (ADCC) are correlated with decreased risk of infection6. However, these responses decay more than 10-fold 6-months post-vaccination, paralleling the decline in vaccine efficacy7. Estimates of HIV-1 specific IgG half-lives range from 25-213 days, depending on the vaccine regimen and antigen (excluding gp41)9. Interestingly, the Vaccine-Induced HIV Seropositivity/Reactivity (VISP/VISR) in non-infected HIV vaccine recipients is commonly observed for vaccines with a Gag or Env component10, indicating that antibody responses can remain detectable for several weeks to a year post-vaccination. However, positive results on diagnostic tests are qualitative in nature and thus not quantitative in estimating antibody longevity. Hence, in this study, we measured antibody responses in the VISP cohort by a quantitative assay, the Binding Antibody Multiplex Assay (BAMA), across vaccine regimens. Furthermore, we model antigen-specific antibody decay kinetics and immunoglobulin subclasses necessary to inform rational design of HIV-1 vaccines with improved durability.While vaccine-elicited antibody durability has been achieved for licensed vaccines such as yellow fever, measles, smallpox, and Hepatitis B, elicitation of long-lived, functional antibody responses with candidate HIV-1 vaccine regimens remains elusive12. Env-specific antibodies after gp120 protein vaccination correlate with the number of circulating short-lived memory B-cells13; however, in participants who control HIV without treatment, memory B cell responses did not correlate with plasma antibody titers14. Since memory B cells differentiate into plasma cells upon boost and studying the plasma cell compartment in human participants is difficult, memory B cells provide the best window into signaling underlying the generation of antibody responses.Vaccine induced antibodies are secreted by plasmablasts within days of exposure, followed by memory B cells and plasma cells. These B cell dynamics are noticeable in analyses of half-lives using antibody-titers24. Specifically, analysis of early innate PBMC signatures associated with antibody levels after one dose of recombinant HIV gp140 with TLR-4 agonist adjuvant revealed a negative correlation of mitotic cell division modules with antibody production and upregulation of plasma cell surface markers a week after vaccination23. Further, a recent study identified a B cell (CD14\u2212CD20+) gene signature that predicts protection from infection after vaccination with Ad26-prime and gp140-boost in Rhesus Macaques and in RV-14424. Collectively, these results suggest that gene expression profiling, particularly from sorted B cells may unveil important insights into mechanisms of B cell differentiation across vaccine types.Additional insights into drivers of vaccine induced antibody responses from genome-wide transcriptional profiling have revealed gene signatures of B cell differentiation and early innate signaling. Early studies of gene expression showed type I interferon, inflamasome, and complement signaling in peripheral blood mononuclear cells (PBMCs) upon vaccinationIn the present study, we assessed the longevity of HIV-1 Env-specific IgG and IgG3 responses across 10 HIV vaccine regimens and quantified the effects of each vaccine type on durability using both linear and nonlinear mixed effects modeling (see Table 10. IgG or IgG3 in serum were assessed by a Binding Antibody Multiplex Assay (BAMA) for binding to a panel of consensus HIV Env proteins including gp120, gp140, gp41, and the V1V2 region of gp120 at peak (2-4 weeks post-last vaccination) at multiple post-vaccination time points. Vaccine immunogens varied by trial with DNA-MVA (HVTN 094 and 205) utilizing a gp150 immunogen, and Canarypox-protein (HVTN 097) and DNA-protein (HVTN 105) utilizing a gp120 immunogen.Five phase I or phase IIa HVTN trials 077 (DNA/Ad35 or Ad5), 094 (DNA/ HIV62B MVA), 097 (ALVAC/AIDSVAX B/E), 105 (DNA/AIDSVAX B/E), 205 (DNA/HIV62B MVA) were investigated in this study. Broadly, HVTN 094 and HVTN 205 were MVA-boosted trials with or without DNA priming, utilizing DNA expressing VLPs displaying gp160 in the prime and matched MVA-expressing VLPs displaying gp150 in the boost, HVTN 077 was Adenovirus (Ad)-boosted, and HVTN 097 and HVTN 105 were protein-boosted with subtype B MN gp120 and subtype A/E A244 gp120. HVTN 097 was primed with canarypox containing clade E 92TH023 gp120 while 105 was primed with DNA containing clade C ZM96 gp140. HVTN 094-T3 and 105-T2 were each primed twice with DNA followed by two MVA or protein boosts, respectively  window of the model.Robust responses against gp140 were elicited in both MVA  and protein-boosted trials . For gp140 and gp120 IgG as well as gp140 IgG3 antibody responses, MVA-boosted trials  displayed longer half lives than protein-boosted trials   baseline Net MFI at peak and 6 month timepoints. Post vaccination data points that were less than (pre-vaccination) baseline were not included. Only viral-boosted trials HVTN 077 (Ad5), HVTN 094 (MVA), and HVTN 205 (MVA) had sufficient post-vaccination data to be modeled. Newer trials  have not yet sampled for a sufficient period post-vaccination to observe long-lived responses, and other trials did not have sufficient participants with maintained antibody responses. DNA/MVA vaccination in HVTN 205 resulted in an asymptote between 103 Fig. . Uniquel103 Fig. , indicatnge Fig.  for MVA nge Fig. , panel DWe next investigated molecular signatures of durable responses in 26) was used with and without addition of FCRL genes to evaluate the role of FCRL genes in the regulation of BCR signaling. The FCRL genes were incorporated in BCR signaling pathways by mining\u00a0primary literature28. Boolean Omics Network Invariant-Time Analysis (BONITA), which performs discrete-state modeling identified BCR pathway with FCRL genes to be significant at p\u00a029 between MVA and protein boosted groups. PTPN6 and PTPN11 inhibit BCR signaling and were identified to have low impact on regulation of BCR pathway by BONITA irrespective of FCRL genes. The regulation identified by BONITA suggests that FCRL2 and FCRL3, which have very high impact scores, are required for activation of PTPN11 and PTPN6 while LYN can be activated by either Fig. Discrete dynamic modeling was employed to better understand changes in BCR signaling and how they relate to changes in FCRL genes. A network of BCR signaling , in agreement with previous vaccine studies33. IL32, shown to act as an immunosuppressant during HIV infection34, was accordingly identified as negatively correlated in both MVA- and protein-boosted trials  genes in B cells40. Taken together, this analysis shows that the durability of antibody responses is induced by a coherent transcriptional signature supported by previous studies of B cell gene expression.An association of B cell gene expression at peak antibody magnitude with antibody half life suggests potential molecular mechanisms responsible for long-lived antibody responses. This association could be dependent on the vaccine type used in each trial, and thus MVA- and protein-boosted\u00a0trials were analyzed separately (see methods). Twenty-four genes were significantly associated with half-life in MVA  and in protein-boosted trials (HVTN 105) . These genes included tetraspanins (SNX13 and TSPAN33) which are involved in several signal transduction events required for B cell activationials Fig . Additio18. Genes highly expressed after MVA-boosted vaccination  were enriched in genes downregulated after LAIV compared to pre-vaccination. Further, genes highly expressed in durable vs transient subjects in protein-boosted 105-T2 (DDPP) were also enriched for genes downregulated after LAIV. Further, a signature of many immunoglobulin genes, TNFSF13B, and FCRL5 was observed seven days after vaccination with TIV or diphtheria toxoid22 as well as in protein-boosted HIV vaccines in our study. Thus, protein-boosted vaccination induces genes regulating BCR signaling.To compare gene signatures identified in this study with previous vaccine signatures we investigated influenza and diphtheria toxoid vaccine response. Comparison between live attenuated (LAIV) or protein (TIV) influenza vaccination revealed that LAIV produced lower magnitudes of HA-specific antibodies and gene expression at 1 week than TIV24 derived two gene signatures of HIV vaccine efficacy, one of 53 genes and the other of 200 genes from B cells following Ad26-gp140 vaccination in non-human primates. These signatures were validated in PBMCs from RV-144. Both signatures were enriched in genes with higher\u00a0expression upon MVA boost than protein boost , which results in responses of the size of those in protein-boosted trials but with extended half lives in multiple years and decades.This study describes differences in half life of antibody response between MVA-boosted and protein-boosted HIV vaccinations. MVA-boosted vaccinations  lead to longer half life with lower-magnitude of antibody responses than protein-boosted HIV vaccines . Nevertheless, protein-boosted vaccines elicited a greater time-averaged mean antibody response to gp120. Promisingly, MVA-boosted vaccines led to half lives longer than those for immunoglobulin molecules53. When gp41 is included in the boost (i.e. gp150 boost vs gp120 boost), gp41 responses were as robust as gp140 responses, with increased response longevity vs gp120 responses. High epitope similarity to gut bacteria may contribute to rapid, large gp41 responses and persistence of gp41-specific antibodies in early HIV infection and HIV vaccination55. Inconclusivity of this study with regard to V1V2 along with putative efficacy of V2-specific antibodies calls for a higher powered study of V2-specific antibody responses57, including ongoing efficacy trials.Antibody responses observed here and in primary trials vary considerably by antigen-specificity. We found responses to gp140 are most frequent and robust followed by gp41 (when included in boost) and gp120 with few responses to V1V260. Taken together, this evidence demonstrates that vector and priming strategies, independent of immunogen, play a critical role in determining the magnitude of the gp120-specific antibody response.In the current study, effects of immunogen on vaccine-elicited antibody durability was not explicitly tested because MVA and protein boosted regimens used gp150 and gp120 respectively. Moreover, protein-boosted regimens (HVTN 097 and HVTN 105) included in this study were boosted with AIDSVAX B/E (gp120 immunogen) whereas MVA-boosted regimens (HVTN 094 and HVTN 205) analyzed in this study were boosted with MVA/HIV62 (gp150 immunogen) matched to the DNA prime. Despite the differences in gp120 sequences previous studies suggest a stronger response regardless of the immunogen used. Moreover, viral-prime, protein-boost vaccine trials have demonstrated robust gp120-specific antibody response following both gp120 and gp140 boost61. Despite the caveats of the HVTN 910 (VISP/VISR) cohort , we have added further evidence that long-lived vaccine-induced antibody responses are achievable with current HIV vaccine regimens. Moreover, this evidence makes clear that future experimental vaccines studies should investigate persistence of antibody responses and underlying B cell transcriptional programs.The nonlinear model demonstrated that two different viral vectors (vaccinia and adenovirus) can lead to long-lasting antibody responses. Nonetheless, 62, their lifespan is likely imprinted at the time of differentiation12. Long term survival of antibodies has been shown to correlate to memory B cell numbers following meningitis vaccine63. Moreover, gp120-specific antibodies correspond to memory B cells following HIV vaccine13. Therefore, study of gene expression in memory B cells is an attempt to understand responses that might have led to lasting memory B cell and plasma cell responses. Though our study can not differentiate into responses driven by specific B cell types, it demonstrates the state of memory B cells that emerge from germinal centers at the same time as longer-lasting MVA-induced plasma cells. Given the difficulty of sampling lymph nodes in human participants and pre-selection of participants, memory B cell signaling provides the most expedient window into germinal center dynamics.Our study profiled gene expression in memory B cells, which develop upon multiple rounds of vaccinations and whose predecessors, at the minimum, would have contributed to the development of long-lived plasma cells. Although plasma cells develop distinct transcriptional programs from memory B cells67. Markedly high FCRL2, FCRL3, and FCRL5 were identified in the context of Hepatitis C vaccination68. T-bet and FCRL5+ memory B cells correlate with longevity of antigen-specific antibody responses69, however there are conflicting data on longevity and FCRL5+ memory B cells71. We found a weak negative correlation between Tbet and half life in MVA trials  and no other correlations. Finally, discrete state modeling shows that FCRL2 and FCRL3 can independently activate LYN which leads to downstream activation of the BCR signaling pathway27. Thus, the current study provides mechanistic insights into regulation of BCR signaling by FCRL.The current gene expression analysis indicated a clear and strong signature of difference between protein and MVA boosted trials including differences in FCRL genes. FCRL molecules have an excitatory role in marginal zone (B2) B cell signalling74. Unfortunately, dendritic cell-based therapeutic HIV vaccination studies demonstrated decreased immunogenicity with TLR4 activation75. Another adjuvant, MF59, used in HVTN 702 (NCT02968849) with gp120-boost following canarypox prime, increases strength, breadth, and durability of response to Influenza vaccine by activating the early innate response76. Thus, the data shown here calls for careful understanding of how TLR4 agonists affect antibody responses on their own and in the context of MF59.The transcriptomic signature of enhanced durability of MVA-boosted regimens  consisted of RUNX1, RUNX2, and TLR4 as well as higher levels of Toll-like Receptor and IL-1R signaling. Thus, use of TLR4 agonists could increase antibody durability. Indeed there are safe TLR4 adjuvants that have already been tested in HIV vaccination24 in MVA-boosted trials along with increased gp120 half-lives is encouraging for the development of MVA as a vector for HIV vaccination. Moreover, enrichment of this signature in subjects with durable responses in AIDSVAX B/E protein-boosted 105-T2 suggests that protective HIV vaccine signatures may also be associated with durable responses. The Ehrenberg et al. gene signatures was identified by measuring gene expression at varied time points in human PBMCs (RV144) and in B cells from non-human primate Ad26-gp140 efficacy studies24. However, there are many differences between our study and Ehrenberg et al24, including antigen specificity , timepoints assessed (2 weeks and 6 months vs multiple), and prime (DNA vs Ad26 or canarypox). Nonetheless, MVA-boosted regimens  produced a B cell gene signature associated with protection, but at lower levels of antibody production than the protein-boosted regimens in Ehrenberg et al. Thus, our data present a rationale for continued investigation of MVA+ protein regimens, including testing immunogen to improve magnitude and durability of antibody responses.Enrichment of HIV vaccine signatures associated with protection47, HVTN 09448, HVTN 09749, HVTN 10550, HVTN 20553 were previously acquired and data were used in this analysis with approval from the HVTN77. Analysis of Levey-Jennings charts indicate the comparability of historical controls across trials and time for positive controls including HIVIG and CH58 mAb. All methods were carried out in accordance with relevant guidelines and regulations, and informed consent was obtained from all subjects.Antibody levels measured by binding antibody multiplex assay (BAMA) were either acquired from the primary analysis or obtained de novo for the current study. We tested serum samples from HVTN trial 910 retrospectively with IRB approval . Longitudinal samples from HVTN 105 at month 12 were obtained with informed consent . Primary data for trials HVTN 07780, Con6 gp120, and gp70_B.CaseA_V1V2 at primary and longitudinal timepoints as described previously82. Briefly, samples were incubated with antigen-coated beads in BAMA assay diluent , 1% milk blotto (w/v), 0.05% Tween-20 (v/v) in PBS), washed, then incubated with either biotinylated anti-human IgG (Southern Biotech) or biotinylated anti-human IgG3  followed by washing and incubation with Streptavidin-PE (BD Biosciences). Binding peak magnitude (Median Fluorescence Intensity) was measured in duplicate using a Bio-Plex 200 (Bio-Rad) and averaged to obtain Mean Fluorescence Intensity (MFI). Positive controls included titrated HIV positive IgG (HIVIG), CH58 mAb  and Purified IgG3 human myeloma protein (Sigma) coupled beads. All experiments were conducted following Good Clinical Laboratory Practice (GCLP) guidelines with tracking of antigen performance through historical Levey-Jennings control charts.Serum samples were tested for IgG and IgG3 binding Ab responses to gp41, Con S gp140lme4 and statistical properties  computed using emmeans which estimates a reference grid based on the factors in the model then averages over other factors and propagates error so that confidence intervals and p-values can be computed for the factor of concern84. Residuals were well-distributed across time . Covariates such as BMI and sex were not included in the model since systematic bias was not observed in these variables , and insufficient sample size existed to consider covariates in our mixed effects models. Time-averaged means were obtained by dividing the area under the response curve by 540 days as log(magnitude) and Antibody decay was assessed by both linear and nonlinear mixed effects modeling of log-transformed or raw Net MFI values from BAMA respectively. Models used random effects to account for inter-individual variability but did not control for demographic variables. Linear mixed effects models included participants with at least two non-zero  was used during cell labeling to prevent gp120 from binding T cells. On average 350 gp120+ cells were obtained per sample.Participants were selected for B cell bulk RNA-sequencing by matching durable 89. BONITA was additionally used to assess the B Cell Receptor Signaling Pathway from which was taken from Wikipathways26 but originally created in NetSlim25. This pathway was manually converted into the format necessary for use with BONITA29. FCRL genes interact through immunoreceptor tyrosine-based activating motif (ITAM) and immunoreceptor tyrosine-based inhibitory motifs (ITIM) with LYN and PTPN molecules, respectively28. To recapitulate this behavior, activating edges were added from FCRL2-5 to LYN and activating edges were added from FCRL1-5 to PTPN6 and PTPN11. Note that PTPN6 and PTPN11 have inhibitory connections to downstream genes; activating them inhibits the rest of the pathway. The resultant networks (with and without FCRL genes) were then subjected to BONITA pathway analysis. Edge sets (.txt) and network files (.graphml) of these BCR networks are now available at https://github.com/Thakar-Lab/BONITA.Genes implicated in durable response were evaluated by measuring association between gene expression at the peak and half-life estimates from linear model by Spearman correlation. The genes with coefficient of variation Supplementary Data\u00a0Table S1.Supplementary Data\u00a0Table S2.Supplementary Data\u00a0Table S3.Supplementary Data\u00a0Table S4.Supplementary Information."}
+{"text": "UBE2O, an E2/E3 hybrid ubiquitin-protein ligase, has been implicated in the regulation of adipogenesis, erythroid differentiation, and tumor proliferation. However, its role in cancer radioresistance remains completely unknown. Here, we uncover that UBE2O interacts and targets Mxi1 for ubiquitination and degradation at the K46 residue. Furthermore, we show that genetical or pharmacological blockade of UBE2O impairs tumor progression and radioresistance in lung cancer in vitro and in vivo, and these effects can be restored by Mxi1 inhibition. Moreover, we demonstrate that UBE2O is overexpressed and negatively correlated with Mxi1 protein levels in lung cancer tissues. Collectively, our work reveals that UBE2O facilitates tumorigenesis and radioresistance by promoting Mxi1 ubiquitination and degradation, suggesting that UBE2O is an attractive radiosensitization target for the treatment of lung cancer. Lung cancer is the deadliest malignancy worldwide, of which non-small cell lung cancer (NSCLC) represents ~85% , 2. LungThe ubiquitin-proteasome system (UPS) mediates more than 80% of protein degradation in eukaryotes, which involves ubiquitin, ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2s), ubiquitin ligase (E3s), and proteasome . Accumul\u2212/\u2212 knockout mice are prone to form squamous cell carcinoma and malignant lymphoma [MAX interactor 1 (Mxi1) is located in the cancer hotspot region 10q24-q25 of human chromosomes . Mxi1 calymphoma , indicatlymphoma . This isIn this study, we first reveal that UBE2O is physically associated with Mxi1 in vitro and in vivo. We then show that UBE2O targets Mxi1 for ubiquitination and degradation at the K46 residue. Functionally, genetical or pharmacological inhibition of UBE2O significantly impairs lung cancer progression and radioresistance in vitro and in vivo. Clinically, UBE2O is remarkedly upregulated and negatively correlated with Mxi1 downregulation in lung cancer tissues.2. All plasmids were subcloned into entry vector and then transferred to destination vector with indicated Myc, HA, SFB, or GST tag for the expression using Gateway Technology (Invitrogen). K46R mutation was generated by the KOD Hot Start DNA Polymerase (Novagen) and validated by DNA sequencing.The human bronchial epithelioid cell line HBE, HEK293T, SMCC-7721, HeLa, and all human lung cancer cell lines including A549, H1299, H292, HCC827, and H1975 cells were purchased from American Type Culture Collection and cultured in RPMI1640 or DMEM with 10% FBS in incubator at 37\u2009\u00b0C with 5% COAnti-UBE2O antibody (GTX119315) for immunohistochemical (IHC) analysis was purchased from GeneTex. Anti-UBE2O antibody (A301-873A) for immunoblotting was purchased from Bethyl Laboratories. Anti-Mxi1 antibody (HPA035319), anti-Flag antibody (F1804), and Cycloheximide (01810) were purchased from Sigma-Aldrich. Anti-Myc (sc-40) and anti-Mxi1 (sc-1042) antibodies were obtained from Santa Cruz Biotechnology. Anti-GAPDH (60004-1-Ig) antibody was obtained from Proteintech. Anti-HA (#3724) and anti-GST (#2624) antibodies were obtained from Cell Signaling Technology. Anti-ATM (A19650) and anti-phospho-ATM-Ser1981 (AP0008) antibodies were obtained from ABclonal. The proteasome inhibitor MG132 (474790) was purchased from Millipore. Arsenic trioxide (ATO) was a clinically available drug which was obtained from Beijing ShuangLu Pharmaceutical Co., Ltd., China .Lipofectamine RNAiMAX reagent (Invitrogen) was used for siRNA transfection. After 48\u2009h of transfection, cells were collected and analyzed by western blotting. The siRNAs ON-TARGETplus SMARTpool for Mxi1 were purchased from Dharmacon. The siRNAs targeting UBE2O and \u03b2-Trcp were as follows:si-UBE2O#1: 5-GGUUGUAGAGUUGAAAGUUTT-3;si-UBE2O#2: 5-CCACCCAGUGUGAAACCAATT-3;si-\u03b2-Trcp: 5-AAGUGGAAUUUGUGGAACAUC-3 , 21.This assay was carried out as described previously , 23. In Cells were lysed in NETN buffer , and then cell lysates were separated by SDS-PAGE. For the exogenous co-immunoprecipitation, cell extracts were incubated with Myc beads (Santa Cruz Biotechnology), S beads (Novagen), or HA-beads (Millipore) overnight. For the endogenous binding, cell lysates were incubated with protein A/G agarose (Santa Cruz Biotechnology) plus IgG or anti-UBE2O/Mxi1 antibody overnight. After being centrifuged and washed with NETN buffer five times, the precipitates were analyzed by immunoblotting.The indicated plasmids and siRNAs were transfected into cells and proteasome inhibitor MG132 was added for 4\u2009h before harvested. Cell lysates were incubated with S protein beads for co-immunoprecipitation, and ubiquitinated Mxi1 was detected by immunoblotting with anti-HA antibody.SFB-Mxi1, Myc-UBE2O, and HA-ubiquitin plasmids were co-transfected in HEK293T cells for 24\u2009h. Cells were then harvested and lysed with NETN buffer. SFB-Mxi1 protein was enriched by S beads co-immunoprecipitation. The ubiquitination-modified lysine (K) sites were identified by mass spectrometry (MS) analysis.This method was carried out as previously described \u201325. In bsh-UBE2O#1: 5-CGATGATTCCTATGGCTTCTA-3;sh-UBE2O#2: 5-CGGGTCTCTTCTTCGATGATT-3 .The Cell-Light EdU Apollo567 in vitro kit was purchased from RIBOBIO, and the EdU assay was conducted as per the instruction manual. Briefly, cells transfected with indicated siRNAs or treated with ATO were suspended and seeded in a 96-well plate. After 2\u2009h incubation with 50\u2009\u03bcM EdU, cells were washed and fixed with 4 % formaldehyde for 30\u2009min. EdU-Apollo was added for 30\u2009min and cells were treated with 0.5% Triton\u2009\u00d7\u2009100/PBS for 10\u2009min. Cells were counterstained with DAPI and then detected by fluorescent microscope.This assay was performed as described previously . Cells tCells were plated in six-well plates for 24\u2009h and incubated with CFSE (carboxy fluorescein succinimidyl ester) for 10\u2009min. The CFSE labeling was stopped by the treatment with 40% cold bovine serum. Flow cytometry analysis was used to measure cell proliferation.Cells were plated in six-well plates and exposed to irradiation with indicated doses. After being cultured for 14 days, cell clones were fixed with 4% formaldehyde and treated with crystal violet solution for 30\u2009min. Colonies with more than 50 cells were evaluated.The reagent kit (4250-050-K) for neutral comet assay was purchased from Trevigen, and this assay was carried out as per manufacturer\u2019s instructions. Briefly, 4\u2009h after irradiation, cells were collected and then combined with molten LMA agarose and immediately pipetted onto a slide. Subsequently, the slides were immersed and placed in 1\u00d7 Neutral Electrophoresis Buffer at 21\u2009V for 45\u2009min and then immersed in DNA precipitation solution for 30\u2009min. Samples were stained with SYBR for 10\u2009min and viewed by a fluorescent microscope. The olive tail moment of at least 100 cells was calculated.Cells were transfected with indicated siRNAs or treated with ATO and then irradiated. After 4\u2009h of irradiation, cells were fixed with paraformaldehyde and then blocked with 5% bovine serum albumin. After being incubated with Rad51 antibody and secondary antibody, samples were washed with PBS buffer for three times. Rad51 foci was detected under a laser confocal microscope. Cells with more than 10 foci were considered positive cells.6 sh-Control, sh-UBE2O#1 or sh-UBE2O#2 H1299 cells. For irradiation treatment, mice were irradiated with 10\u2009Gy when the tumor volume reached 100 \u2009mm3. For ATO treatment, ATO  were administered to mice every day when the tumor volume reached 130\u2009mm3. Tumor sizes and weights were monitored every 2 or 3 days.Experiments in xenograft mouse models were approved by the Medical Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. This assay was carried out as previously described . BrieflyThe lung adenocarcinoma tissue microarray, which contained 82 paired carcinoma tissues and adjacent tissues, was purchased from Shanghai Outdo Biotech . IHC analysis was performed as previously described , 27, 28.t test. Correlations of UBE2O and Mxi1 expression in tumor tissues was analyzed using the Pearson chi-square test. Overall survival analysis was evaluated by the Kaplan\u2013Meier method. P value of <0.05 was considered significant.Each experiment was performed at least three times independently, and the data were shown as mean\u2009\u00b1\u2009SD unless stated otherwise. The statistical significance between two independent groups was analyzed by unpaired two-tailed Student\u2019s To better understand how Mxi1 is controlled at the posttranslational level, we previously conducted a tandem affinity purification/MS analysis to identify Mxi1-binding proteins . Of themGiven that UBE2O is an E2/E3 hybrid ubiquitin-protein ligase and has been reported to mediate ubiquitination of several proteins, we therefore speculated that Mxi1 might be a ubiquitination substrate of UBE2O. As shown in Fig.\u00a0To identify the potential ubiquitination site of Mxi1 by UBE2O, we performed a MS analysis and found the lysine 46 (K46) site in Mxi1 had a di-glycine modification Fig.\u00a0, indicatOur previous study has clearly shown that Mxi1 is downregulated and inhibits lung cancer cell proliferation . As mentWe have previously found that overexpression of Mxi1 enhanced the radiosensitivity in lung cancer cells . Since UThe biological functions showed that UBE2O plays oncogenic roles in lung cancer and is expected to be an attractive target for anticancer and radiosensitization therapeutics. It has been reported that ATO can inhibit UBE2O activity by crosslinking adjacent cysteines in its catalytic domain . TherefoGiven that UBE2O depletion enhanced the radiosensitivity of lung cancer, we therefore wondered whether pharmacological inhibition of UBE2O with ATO had a similar effect. To verify our hypothesis, we performed the neutral comet and Rad51 foci experiments and found that ATO treatment significantly increased the length and DNA content in the comet tails as well as impaired the formation of Rad51 foci Fig.\u00a0. In agreSince Mxi1 is a ubiquitination substrate of UBE2O, we determined whether Mxi1 is required for the functions of UBE2O in lung cancer. H1299 cells were transfected with siRNAs targeting UBE2O, Mxi1 or both Fig.\u00a0. As seenP\u2009<\u20090.05). Importantly, we analyzed the association between UBE2O and Mxi1 expression in the paired lung adenocarcinoma tissues and found that there was a negative correlation between UBE2O and Mxi1 protein levels (P\u2009<\u20090.001)  Fig.\u00a0. Taken tThe downregulation of Mxi1 expression has been found in human lung cancer , but theA growing body of evidence has shown that Mxi1 acts as an antagonist of proto-oncogene c-Myc through competing for Max and repressing the transcriptional activity of c-Myc . We prevUBE2O has been reported to play essential roles in cell proliferation and apoptosis, cellular clock function, and metabolic homeostasis , 31\u201333. In summary, our work identifies UBE2O as a novel E3 ubiquitin ligase targeting Mxi1 for ubiquitination and destruction, leading to tumorigenesis and radioresistance in vitro and in vivo, as proposed in Fig.\u00a0Supplemental MaterialSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4"}
+{"text": "Motoneurons (MNs) control muscle contractions, and their recruitment by premotor circuits is tuned to produce accurate motor behaviours. To understand how these circuits coordinate movement across and between joints, it is necessary to understand whether spinal neurons pre-synaptic to motor pools have divergent projections to more than one MN population. Here, we used modified rabies virus tracing in mice to investigate premotor interneurons projecting to synergist flexor or extensor MNs, as well as those projecting to antagonist pairs of muscles controlling the ankle joint. We show that similar proportions of premotor neurons diverge to synergist and antagonist motor pools. Divergent premotor neurons were seen throughout the spinal cord, with decreasing numbers but increasing proportion with distance from the hindlimb enlargement. In the cervical cord, divergent long descending propriospinal neurons were found in contralateral lamina VIII, had large somata, were neither glycinergic, nor cholinergic, and projected to both lumbar and cervical MNs. We conclude that distributed spinal premotor neurons coordinate activity across multiple motor pools and that there are spinal neurons mediating co-contraction of antagonist muscles. We are able to walk, run and move our bodies in other ways thanks to circuits of neurons in the spinal cord that control how and when our muscles contract and relax. Neurons known as premotor neurons receive information from other parts of the central nervous system and control the activities of groups (known as pools) of motor neurons that directly activate individual muscles.To bend a joint or move our limbs, the movement of different muscles needs to be coordinated. Previous studies have focused on how premotor neurons activate a pool of motor neurons to contract a single muscle, but it remains unclear if and how some of these premotor neurons can co-activate different pools of motor neurons to control more than one muscle at the same time. Here, Ronzano, Lancelin et al. injected mice with modified rabies viruses labelled with different fluorescent markers to build a map of the premotor neurons that connect to motor neurons controlling the leg muscles.The experiments revealed that many of the individual premotor neurons in the spinal cords of mice connected to different pools of motor neurons. In the upper region of the spinal cord \u2013 which is primarily responsible for controlling the front legs \u2013 some large premotor neurons activated motor neurons in this region as well as other motor neurons in a lower region of the spinal cord that controls the back legs. This suggests that these large premotor neurons may be important for coordinating muscles contraction within and between limbs.Many neurological diseases are associated with difficulties in contracting or relaxing muscles. For example, individuals with a condition called dystonia experience disorganized and excessive muscle contractions that prevent them from being able to bend and straighten their joints properly. By helping us to understand how the body coordinates the activities of multiple limbs at the same time, the findings of Ronzano, Lancelin et al. may lead to new lines of research that ultimately improve the quality of life of patients with dystonia and other similar neurological diseases. The spinal cord is ultimately responsible for organizing movement by controlling the activation pattern of motoneurons (MNs), which in turn produce appropriate patterns of muscle contractions to produce limb movement. Across any single limb joint, there are fundamentally three types of control \u2013 or three \u2018syllables of movement\u2019 \u2013 possible. The three basic syllables are: (1) changing a joint angle, (2) stiffening a joint, and (3) relaxing a joint. The concatenation of these syllables across joints within and between limbs ultimately produces behaviour .To change a joint angle, MNs innervating synergist muscle fibres are activated whilst those that innervate antagonist muscle fibres are inhibited. This \u2018reciprocal inhibition\u2019  is mediaThe other two syllables are less well studied, but it is clear that behavioural joint stiffening requires co-activation of MNs innervating antagonist muscle groups, while joint relaxation would require co-inhibition of these MNs. Co-contraction has largely been thought to result from brain activity , whereasTo identify whether these syllables are produced by spinal circuits, several questions can be asked: Does the spinal cord contain circuits that lead to co-activation or co-inhibition of different pools of MNs \u2013 either synergists or antagonists? Does each motor pool have its own dedicated population of premotor INs, and are these INs interconnected in such a way that they can produce contraction of different muscle groups? Or are there populations of INs that project to multiple motor pools in order to effect contraction (or relaxation) of multiple muscles? Indeed, INs that have activity in keeping with innervation of multiple synergists, leading to motor \u2018primitives\u2019 or synergies  have beeNormal behaviours in quadrupeds as well as bipeds require coordination of syllables across joints between forelimbs and hindlimbs. This coordination relies on populations of propriospinal neurons projecting in either direction between the lumbar and cervical enlargements . Long deIn the present study, we examine circuits underlying co-activation and co-inhibition in the spinal cord by assessing premotor neurons through the use of RabV tracing techniques . We usedn = 4, two extensor and two flexor pairs). Notably, five MNs were double labelled, most likely due to secondary infection of synaptically connected MNs , one of them being double labelled  or antagonist (n = 3 pairs) pairs of muscles, double-labelled premotor INs were distributed similarly  and extensor (LG) muscles, 260 MNs were labelled  boutons in apposition to L1 , as welln = 4, 2 extensor and two flexor pairs, n = 3 antagonist pairs, In order to maintain posture and stability, trunk muscles are coordinated with hindlimb movements. Neurons in the thoracic cord that are premotor to lumbar MNs have previously been described ; we thusn = 4 synergist and 42/59, n = 3 antagonist pairs, In all animals (7/7), most divergent premotor neurons in the thoracic cord were located in the ipsilateral dorsal quadrant  have been shown to modulate interlimb coordination to provide stability . Given tn = 7, four synergist and three antagonist pairs, n = 4 synergist pairs and 47.9% \u00b1 7.1 % per animal, total of 19/37 neurons, n = 3 antagonist pairs, 2, n = 38 premotor LDPNs) compared to the double-labelled premotor neurons in the thoracic and lumbar cords . On average, the cross-sectional area of divergent cervical LDPNs was comparable to that of cervical MNs  and extensor (LG and MG) MNs were localized throughout the rostro-caudal extent of the ventral cervical cord with an enrichment between C6 and T1 . Of 92 pn = 3 LG injections, n = 3 LG injections, To determine the neurotransmitter phenotype of the premotor LDPNs, we used single \u0394G-Rab-mCherry injections in ChAT-Cre;R\u03a6GT mice crossed with mice expressing eGFP under the control of the promoter for the neuronal glycine transporter GlyT2 . GlyT2 iC, cholinergic  and may be detectable at this early postnatal stage  with \u0394G-Rab-mCherry, and extensor hindlimb GS with \u0394G-Rab-eGFP.2, p = 0.056, n1 = 16 premotor LDPNs infected from both homolateral forelimb and hindlimb injections vs n2 = 38 divergent premotor LDPNs infected from dual hindlimb injections (see above), Mann\u2013Whitney test; Since it has been suggested that LDPNs participate in ipsilateral control of forelimb and hindlimb , we sougGiven the involvement of LPDNs in the diagonal synchronization of forelimb and hindlimb during locomotion , we alsoWhile sharing similar features with the LDPNs infected from dual hindlimb injections, it remains to be determined whether these neurons premotor to hindlimb and forelimb muscles form a homogenous population with the divergent LDPNs.n = 3, Having identified a population of divergent premotor LDPNs with projections from the cervical to the lumbar region, we next investigated whether ascending propriospinal neurons projecting from the lumbar or thoracic segments to cervical MNs could be identified. Following FMs injections, ascending premotor INs were observed throughout the cord . There were very few (<1%) bifurcating (ascending/descending) premotor neurons in the thoracic cord after injections in homolateral GS and FMs . This distribution of lumbar premotor LAPNs is different from that of cervical premotor LDPNs, which were almost exclusively ventral . Of the 117 lumbar premotor LAPNs identified, 10 were also labelled from GS injections, indicating that some neurons projected both to local lumbar MNs as well as to cervical MNs  in the lumbar cord, about half of which were localized in the dorsal ipsilateral quadrant . Of these, however, 10/12 were in the ventral contralateral quadrant , a stiffening of a joint (requiring co-activation of flexors and extensor MNs), and a relaxation of a joint (requiring co-inhibition of flexor and extensor MNs). While neural circuits for reciprocal inhibition have been well studied over many decades , circuitn = 27) were performed according to the Animals (Scientific Procedures) Act UK (1986) and certified by the UCL AWERB committee, under project licence number 70/7621. Homozygous ChAT-IRES-Cre mice . The supernatant was collected and medium was added for another cycle (three cycles maximum), after which the supernatant was filtered (0.45 \u00b5m filter) and centrifuged 2 hr at 19,400 rpm (SW28 Beckman rotor). The pellets were re-suspended in phosphate-buffered saline (PBS) and centrifuged together at 21,000 rpm, 4\u00b0C, 4 hr in a 20 % sucrose gradient. Pellets of each collection were then re-suspended and stored in 5\u201310 \u00b5l aliquots at \u201380\u00b0C.We used the glycoprotein G-deleted variant of the SAD-B19 vaccine strain rabies virus (a kind gift from Dr M. Tripodi). Modified RabV (\u0394G-Rab) with the glycoprotein G sequence replaced by mCherry or eGFP (\u0394G-Rab-eGFP/mCherry) was produced at a high concentration with minor modifications to the original protocol . BHK cel5 cells/ml and incubated overnight at 37\u00b0C and 10 % CO2 (growth). The virus was prepared for two serial dilutions with two different aliquots and added in the well after an equal volume of medium had been removed  and incubated 48 hr at 35\u00b0C and 3 % CO2. The titre was determined from the count of cells in the higher dilution well and was between 109 and 1010 infectious units (IU)/ml.Virus titration was performed on BHK cells plated in 10 % FBS medium at 1.5 \u00d7 109 and 1010 IU/ml. The incisions were closed with vicryl suture, and the mice were closely monitored for 24 hr post-surgery. Mice were perfused 9 days after the injections. Due to the proximity of synergist pairs of muscles, prior to spinal tissue processing, we dissected the injected leg and confirmed that there was no contamination of virus across the injected muscles or in adjacent muscles below or above the knee. When injecting FMs, we could not target a single muscle. To visualize which muscles had been infected, we carefully dissected each FM and assess for the presence of fluorescent signal  was given to the neonatal pups (P1\u2013P3) prior to surgery and all procedures were carried out under general isoflurane anaesthesia. After a skin incision to expose the targeted muscle, the virus (1 \u00b5l) was injected intramuscularly using a Hamilton injector (model 7652-01) mounted with a bevelled glass pipette (inner diameter 50\u201370 \u00b5m). The mice were injected in TA and PL (ankle flexor pair), LG and MG (ankle extensor pair) for synergist pairs and TA and LG for antagonist pairs. In hindlimb/forelimb double injections, the LG and MG were both injected with 1 \u00b5l of one RabV to increase the number of long projecting cells infected. In addition, 1 \u00b5l of the second RabV was injected in FMs see  without gnal see . Three hgnal see .The mice were perfused with PBS (0.1 M) followed by PBS 4 % paraformaldehyde under terminal ketamine/xylazine anaesthesia . The spinal cords were then collected through a ventral laminectomy and post-fixed for 2 hr. The cords were divided into the different parts of the spinal cord , cryoprotected overnight in 30 % sucrose PBS, embedded in optimal cutting temperature compound (Tissue-Tek) and sliced transversally (30 \u00b5m thickness) with a cryostat . Sections were incubated with primary antibodies for 36 hr at 4\u00b0C and with secondary antibodies overnight at 4\u00b0C in PBS double salt, 0.2 % Triton 100-X (Sigma), 7 % donkey normal serum (Sigma). The primary antibodies used were: goat anti-choline acetyltransferase , chicken anti-mCherry , rabbit anti-GFP , guinea pig anti-vGluT2 , and rabbit anti-Lhx1 ; and the secondary antibodies: donkey anti-rabbit Alexa 647 , donkey anti-goat preadsorbed Alexa 405 , donkey anti-rabbit Alexa 488 , and donkey anti-chicken Cy3 . The slides were mounted in Mowiol  and coverslipped  for imaging.x,y) Cartesian system and using the \u2018Spots\u2019 function of Imaris. The y-axis was set to the dorso-ventral axis. Positive values were assigned for dorsal neurons in the y-axis and ipsilateral (to the hindlimb injection) neurons in the x-axis. Coordinates were collected on every section and normalized through the cervical, thoracic, lumbar, and sacral parts separately using grey matter borders and fixing the width and the height of the transverse hemisections. To calculate divergence rates, given the high density of premotor INs infected in the lumbar cord all infected premotor INs  were quantified in one of every three sections which further allowed to avoid counting the same cells twice on consecutive sections. In the cervical, thoracic, and sacral regions, all cells were quantified, as their low density allowed for manually excluding premotor neurons found in consecutive sections. Since MNs are big cells localized as a restricted column of the ventral spinal cord, we quantified them on every other sections, to avoid counting the same cell twice on consecutive sections.Images of the entire sections were obtained using a Zeiss LSM800 confocal microscope with a \u00d720 air objective (0.8 NA) and tile advanced set up function (ZEN Blue 2.3 software). A \u00d763 oil objective was used for Airy scan imaging of somata and excitatory boutons. Tiles were stitched using Zen Blue and analyses were performed using Zen Blue and Imaris  software packages. Location maps were plotted setting the central canal as  in the  and GraphPad PRISM (version 7.0). To compare cell sectional areas, non-parametric rank tests were used as specified in each related result. The numbers of animals/cells in each experiment and statistical tests used are reported in the figure legends or directly in the text. Results and graphs illustrate the mean \u00b1 standard deviation. Statistical significance levels are represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: not significant.All statistical analyses and plots were made using R  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Ronald Calabrese as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Simon Gosgnach (Reviewer #1).Thank you for submitting your article \"Spinal neurons innervating multiple local and distant motor pools\" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.The three reviewers acknowledge that the paper is of a great interest for researchers working within the field of motor control, and more specifically at the level of the spinal cord. Despite the rather descriptive aspect of the study it might indeed set the foundation for future studies regarding the specific identities and functions of the premotor neurons investigated here. Their detailed comments are described below, but I would like to emphasize a few important points harvested from their reviews:1) You should tone down the part on the V0v origin identification, or better do the appropriate characterization as suggested below;2) You should further discuss the neurotransmitter characterization and eventually address this point experimentally if possible;3) You should further describe the rostro-caudal extension of the labelled pre-motoneurons.4) Also, a functional aspect is clearly missing, the more you can address this in your revised version the more it will strengthen the paper, but we are aware that fully investigating this would require too much time and represents a significant amount of work if the data are not available yet.Reviewer #1 (Recommendations for the authors):1. More detail regarding rostra-caudal location in the spinal cord should be given in all cases. First I think a Figure describing the location of the motor pools for the 4 injected muscles should be included- this could be in the supplementary data or it could go as a panel in Figure 1. This is needed so we have a clear idea of how far the axons of the PMIs project. In my experience a motor pool can span several lumbar segments. More specific information should also be provided regarding the location of the sections which contain the PMIs being described. I realize this is not an exact science after cutting tissue, but given your care in counting every third section, you should have a pretty good idea of which segment houses the neurons. This would be preferred over the current description of \"rostral cervical\" or equivalent.In the Figures the number of MNs infected with either virus is typically quite low so I assume the sections are not particularly close to the middle of the infected motor pools?2. The amount of raw data shown is light. There is one immuno panel shown per Figure. I realize the take home message comes from the schematics, but it would be nice to see images of spinal sections with more single and double infected cells. Each immuno image contains just one divergent PMI and other than Figure 1 and 6 there are no other cells that seem to be infected with either the red or green expressing virus in the immuno image.3. I get a little uncomfortable with the data interpretation at the back end of the Results- particularly with the section titled Cervical LDPNs are excitatory- this is speculation and should not be in the title. The data simply shows 95% of these cells are not glycinergic.4. Related to point 3 \u2013 The next section is titled \"cervical premotor LDPNs likely arise from the V0v domain\". In addition to V0 and V1 cells, Lhx1 is also expressed postmitotically in the dI2, dI4, dI6, and V2 populations, and even some V3 cells . It is thus extremely speculative to suggest that the V0v cells are the ones we are looking at here. Even more so when we factor in the finding that Lhx1 plays a key role in establishing GABAergic neuronal identity .Incidentally the current thinking on V0v cells is not that they contact MNs directly, but rather disynaptically via an inhibitory IN (see Kiehn 2016 Nat Rev Nsci review). Some V0 and V3 neurons have been shown to project to MNs , but V3 cells also have been shown to project to non MN targets recent (Chopek Cell Rep paper), and V0 cells very possibly have many other downstream targets, this simply has yet to be investigated.I really think this part of the manuscript should not be included due to its preliminary nature. In order to be included there should be some sort of Evx1 staining, or a functional assay in the V0v deficient mice used in the Talpalar 2012 paper in which issues with muscle stiffness are seen in the absence of V0v cells.The reason I have such an issue with this is that the identity of these cells as belonging to the V0v population is so very circumstantial based on the data here. If it were to be published as is, V0v cells would immediately be cited, in the work of others, as key regulators of muscle stiffness. Even in this manuscript \u2013 line 398 of the discussion the authors say that a subset of labeled LDPNs \"are clearly V0v derived\". This is a problem.Reviewer #2 (Recommendations for the authors):Could the author present data or mention that they have tested that pure R\u03a6GT mice injected with virus injected do not labelled any premotor neurons?Reviewer #3 (Recommendations for the authors):\u2013 It should be mentioned earlier in the Results that this study was performed in neonates.\u2013 Green and blue in the immuno figures  are difficult to differentiate between due to low contrast between the two.\u2013 Line 9-11: Divergent is used before it is defined. It works in the initial sentence (line 9) but is a bit deceptive in the next (lines 10-11) as it seems to imply that divergent is referring only to antagonist motor pools.\u2013 Lines 37-42: I suggest adjusting the framing of this section. Most of those questions have answers, as mentioned in the last line of the paragraph and in the Discussion. Instead, highlighting that the location and identities of many of these neurons is unknown may better set up the results to follow.\u2013 Lines 146-148: It is not clear what the 22% refers to. It may be \"where they make up 22% of the neurons labelled\"?\u2013 The schematic in Figure S4 A implies that the ventral divergent interneuron in cervical cord is a double jump from the forelimb motor pool. It is likely that there is a 'mis-connection' in the schematic \u2013 the ventral neuron should synapse with the forelimb motor neurons, rather than the dorsal neuron to the ventral neuron. 1) You should tone down the part on the V0v origin identification, or better do the appropriate characterization as suggested below;We have extensively edited the sections related to the identification of V0v interneurons. We agree with the referees that we do not have sufficient evidence to pinpoint the genetic lineage of the long range descending interneurons and have toned this down considerably. As suggested by Reviewer#1 we performed further experiments using an Evx1 antibody to selectively label V0v interneurons. However, while good labeling was obtained in neonatal (P0) tissue, no labelling was detected in tissue of the age we used in the present study (P9-P10), in line with downregulation of transcription factors during development \u2013 a feature that also reduced our sensitivity in the Lhx1 assay, albeit to a lesser extent. To compound our uncertainty, a recent study was published while we were revising our manuscript identifying a class of dI2 derived interneurons with morphological features similar to the LDPNs described in our study. And we note that dI2 INs also express Lhx1. Given this further uncertainty and the reduced level of detection of Lhx1 in more mature tissue, we have toned down any conclusion that may suggest we have identified the cardinal class of divergent LDPNs. But we prefer to keep the data in the manuscript since we give evidence that at least some LDPNs are Lhx1-expressing (and thus likely V0v or dI2 as discussed). Specific changes in the text are detailed in the response to reviewers #1 and #3.2) You should further discuss the neurotransmitter characterization and eventually address this point experimentally if possible;Identifying the transmitter phenotype of all the divergent interneurons would have required a different protocol, e.g. with a genetically (non-cre) labelled mouse. While the excitatory nature of the divergent LDPNs is sufficiently established, divergent premotor INs in the lumbar and thoracic region are certainly both inhibitory and excitatory. We now demonstrate this point further by adding supplementary figures showing double labelled terminals that are positive or negative for VGlut2 and are localized throughout the thoracic and lumbar cord . Further work will be required to understand whether divergent excitatory and inhibitory premotor interneurons are segregated and whether they belong to homogeneous cardinal classes or not.3) You should further describe the rostro-caudal extension of the labelled pre-motoneurons.We have added new figures where the distribution of premotor interneurons is shown along the rostrocaudal axis for lumbar, thoracic and sacral cord .4) Also, a functional aspect is clearly missing, the more you can address this in your revised version the more it will strengthen the paper, but we are aware that fully investigating this would require too much time and represents a significant amount of work if the data are not available yet.The question on the function of these LDPNs is clearly one of our top priorities. Our manuscript is focused on the discovery of these peculiar cells, but determining their function is a necessary next step. The finding of a precise anatomical location of LDPNs and the availability of tracing methods will now allow us to access and manipulate these cells, but this is clearly a separate project and in our opinion could not be reduced to an appendage to an already dense manuscript.Reviewer #1 (Recommendations for the authors):1. More detail regarding rostra-caudal location in the spinal cord should be given in all cases. First I think a Figure describing the location of the motor pools for the 4 injected muscles should be included- this could be in the supplementary data or it could go as a panel in Figure 1. This is needed so we have a clear idea of how far the axons of the PMIs project. In my experience a motor pool can span several lumbar segments. More specific information should also be provided regarding the location of the sections which contain the PMIs being described. I realize this is not an exact science after cutting tissue, but given your care in counting every third section, you should have a pretty good idea of which segment houses the neurons. This would be preferred over the current description of \"rostral cervical\" or equivalent.We have now added as supplementary figures the rostro-caudal distribution of divergent premotor INs of lumbar segments, and the rostral-caudal distribution of all premotor INs of the thoracic and cervical regions . We also provided the number of motoneurons infected from each pair of muscles injected, a number that we note is underestimated due to the toxicity of the virus for starter cells (supplementary file 1).In the Figures the number of MNs infected with either virus is typically quite low so I assume the sections are not particularly close to the middle of the infected motor pools?In Figure 1, the section presented is close to the middle of the infected motor pools. Indeed, 2 MNs infected on one single section, as in the example of Figure 1, represents the typical count of MNs within a single section within the labelled motor column. This is due partly to the sparseness or rabies infection, but, more importantly, to the fact that each section is only 30 \u03bcm thick. We have also added supplementary figures  showing more examples of lumbar sections following injections in the different motor pools. Amongst the examples shown, there are up to 8 MNs infected on a single section, but this is a rare occurrence .2. The amount of raw data shown is light. There is one immuno panel shown per Figure. I realize the take home message comes from the schematics, but it would be nice to see images of spinal sections with more single and double infected cells. Each immuno image contains just one divergent PMI and other than Figure 1 and 6 there are no other cells that seem to be infected with either the red or green expressing virus in the immuno image.On Figure 1, we highlighted the 3 divergent interneurons on the image shown. Due to the relative low density of divergent premotor neurons and the thinness of the sections, it is impossible to have more divergent neurons in a single section, particularly in the thoracic and cervical cord. To provide a broader perspective of divergent premotor neurons, we added more examples of stained sections from lumbar, thoracic, and cervical spinal cord showing divergent neurons. These are presented in figure supplements 1,2,3 linked to figure 1, 2, and 3.3. I get a little uncomfortable with the data interpretation at the back end of the Results- particularly with the section titled Cervical LDPNs are excitatory- this is speculation and should not be in the title. The data simply shows 95% of these cells are not glycinergic.We have modified the title of this section: \u201cCervical LDPNs are neither inhibitory nor cholinergic\u201d (line 201) since only 1/21 cervical LDPNs was eGFP positive and none of them were ChAT positive.4. Related to point 3 \u2013 The next section is titled \"cervical premotor LDPNs likely arise from the V0v domain\". In addition to V0 and V1 cells, Lhx1 is also expressed postmitotically in the dI2, dI4, dI6, and V2 populations, and even some V3 cells . It is thus extremely speculative to suggest that the V0v cells are the ones we are looking at here. Even more so when we factor in the finding that Lhx1 plays a key role in establishing GABAergic neuronal identity .Incidentally the current thinking on V0v cells is not that they contact MNs directly, but rather disynaptically via an inhibitory IN (see Kiehn 2016 Nat Rev Nsci review). Some V0 and V3 neurons have been shown to project to MNs , but V3 cells also have been shown to project to non MN targets recent (Chopek Cell Rep paper), and V0 cells very possibly have many other downstream targets, this simply has yet to be investigated.I really think this part of the manuscript should not be included due to its preliminary nature. In order to be included there should be some sort of Evx1 staining, or a functional assay in the V0v deficient mice used in the Talpalar 2012 paper in which issues with muscle stiffness are seen in the absence of V0v cells.The reason I have such an issue with this is that the identity of these cells as belonging to the V0v population is so very circumstantial based on the data here. If it were to be published as is, V0v cells would immediately be cited, in the work of others, as key regulators of muscle stiffness. Even in this manuscript \u2013 line 398 of the discussion the authors say that a subset of labeled LDPNs \"are clearly V0v derived\". This is a problem.We thank the reviewer and agree that our evidence for the identification of V0 interneurons is not definitive. While preparing the revision, we attempted to identify Evx1 derived interneurons using a specific antibody (a gift from Columbia University). However, while we obtained good staining in young (P0) tissue, no staining was observed in P9 tissue, the age at which the tissue was analyzed in this paper. Having tried many different concentrations and incubation times, with similar results, we conclude that Evx1 expression is downregulated, even more than Lhx1  and therefore, a posteriori identification of the long descending projection neurons is not possible. Only genetic labelling could resolve the issue, but this would not be possible since we do not have the relevant mouse line. To compound our uncertainty, a recent study was published while we were revising our manuscript identifying a class of dI2 derived interneurons with morphological features similar to the LDPNs described in our study. And we note that dI2 INs also express Lhx1. Given this further uncertainty and the reduced level of detection of Lhx1 in more mature tissue, we have toned down any conclusion that may suggest we have identified the cardinal class of divergent LDPNs and re-written the related section, (now starting at line 219). But we prefer to keep the data in the manuscript, since we give evidence that at least some LDPNs are Lhx1-expressing (and thus likely V0v or dI2 as discussed). See also answer to reviewer #2 related to the same point.Reviewer #2 (Recommendations for the authors):4) Could the author present data or mention that they have tested that pure R\u03a6GT mice injected with virus injected do not labelled any premotor neurons?We tested for leaks of the rabies glycoprotein by performing injection of an EnvA pseudotyped rabies virus in heterozygous R\u03a6GT mice. We chose to use a pseudotyped rabies in order to test for ectopic expression of both the rabies glycoprotein and the TVA receptor . The example data are shown in our preprint in which we describe a similar approach . In summary, following the injections we observed scant motoneuron labelling , but no interneurons. Since we used pseudotyped rabies, the infection of some motoneurons shows that there is some ectopic expression of TVA in motoneurons that gave rise to motoneuron infection due to the extremely high affinity of EnvA for the TVA receptor. However, no trans-synaptic labelling was observed, indicating that any leak of the G protein, was not sufficient to sustain transsynaptic jumps. We now state this at the end of Methods section on intramuscular injections (lines 504-508).Reviewer #3 (Recommendations for the authors):\u2013 It should be mentioned earlier in the Results that this study was performed in neonates.This has been clarified and is now mentioned line 95 of the results.\u2013 Green and blue in the immuno figures  are difficult to differentiate between due to low contrast between the two.Colors are unfortunately difficult to pick when taking into account the many ways people can actually see them. We spend far too much time in lab meetings discussing colours. We had to pick two colors that everyone could easily differentiate for eGFP (green) and mCherry (pink), while keeping colors close to their fluorescent emission spectrum to avoid misunderstanding. Also, since we are focusing on double labelled neurons, the colocalisation of the two colors needed to give a completely different tint (white in our case). Blue remained the only option as the fourth color so that it could readily be differentiated from the three others. It is true that blue is more difficult to visualize, however, since blue is used for motoneuron staining, which is not the focus of the study, we thought that the combination used was the best option.\u2013 Line 9-11: Divergent is used before it is defined. It works in the initial sentence (line 9) but is a bit deceptive in the next (lines 10-11) as it seems to imply that divergent is referring only to antagonist motor pools.Divergent is now defined clearly in the abstract before its use.\u2013 Lines 37-42: I suggest adjusting the framing of this section. Most of those questions have answers, as mentioned in the last line of the paragraph and in the Discussion. Instead, highlighting that the location and identities of many of these neurons is unknown may better set up the results to follow.We have included the referee\u2019s suggestion and, while keeping the list of questions, we have stressed that what is not known are the location and identities of the interneurons mediating synergies (lines 46-48).\u2013 Lines 146-148: It is not clear what the 22% refers to. It may be \"where they make up 22% of the neurons labelled\"?This has been clarified (line 164).\u2013 The schematic in Figure S4 A implies that the ventral divergent interneuron in cervical cord is a double jump from the forelimb motor pool. It is likely that there is a 'mis-connection' in the schematic \u2013 the ventral neuron should synapse with the forelimb motor neurons, rather than the dorsal neuron to the ventral neuron.We thank the reviewer for this comment and apologize. Indeed, we had made a mistake in the schematic of what is now Figure 6\u2014figure supplement 1A. This has now been corrected."}
+{"text": "Oral cancer is one of the most common human malignancies, and its incidence is increasing worldwide. In particular, oral squamous cell carcinoma (OSCC) is characterized by high rates of proliferation, invasiveness, and metastasis. Currently, standard treatment for OSCC includes surgical removal, chemotherapy, and radiotherapy; however, the survival rate of patients with OSCC remains low, thus new therapies are needed. It has been proven that excessive NLRP3 inflammasome activation and apoptosis alteration may contribute to oral cancer progression. This study aimed to investigate the effect of BAY-117082, an NLRP3 inflammasome inhibitor, in an in vitro and in vivo xenograft model of oral cancer. In vitro results revealed that BAY-117082 at concentrations of 5, 10, and 30 \u00b5M was able to reduce OSCC cell viability. BAY-117082 at higher concentrations significantly reduced NLRP3, ASC, caspase-1, IL-1\u03b2, and IL-18 expression. Moreover, Bax, Bad, and p53 expression were increased, whereas Bcl-2 expression was reduced. Furthermore, the in vivo study demonstrated that BAY-117082 at doses of 2.5 and 5 mg/kg significantly decreased subcutaneous tumor mass, and also reduced NLRP3 inflammasome pathway activation. Therefore, based on these results, the use of BAY-117082 could be considered a promising strategy to counteract oral cancer progression, thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways. Oral cancer is one of the most common malignancies in the world . Oral caRecent studies demonstrated that the NLRP3 inflammasome pathway is upregulated in OSCC animal models and OSCC patients , suggestMTT assay was used to assess CAL27, HSC-2, and SCC-4 cell viability following 24 h of treatment with BAY-117082 at different concentrations . Our results show that BAY-117082 treatment only at concentrations of 5, 10, and 30 \u00b5M significantly reduced CAL27, HSC-2, and SCC-4 cell viability in the same way, as shown in Based on the MTT results, we decided to investigate in another analysis only BAY-117082 at concentrations of 5, 10, and 30 \u00b5M, because these represented the most cytotoxic concentrations. Furthermore, since BAY-117082 showed similar effects on cell viability in all three cell lines, we decided to continue to investigate its effect on only the CAL27 cell line, because it represented a frequently used cell line in the field of OSCC ,19.Inflammasome is a cytoplasmic multi-protein complex that regulates innate immunity response through ASC and caspase-1 activation . NLRP3 iOnce activated, NLRP3 inflammasome induces the release of pro-inflammatory cytokines IL-1\u03b2 and IL-18, which can contribute to cancer progression ,22. ThusApoptosis is a physiological process of programmed cell death that is essential for normal tissue development and cell hemostasis . DysreguRecent studies highlighted the role of pro-apoptotic p53 protein in modulating various cellular processes such as metabolism, metastasis, and communication within the tumor microenvironment . p53 defOur results demonstrate that the control group was characterized by low p53 expression, while treatment with BAY-117082 at concentrations of 10 and 30 \u00b5M significantly increased pro-apoptotic p53 expression E. To evaluate the effect of BAY-117082 on the growth of OSCC cells in vivo, the CAL27 xenograft model was established in nude mice. In this context, our results show that treatment with BAY-117082 at doses of 2.5 and 5 mg/kg significantly reduced subcutaneous tumor mass and neutrophilic infiltration in a dose-dependent manner A\u2013C. MoreTo confirm the effect of BAY-117082 on the NLRP3 inflammasome pathway, we decided to also investigate this pathway in the xenograft model by Western blot analysis. The results show that the control group was characterized by high expression of NLRP3, ASC, and caspase-1; however, treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to significantly reduce their expression . Moreover, we decided to investigate, by immunohistochemistry staining, the expression of IL-1\u03b2, a pro-inflammatory cytokine related to cancer progression. Our results show that the control group was characterized by marked expression of IL-1\u03b2, while treatment with BAY-117082 at doses of 2.5 and 5 mg/kg was able to reduce its expression in a dose-dependent manner , confirmAccumulating evidence has suggested that the nuclear factor-\u03baB (NF-\u03baB) signaling pathway plays a critical role in oral carcinogenesis ,25. It hCancer development and its response to therapy are strongly influenced by innate and adaptive immunity, which can promote or attenuate tumorigenesis with opposite effects on the therapeutic outcome . It has Apoptosis plays a key role in cancer progression, therefore targeting apoptosis could be a relevant therapeutic approach in anti-cancer drug development . Thus, wKi-67 is a DNA-binding nuclear protein involved in cell proliferation , and is Oral squamous cell carcinoma (OSCC), the most common oral cancer, arises from the mucosal lining of the oral cavity, with an incidence of 450,000 new cases per year . Major rAlthough the pathophysiology of oral cancer remains unclear, in vivo and in vitro studies have demonstrated that aberrant and excessive NLRP3 inflammasome activation significantly contributes to the initiation and progression of oral cancer ,20. MoreFirst of all, we evaluated the cytotoxic effect of BAY-117082 at different concentrations in an in vitro model of OSCC using CAL27, HSC-2, and SCC-4 cell cultures. Clearly, our results demonstrate that BAY-117082 treatment only at higher concentrations significantly reduced CAL27, HSC-2, and SCC-4 cell viability in the same way. Many papers have proven that NLRP3 inflammasome plays a key role in oral carcinogenesis ,21. NLRPHowever, recent studies have demonstrated that excessive and aberrant NLRP3 inflammasome activation may contribute to oral cancer progression ,20. ThusOnce activated in response to intracellular stimuli, NLRP3 inflammasome promotes the proteolytic processing of pro-IL-1\u03b2 and pro-IL-18 into their bioactive forms IL-1\u03b2 and IL-18, respectively . OverexpAmong the mechanisms involved in cancer pathogenesis, apoptosis also has a fundamental role ,35. ProgDespite the promising results obtained with an in vitro model on CAL27 cell culture, we decided to construct an in vivo xenograft model of oral cancer to confirm the beneficial effect of BAY-117082. Our data demonstrate that BAY-117082 treatment significantly reduced subcutaneous tumor mass and tumor necrosis compared to the control group in a dose-dependent manner. Additionally, treatment with BAY-117082 significantly decreased tumor burden and tumor weight compared to the control group in a dose-dependent manner, without changing the animals\u2019 weight. Moreover, considering the key role of NLRP3 inflammasome in oral carcinogenesis , we alsoIt has been demonstrated that the NF-\u03baB signaling pathway has a fundamental role in oral cancer pathogenesis . NF-\u03baB iIn addition, NF-\u03baB plays a critical role in regulating the activation and differentiation of innate immune cells and inflammatory T cells . In a maFurthermore, to confirm the data obtained in the in vitro model with regard to the apoptosis pathway, we decided to also investigate the effect of BAY-117082 on programmed cell death in the xenograft model, demonstrating that it was able to significantly increase pro-apoptotic Bax protein expression, while anti-apoptotic Bcl2 and Bcl-xL were significantly reduced following BAY-117082 treatment in a dose-dependent manner, in contrast to cell proliferation. Another important hallmark of cancer is cell-cycle dysregulation . During Therefore, in this study we decided to investigate Ki-67 expression, showing that the control group was characterized by elevated Ki-67 expression, whereas treatment with BAY-117082 was able to significantly reduce its appearance. Thus, based on the results obtained, using BAY-117082 could be considered as a valid therapeutic strategy to reduce or counteract OSCC progression thanks its ability to modulate the NLRP3 inflammasome and apoptosis pathways. However, further investigations are needed to better understand the involvement of these pathways in oral carcinogenesis. 2.Human OSCC cell lines CAL27, HSC-2, and SCC-4 were obtained from ATCC . Cells were grown in Dulbecco\u2019s Modified Eagle\u2019s Medium  for CAL27 and Minimum Essential Eagle\u2019s Medium  for HSC-2 and SCC-4 cells, supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and 100 U/mL penicillin and 100 \u03bcg/mL streptomycin  at 37 \u00b0C with 5% CO4 cells/well to a final volume of 150 \u03bcL. After 24 h, cells were treated with BAY-117082 (Sigma-Aldrich\u00ae) for 24 h at increasing concentrations 0.1, 0.5, 1, 3, 5, 10, and 30 \u03bcM dissolved in PBS. Then, cells were incubated at 37 \u00b0C with MTT (0.2 mg/mL) for 1 h. The medium was removed and cells were lysed with dimethyl sulfoxide (DMSO) (100 \u00b5L). The extent of reduction in MTT to formazan was quantified by measuring optical density at 550 nm with a microplate reader.A 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to assess cell viability, as previously described . CAL27, Control group (Ctr): Human OSCC cell lines CAL27, HSC-2, and SCC-4BAY-117082 0.1 \u03bcM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 0.1 \u03bcM for 24 hBAY-117082 0.5 \u03bcM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 0.5 \u03bcM for 24 hBAY-117082 1 \u03bcM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 1 \u03bcM for 24 hBAY-117082 3 \u03bcM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 3 \u03bcM for 24 hBAY-117082 5 \u03bcM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 5 \u03bcM for 24 hBAY-117082 10 \u03bcM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 10 \u03bcM for 24 hBAY-117082 30 \u03bcM group: CAL27, HSC-2, and SCC-4 cells treated with BAY-117082 30 \u03bcM for 24 h6 CAL27 cells were plated in 6-well plates  and incubated with BAY-117082 for 24 h [g for 15 min at 4 \u00b0C. Protein concentration was estimated by the Bio-Rad protein assay using bovine serum albumin as standard. Samples were heated at 95 \u00b0C for 5 min, and the same amounts of protein were separated on 12% SDS-PAGE gel and blotted to PVDF membrane (Immobilon-P). Membranes were incubated overnight at 4 \u00b0C with the following primary antibodies: anti-NLRP3 , anti-ASC , anti-caspase-1 , anti-IL-1\u03b2 , anti-IL-18 , anti-Bax , anti-Bcl2 , and anti-Bad . Then, membranes were incubated with peroxidase-conjugated bovine anti-mouse or goat anti-rabbit or anti-mouse IgG  for 1 h at room temperature. To ascertain that the blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against \u03b2-actin protein for cytosolic fraction  and Lamin A/C for nuclear fraction . The signals were detected with a chemiluminescence detection system reagent according to the manufacturer\u2019s instructions . The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc  using ImageLab software.For the Western blot, 1 \u00d7 10for 24 h . Then, c2HPO4, pH 7.4), permeabilized in 0.2% Triton X-100/PBS, and blocked with 10% bovine albumin serum. Cells were incubated overnight (O/N) at 4 \u00b0C with primary antibodies: anti-p53  and anti-caspase-1 . After being washed in PBS, cells were incubated with secondary antibody Alexa Fluor 488 goat anti-mouse  for 1 h at 37 \u00b0C. Sections were washed in PBS and 2 \u03bcg/mL 4\u2032,6\u2032-diamidino-2-phenylindole  in PBS was added for nuclear staining. Sections were observed and photographed at 40\u00d7 magnification using a Leica DM2000 microscope . Immunofluorescence assay was performed as previously described by Donaldson . CAL27 cBALB/c nude male mice were obtained from Jackson Laboratory  and housed in microisolator cages under pathogen-free conditions with 12 h light/12 h dark. Animals were fed with a standard diet and water ad libitum. This study was approved by the University of Messina Review Board, under project identification code 137/2017-PR, released on 9 February 2017. Animal care was in compliance with Italian regulations on the protection of animals used for experimental and other scientific purposes (DM 116192) as well as EU regulations (OJ of EC L 358/1 18 December 1986).6 CAL27 cells per tumor in 0.2 mL of PBS and 0.1 mL Matrigel  as previously described [The xenograft tumor model was established by subcutaneously inoculating 3 \u00d7 10escribed . After t3, mice were treated with BAY-117082 at doses of 2.5 and 5 mg/kg every 3 days according to [2 \u00d7 L/2, where W and L represent minor and major length. After 30 days, mice were sacrificed and tumors were excised and processed for analysis. After 1 week of tumor induction, mice were divided randomly into 3 groups. When tumor size reached about 200\u2013300 mmrding to . BAY-117Control group (vehicle): weekly intravenous (IV) administration of salineControl group + BAY-117082 2.5 mg/kg: intraperitoneal administration of BAY-117082 2.5 mg/kg dissolved in PBSControl group + BAY-117082 5 mg/kg: intraperitoneal administration of BAY-117082 5 mg/kg dissolved in PBSMice were randomly divided into 3 groups:n = 14 mice for each technique. The minimum number of mice for each technique was estimated with one-way fixed effects ANOVA with G-power software. This statistical test generated a sample size equal to Histological evaluation was performed as previously described by Paterniti et al. . Tumor sImmunohistochemical localization was carried out as previously described by Scuderi et al. . Tumor sw/v) dried nonfat milk in buffered saline (PM) for 45 min at room temperature and subsequently probed with specific antibodies: anti-NLRP3 , anti-ASC , anti-caspase-1 , anti-Bax , anti-Bcl2 , anti-Bcl-xL , anti-NF-\u03baB , and anti-I\u03baB\u03b1  in 1\u00d7 PBS, 5% w/v dried nonfat milk and 0.1% Tween-20 (PMT) at 4 \u00b0C overnight. Membranes were incubated with peroxidase-conjugated goat anti-mouse IgG secondary antibody  or peroxidase-conjugated goat anti-rabbit IgG secondary antibody  for 1 h at room temperature. To establish that blots were loaded with equal amounts of proteins, they were also incubated in the presence of the antibody against \u03b2-actin protein . Signals were revealed with an enhanced chemiluminescence (ECL) detection system reagent according to the manufacturer\u2019s instructions . The relative expression of protein bands was quantified by densitometry with Bio-Rad ChemiDoc XRS+ software and standardized to \u03b2-actin levels as an internal control.Protein levels in tumor samples were quantified as previously described . CytosolAll compounds and other chemicals were obtained from Sigma-Aldrich . All stock solutions were prepared in non-pyrogenic saline .t-test analysis. All values are indicated as mean \u00b1 standard error of the mean (SEM) of N observations. Data were analyzed with GraphPad Prism 7.04 software using In conclusion, the results obtained offer new insights into the role of the NLRP3 inflammasome and apoptosis signaling pathways in oral carcinogenesis, showing that the use of BAY-117082, a strong NLRP3 inflammasome inhibitor, could represent a potential therapeutic treatment to counteract or reduce the progression of OSCC, which has been showing an increasing mortality rate."}
+{"text": "Mangifera casturi Kosterm., a mango plant from Kalimantan Selatan, Indonesia, has limited genetic information, severely limiting the research on its genetic variation and phylogeny. We collected M. casturi\u2019s genomic information using next-generation sequencing, developed microsatellite markers and performed Sanger sequencing for DNA barcoding analysis. These markers were used to confirm parental origin and genetic diversity of M. casturi hybrids. The clean reads of the Kasturi accession were assembled de novo, producing 259\u00a0872 scaffolds (N50\u2009=\u20091\u00a0445\u00a0bp). Fourteen polymorphic microsatellite markers were developed from 11\u00a0040 microsatellite motif-containing sequences. In total, 58 alleles were produced with a mean of 4.14 alleles per locus. Microsatellite marker analysis revealed broad genetic variation in M. casturi. Phylogenetic analysis was performed using internal transcribed spacers (ITS), matK, rbcL, and trnH-psbA. The phylogenetic tree of chloroplast markers placed Kasturi, Cuban, Pelipisan, Pinari, and Hambawang in one group, with M. indica as the female ancestor. Meanwhile, the phylogenetic tree of ITS markers indicated several Mangifera species as ancestors of M. casturi. Thus, M. casturi very likely originated from the cross-hybridization of multiple ancestors. Furthermore, crossing the F1 hybrids of M. indica and M. quadrifida with other Mangifera spp. may have generated much genetic variation. The genetic information for M. casturi will be a resource for breeding improvement, and conservation studies. Mangifera casturi Kosterm., or Kalimantan mango, is an endogenous fruit plant in Kalimantan Selatan, Indonesia; it is classified as extinct in the wild according to the IUCN Red List1. M. casturi belongs to the Mangifera genus within the Anacardiaceae family2 and is classified as a common ancestor of the Mangifera species in Indonesia3. M. casturi is proposed to be the natural hybrid of M. indica and M. quadrifida according to single nucleotide polymorphisms (SNPs) analysis4. In Kalimantan Selatan, M. casturi are known by various local names, such as Kasturi, Cuban, Pelipisan, Pinari, and Rawa-rawa  in nucleotide repositories such as the NCBI, and one SRA study (SRP183190) reported.7. Also, from NGS data, it is easier to obtain genetic information such as microsatellite markers, which are superior to other markers like RAPD and AFLP and are already used in other Mangifera species9. Microsatellite markers can determine distinct variations at the level of species as they are codominant; as a result, they are widely used in population and genetic studies10. Microsatellite markers have also been used to determine the genetic variation in M. indica11. Although microsatellites are important for taxonomy and the study of genetic conservation, no M. casturi-specific microsatellite markers have been reported.Recently, sequencing has advanced significantly from Sanger sequencing to next-generation sequencing (NGS). For example, whole-genome sequencing can produce comprehensive genomic information on a speciesrbcL, matK12, and trnH-psbA13, internal transcribed spacers (ITS), and second internal transcribed spacers (ITS2) from nuclear ribosomal DNA14, have been widely used for phylogenetic analysis at various taxonomic levels. These DNA barcoding markers from chloroplast regions can also be determined at the genus or family level because of their inheritance from a maternal ancestor. On the other hand, the ITS region can determine the barcoding of the paternal and maternal ancestor. However, DNA barcoding sequences for M. casturi have not been recorded in any public database. As a result, there have been no phylogenetic studies of M. casturi using DNA barcoding to achieve accurate identification at the taxonomy level.In recent years, Sanger sequencing approaches have been utilized for DNA barcoding. DNA barcoding methods based on chloroplast regions, such as M. casturi using NGS and Sanger and to analyze and determine the genetic variation among the M. casturi hybrids. Microsatellite markers were used to assess genetic variation among M. casturi hybrids, Kasturi, Cuban, Pelipisan, and Pinari. Furthermore, at a higher taxonomy level, phylogenetic analysis of M. casturi hybrids and Mangifera species was performed using DNA barcoding. In addition, there is no clear information and proof about the genetic variation and relationship among M. casturi hybrids. In this study, we propose a candidate ancestor from a natural hybridization of M. indica and M. quadrifida.This study aimed to collect genomic information from M. casturi DNA was obtained with high-throughput sequencing using an Illumina HiSeq 4000 system with paired end 150\u00a0bp reads. The raw data were registered in the DDBJ with accession number DRA011022. Clean reads were obtained via filtering, and 10.95 Gbp of de novo genome assembly was performed using a Ray Assembler. We obtained 259\u00a0872 scaffolds with an N50 value of 1\u00a0445\u00a0bp and a maximum scaffold length of 144\u00a0601\u00a0bp  ranged from 0 for 4 markers  to 0.62 for the mc8693 locus with a mean Ho of 0.26. The expected heterozygosity (He) ranged from 0.40 for the mc167596 locus to 0.80 for the mc230178 locus, with a mean of 0.65. The fixation index (FST), reflecting the degree of genetic differentiation, ranged from 0.08 to 1.00 with an average of 0.60 per locus. Lastly, Shannon\u2019s information index ranged from 0.69 and 1.75 with a mean of 1.21.Eight samples, namely Pelipisan, Cuban, Pinari, Kasturi, Rawa-rawa, M. casturi accessions in a natural population, as it is dissimilar to other Mangifera species.The mc230178 and mc58089 loci produced seven alleles from eight samples, while the mc122955 and mc88387 loci produced two alleles. Some loci, namely mc176197, mc21672, and mc88075, displayed the same alleles between Kasturi and Cuban. In the mc88387 locus, only the Kasturi sample was not amplified; thus, this locus might have a null allele in Kasturi. Therefore, mc88387 locus can be used to identify M. foetida), Pelipisan, Pinari, M. indica in one cluster, while Kasturi and Cuban in the second cluster, and Rawa-rawa and M. quadrifida in the third cluster.Furthermore, principal coordinate analysis was performed using the GENALEX 6.501, indicating that 33.85% of the variance within the microsatellite data was graphed by the first axis, and 19.43% by the second axis Fig.\u00a0. AdditioM. quadrifida and Rawa-rawa were placed in the same clade. All the M. casturi accessions were in the same clade as M. indica and Hambawang (M. foetida). Cuban were most closely related to M. indica. However, Kasturi and Pelipisan were in the same clade where some loci showed similar alleles. Thus, these accessions had a closer genetic relationship to each other than to Cuban. However, Pinari also exhibited distinct genetic differences from the other M. casturi accessions, even though Cuban was quite distant from other M. casturi accessions.The result was also presented in a UPGMA dendrogram Fig.\u00a0. M. quadmatK, rbcL, and trnH-psbA  and were deposited in the DDBJ Nucleotide Sequence Submission System under the accession number LC602976- LC602993. However, the matK, rbcL, and trnH-psbA sequences from other Mangifera species were downloaded from the public nucleotide database of NCBI , M. griffthii, M. quadrifida, M. kemanga, M. torquenda, and M. sumatrana.On the other hand, the ITS phylogenetic tree produced three large groups: Indica 1, indica 2, and a group containing Kasturi, Cuban, Pelipisan, and Pinari. Hambawang was included in the indica 2 group. Meanwhile, Pinari was placed in a sub-group with Mangifera genus originates from southeast Asia and has polyembryonic seeds, derived from gametes or nucellar cell components2. Most Mangifera flowers are either hermaphrodites or males32; thus, self-crossing can occur in various species. However, self-incompatibility in the Mangifera genus has been reported in several types of mangos33, suggesting that various Mangifera species can cross-hybridize34. As a result, cross-hybridization in the natural populations has produced many interspecies, including M. odorata (Kuini), a natural hybrid between M. indica and M. foetida9.The 36. Microsatellites in Mangifera species that were previously identified in M. indica have been useful in the genetic analysis of genus Mangifera and its related genera38. In This study, genetic analysis has revealed markers in 14 microsatellite loci and that different allele sizes have arisen from four accessions of M. casturi namely Kasturi, Cuban, Pelipisan, and Pinari. The expected heterozygosity (He) value of the microsatellite markers used in the M. casturi analysis ranged between 0.40 and 0.80, with an average of 0.65, which indicated that the highly informative microsatellite markers could be employed in genetic diversity studies of M. casturi. In this study, a high level of genetic variation was discovered in M. casturi accessions, likely arising from repetitive interspecific hybridization. In Petunia, microsatellite markers have determined genetic differentiation and hybrid identification39. The accessions of Kasturi, Cuban, and Pelipisan were more closely related than Pinari. Kasturi and Cuban are very similar in fruit size. However, morphologically, the fruit shape of Kasturi is more oval than Cuban. In contrast, a Pelipisan fruit is more oval and slightly larger than Kasturi and Cuban. Pinari has the largest fruit size among the M. casturi accessions. Lastly, Pinari is classified into the M. casturi group by the locals, based on its purplish skin similar to that of other M. casturi accessions 4.Microsatellite markers have been used successfully to determine genetic variation among many plantsJuglans regia and J. cathayensis indicated a rare phenomenon and backcrosses between hybrids and either of the parental species40. In addition, Kuini (M. odorata), a natural hybrid between M. indica and M. foetida was revealed by AFLP analysis to represent a simultaneous backcross between the F1 hybrids of Kuini and M. foetida9. On the other hand, SNP analysis using double-digest restriction-site-associated DNA (ddRAD)4, revealed M. casturi to be a natural hybrid between M. indica and M. quadrifida, whereas their F1 hybrid was a backcross with M. indica. Morphologically, M. casturi is very close to M. quadrifida, with the same purplish skin and small fruit size4. Therefore, M. casturi has several types known as Kasturi, Cuban, Pelipisan, and Pinari. These hybrids are believed to be hybrids between M. indica and M. quadrifida and backcrosses between hybrids and either of the ancestors.Intraspecies genetic variation can occur because of multiple cross-hybridizations among several species. Using microsatellite markers, hybridization between Garcinia mangostana), microsatellite markers indicate cross-hybridization with multiple ancestors, including G. malaccensis, G. celebica, and G. porrecta22. In this study, microsatellite analysis results showed that four accessions of M. casturi, namely Kasturi, Cuban, Pelipisan, and Pinari had allelic differences in all the microsatellite loci. However, allele sharing between the four accessions was detected in the mc8693 locus with an allele size of 160/182, indicating that these accessions were derived from the same ancestor, M. indica. In contrast, the allele differences in the microsatellite loci suggested that the four M. casturi accessions underwent cross-hybridization with multiple ancestors. In Oaks (Quercus spp.), microsatellites indicate sharing most alleles in their hybrids than recurrent gene flow41.In an allopolyploid plant such as mangosteen  region has also been indicated as a barcoding regionM. casturi hybrids. Based on genomic data, we have identified that M. casturi, which is endemic to Kalimantan Selatan, consists of 4 types, namely Kasturi, Cuban, Pelipisan, and Pinari. In Addition, based on the combination of microsatellite data, and DNA barcoding, M. casturi hybrids are natural hybrids between M. indica and M. quadrifida. Moreover, the genomic data represent an important genetic resource for breeding and improving the characteristics of this local mango in the future. The habitat of M. casturi is severely threatened; as a result, it is classified as extinct in the wild. Thus, more intensive conservation efforts are necessary. Moreover, since M. casturi varieties have never been confirmed or registered by authorities, the results of this study can help breeders and the local government to officially document this local mango and one of their elite germplasm.In this study, the genomic data revealed the genetic variation and ancestral origin of M. casturi accessions were collected from the Banjar district, Kalimantan Selatan, in the southern region of Kalimantan, three Mangifera species were collected from Banua Botanical Garden  and Dramaga, Bogor, West Java (M. indica) . 15, and clean reads were filtered using the Fastp version 0.20.1 (https://github.com/OpenGene/fastp) with default parameters16. Clean reads were assembled using a Ray version 2.1.0 (https://github.com/sebhtml/ray) with default parameters (-k 63 -minimum-contig-length 200)17 under the Maser Platform (https://cell-innovation.nig.ac.jp/). 18. The assessment of genome assembly was performed to check the assembled contig quality using BUSCO version 3.0.2 (https://busco.ezlab.org). 19 on the Maser Platform with default parameters.ca) Fig.\u00a0. To analhttps://webblast.ipk-gatersleben.de/misa/). 20 from M. casturi scaffolds, the parameters being set to the following minimum repeat levels: six for two bases, and five for three, four, five, and six bases. The difference between microsatellite motifs was 100 bases. The microsatellite motif-containing sequences were selected based on parameters; (1) the flanking region are at least 150\u00a0bp long in both directions (2) microsatellite repeats have the longest repeat motifs. The primer was designed using the web version of Primer 3 with default parameters21.Microsatellite markers were extracted using the MISA version 2.1 . A Type-it microsatellite PCR kit (Qiagen) was used to analyze the microsatellite markers. PCR master mix was prepared with a mixture of 3.2 \u03bcL RNase-free water, 5 \u03bcL 2\u2009\u00d7\u2009Type-it Multiplex PCR Master Mix, 0.4 Q solution, 0.2 \u03bcL of 10\u00a0\u03bcM forward primer, and 0.2 \u03bcL of 10\u00a0\u03bcM reverse primer. PCR was performed using a SimpliAMP Thermo Cycler (Applied Biosystems). The PCR conditions were as follows: initial conditions of PCR pre-denaturation at 95\u00a0\u00b0C for 5\u00a0min, followed by 32 cycles of denaturation at 95\u00a0\u00b0C for 30\u00a0s, annealing at 57\u00a0\u00b0C for 1\u00a0min 30\u00a0s, extension at 70\u00a0\u00b0C for 30\u00a0s, and final extension at 60\u00a0\u00b0C for 30\u00a0min. The amplicons were checked using 1% electrophoresis gel in TAE buffer for 20\u00a0min at 100 v. Before loading the sample into QIAxcel capillary electrophoresis (Qiagen), the sample was diluted twice and then run using a QIAxcel DNA High Resolution Kit (Qiagen). Allele size data were confirmed and processed manually using QIAxcel ScreenGel version 1.4.0 .Genomic DNA was isolated using the modified CTAB method, with a slight modificationhttps://biology-assets.anu.edu.au/GenAlEx/) for each microsatellite marker, including the number of alleles (Na) per locus, and both observed (Ho), and expected (He) heterozygosity, fixation index (FST), and Shannon's information index (I). The principal coordinate analysis (PCoA) via Covariance matrix with data standardization was also performed using GENALEX 6.501. The microsatellite data were processed using the Phylip version 3.695 (https://evolution.genetics.washington.edu/phylip.html) with the unweighted pair group method and arithmetic mean (UPGMA) method. The resulting dendrogram was edited using the program MEGA-X Software (https://www.megasoftware.net)23.Descriptive statistics were calculated using GENALEX version 6.501 27. PCR barcoding was performed using KOD Plus (Toyobo) according to the manufacturer's protocol. The PCR products were cleaned using ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems). Then, PCR sequencing was carried out with a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), followed by purification using a BigDye XTerminator Purification Kit (Applied Biosystems) according to the manufacturer protocol. The sequencing products were performed using a 3500 Genetic Analyzer (Applied Biosystems). Sequence data were analyzed using Sequencing Analysis Software version 6.0 , and the data were processed with ATGC-MAC version 7 (https://www.genetyx.co.jp) and MEGA-X software (https://www.megasoftware.net). 23.For the DNA barcoding analysis, we used three chloroplast genes: 28. The Mangifera sequences of matK, rbcL, and trnH-psbA, and ITS complete sequences were downloaded from NCBI  from Plant Quarantine Division of National Agency for Agricultural Quarantine in Banjarbaru, South Kalimantan following permit approvals from South Kalimantan Natural Resources Conservation Agency/BKSDA of the Ministry of Environment and Forestry of the Republic of Indonesia (KLHK) as agency in charge of managing conservation areas including protected plant in the territory, particularly the nature reserve forests  and national park. The M. casturi samples from Banjar, South Kalimantan as herbaria voucher (received by Agung Sriyono) were duplicated and stored in Banua Botanical Garden, Province of South Kalimantan, Banjarbaru,\u00a0Indonesia.All experiments were performed in accordance with relevant guidelines and regulations. The experimental research has complied with Bogor Agricultural University research regulation (No: 11/SA-IPB/P/2016 on research and publication ethics), and the field study was in accordance with the national legislations of Indonesian Law Number 5/1990 on biological diversity conservation and Indonesia Law Number 11/2013 on the ratification of the Nagoya Protocol. Supplementary Information 1.Supplementary Information 2."}
+{"text": "Individuals with greater genetic risk for Schizophrenia or Bipolar Disorder are more vulnerable to the adverse effects of birth at early gestational age on brain development, as assessed by cognition at age four. Better understanding of gene-environment interactions will inform more effective risk-reducing interventions for this vulnerable population.Preterm birth is an extreme environmental stress associated with an increased risk of later cognitive dysfunction and mental health problems. However, the extent to which preterm birth is modulated by genetic variation remains largely unclear. Here, we test for an interaction effect between psychiatric polygenic risk and gestational age at birth on cognition at age four. Our sample comprises 4934 unrelated individuals . Genome-wide polygenic scores (GPS\u2019s) were calculated for each individual for five different psychiatric pathologies: Schizophrenia, Bipolar Disorder, Major Depressive Disorder, Attention Deficit Hyperactivity Disorder and Autism Spectrum Disorder. Linear regression modelling was used to estimate the interaction effect between psychiatric GPS and gestational age at birth (GA) on cognitive outcome for the five psychiatric disorders. We found a significant interaction effect between Schizophrenia GPS and GA ( Preterm infants have higher rates of cognitive impairment, cerebral palsy, autism spectrum disorders and psychiatric disease5, not all of which can be explained by the degree of prematurity and severity of clinical problems around the time of birth.Preterm birth is an extreme perinatal stress and is associated with a significantly increased risk of later cognitive dysfunction and psychiatric disease. Preterm birth is common, accounting for around 11% of all births and is a leading cause of infant mortality and morbidity worldwideMagnetic resonance imaging studies indicate that preterm brain injury is a multifactorial disorder which encompasses alterations to cortical and subcortical gray matter and white matter. These studies have contributed to our understanding of the pathophysiology underlying neurodevelopmental disability and cognitive impairments in the preterm population, however, a great deal remains unknown. Specifically, the extent to which genetic factors play a role in the variable cognitive outcomes of preterm infants and the possible interaction between genetic risk and the environmental exposure to preterm birth has not been examined in detail. Here, we explore whether common genetic variation is important in determining an individual\u2019s susceptibility to the environmental stress of prematurity and thus plays a role in the diverse neurodevelopmental and cognitive outcomes we observe in preterm infants.6. We therefore anticipate that gene-environment interactions involving these complex pathologies will be most effectively studied by looking at multiple genetic variants rather than focussing on a single genetic locus. As samples sizes have grown, genome-wide association studies (GWAS) have become increasingly informative, allowing detection of small effects of single nucleotide polymorphisms (SNPs). Using the summary statistics from these studies it is possible to generate individual-specific genotypic scores to predict phenotypic variance, genome-wide polygenic scores (GPS). A genome-wide polygenic score, which is an aggregate of trait-related effect sizes of SNPs across the genome in independent samples can be used to test the predictive power of multiple genetic variants simultaneously. Genome-wide polygenic scores provide a useful approach to exploring gene-environment interactions in complex traits.Research into the genetics of psychiatric disease has shown it to be polygenic; psychiatric disorders are influenced by many genetic variants, each of small effect7. Under this notion, individuals with a greater polygenic risk for psychiatric disease, when born preterm, would be more likely to suffer adverse developmental consequences. We showed that in a cohort of preterm infants, psychiatric GPS was negatively associated with lentiform volume; genetic predictors of neuropsychiatric disease increase vulnerability to abnormal lentiform development after perinatal stress.In previous work, we hypothesised that genes associated with psychiatric disease might increase vulnerability to abnormal brain development in infants subjected to the environmental stress of prematurityWe now extend this work by looking at the possible influence of genome-wide polygenic risk for psychiatric disease in preterm individuals, as measured by their cognitive abilities in early childhood. In a large cohort of unrelated twins, we test for a possible gene-environment interaction between the GPS\u2019s for five different psychiatric disorders (Autism Spectrum Disorder (ASD), Attention Deficit Hyperactivity Disorder (ADHD), Bipolar Disorder, Major Depressive Disorder and Schizophrenia) and gestational age at birth on cognition at age four.Linear regression modelling was used to test for a possible gene-environment interaction between the GPS\u2019s for five different psychiatric disorders  and gestational age at birth on cognition at age four. Genome-wide polygenic scores were adjusted for the first five ancestry principle components, genotype chip and plate and each regression model included the covariates sex and socio-economic status (SES) (Eq. ).Genotype, phenotype and covariate information was available for 4934 individuals . Of these 2066 were born before 37 weeks completed gestation and 918 were born at or below 34 weeks completed gestation. A table showing the number of individuals at different gestational age cut-offs is included in the Supplementary Information  and an interaction effect for gestational age at birth and Bipolar genetic risk . There was no evidence for an interaction effect between gestational age at birth and genetic risk for Autism Spectrum Disorder, ADHD or Major Depressive Disorder. For both Schizophrenia and Bipolar Disorder, the effect of gestational age at birth on cognitive outcome was greater for those individuals with a larger psychiatric genetic risk burden. Results for the full regression model for all five psychiatric pathologies are given in Supplementary Table Results indicated a significant interaction between psychiatric genetic risk and gestational age at birth for two of the five pathologies investigated Table  after Bo\u03b2\u2009=\u20090.038, se\u2009=\u20090.013, p\u2009=\u20097.07\u2009\u00d7\u200910\u20133) and gestational age at birth and Bipolar genetic risk  on cognitive outcome at four. Further, there was no evidence for an association of cognition with twin birth order and the addition of this as a covariate to our model did not significantly alter the results. There remained a significant interaction effect of gestational age at birth and Schizophrenia genetic risk  and gestational age at birth and Bipolar genetic risk  on cognitive outcome at four.Removing the covariate socio-economic status from the model did not markedly alter the results. There remained a significant interaction effect of gestational age at birth and Schizophrenia genetic risk .\u20133). The adverse effect of birth at earlier gestational age (<\u2009=\u200934 weeks) on cognition is greater for individuals with a higher genetic risk burden for Schizophrenia than it is for those with the lowest risk  including sex and socio-economic status as covariates. None of the psychiatric genetic risk scores showed a significant association with cognition at age four .The similar results obtained for both Bipolar Disorder and Schizophrenia are consistent with evidence for shared genetic aetiology for these disorders. In our cohort there is a significant correlation between genetic risk for Bipolar Disorder and Schizophrenia  and stressful life events, provides an early example of such a study. The authors showed that the level of depressive symptoms experienced by individuals in response to stressful life events was dependant on the allele they carried at this polymorphism.Gene\u2013environment interactions are the result of individuals responding differently to environmental stimuli, depending on their genotype. Many studies have explored gene-environment interactions for psychiatric pathologiesWhilst early studies largely focussed on single polymorphisms, evidence suggests that a polygenic approach is likely to be more fruitful in understanding how genetic risk for psychiatric pathologies may increase susceptibility to environmental risk factors. More recent studies frequently investigate gene-environment interactions on a genome-wide scale, leveraging results from genome-wide association studies to construct polygenic scores. It is hoped that this approach could reveal interactions that may have gone undetected using candidate SNP analysis.15 and early life adversity17 with a few studies exploring perinatal factors. Studies with a perinatal focus include work indicating a significant interaction between maternal CMV infection and a polymorphism in the CTNNA3 gene on schizophrenia risk18 and work indicating that low birth weight predicts poorer academic and physical performance in individuals at high risk of Schizophrenia but not for those at low risk19.Gene-environment interaction studies exploring Schizophrenia have largely focussed on stressful life events13. The majority of work on Bipolar Disorder has been using candidate genes and to the best of our knowledge there have been no previous studies using summary statistics for Bipolar Disorder from the Psychiatric Genomics Consortium.Studies exploring Bipolar Disorder have followed similar themes to those for Schizophrenia focussing on environmental exposure to stressful life events20.Preterm birth itself may be a genetically inherited trait. However, it is difficult to know the extent to which this could contribute to confounding in this work. Despite evidence for a modest contribution of genetics to the risk of preterm birth, to-date, there exist few robust genetic associations identified through genome-wide association studies for prematurity. A large recent study identified two intergenic loci associated with preterm birth but failed to replicate these resultsPreterm birth is complex with heterogenous aetiology. Data that would allow us to distinguish between different aetiologies such as information on maternal infection or maternal pathology were not available in this study. It is possible that genetic risk alleles that confer an increased risk of Schizophrenia or Bipolar Disorder might also confer an increased risk of pathologies associated with preterm birth . Whilst we believe that such shared genetic aetiology, if present, is unlikely to fully explain our results, we cannot exclude the possibility that it could introduce confounding.21. That said, we cannot exclude the possibility that we are underpowered to detect such an association.We have explored the possibility that genetic risk for psychiatric disease is associated with gestational age at birth within our cohort. For the five psychiatric pathologies examined in this study we found no significant association between genetic risk for psychiatric disease and gestational age at birth . As children grow older, smaller differences in ability might be easier to measure but they will similarly be subjected to a larger diversity of additional environmental exposures.Finally, this study focused on cognitive outcome at age four. We hypothesised that the interaction between genetic risk for psychiatric disease and gestational age at birth would explain a significant fraction of the variability in cognitive outcome at age four. Work looking at preterm outcomes has shown stability of impairment in early childhood and confirmed that the most commonly observed impairment is in developmental or cognitive functionIn summary, individuals with greater genetic risk for either Schizophrenia or Bipolar Disorder were shown to be more vulnerable to the negative effects of birth at earlier gestational age on cognitive outcome. The pathophysiology underlying preterm neurodevelopmental impairment may therefore be vulnerable to genetic variability associated with adult psychiatric disease.23. Written informed consent was obtained from parents prior to data collection. Project approval was granted by King\u2019s College London\u2019s Research Ethics Committee for the Institute of Psychiatry, Psychology and Neuroscience (PNM/09/10-104) and the research was performed in compliance with the Declaration of Helsinki.Participants were drawn from the Twins Early Development Study (TEDS). Between 1994 and 1996 TEDS recruited over 15,000 twin pairs born in England and Wales, who have been assessed in multiple waves across their development up until the present date. Roughly 10,000 twin pairs still actively contribute to TEDS, providing genetic, cognitive, psychological and behavioural data. TEDS participants and their families are representative of families in the UKThis study included 4934 unrelated individuals of European ancestry from the TEDS cohort. The dataset was subject to standard exclusions. This included exclusion of individuals with serious medical conditions which would likely affect their ability to take part in the TEDS assessments or conditions known to be associated with significant cognitive impairment. It also included exclusion of individuals in whom extreme circumstances were identified in the perinatal period such as prolonged hospital admission following birth. An initial filtering was then undertaken to select a cohort of unrelated individuals; this involved selection of all the unpaired twins plus one twin from each genotyped pair. Individuals were retained that had genotyping for the GPS analysis, data for cognitive outcome at age four, recorded gestational age at birth and recorded parental socio-economic status.The final sample comprised 4934 individuals. Of these, 1858 were from monozygotic (MZ) twin pairs and 3076 were from dizygotic (DZ) twin pairs (1596 same-sex DZ pairs and 1480 opposite-sex DZ pairs). 2066 of the 4934 individuals were born at less than 37 weeks completed gestation and 918 were born at or below 34 weeks gestation.https://www.wtccc.org.uk/ccc2/). The remaining 3190 individuals were genotyped on HumanOmniExpressExome-8v1.2 arrays at the Molecular Genetics Laboratories of the Medical Research Council Social, Genetic Developmental Psychiatry Centre, using DNA that was extracted from saliva samples.Genotyping was undertaken at two separate timepoints with two different genotyping platforms. 1744 of the 4934 individuals were genotyped on the Affymetrix GeneChip 6.0 SNP array at Affymetrix, Santa Clara  using buccal cell DNA samples. Genotypes were generated at the Wellcome Trust Sanger Institute  as part of the Wellcome Trust Case Control Consortium 2  through the Sanger Imputation Service before merging genotype data from both platforms .After quality control, 635,269 SNPs remained for Affymetrix GeneChip 6.0 genotypes, and 559,772 SNPs for HumanOmniExpressExome genotypes. Genotypes from the two platforms were separately phased using EAGLE22\u2009>\u20090.1) and excluding high linkage disequilibrium genomic regions.The final data contained 7,363,646 genotyped or well imputed SNPs. Principal component analysis was performed on a subset of 39,353 common (MAF\u2009>\u20095%), perfectly-imputed (info\u2009=\u20091) autosomal SNPs, after stringent pruning to remove markers in linkage disequilibrium (r26. The cognitive ability measure was computed as the mean of the four standardised elements.The standardised cognitive ability measure at four was a composite of \u2018Parent Report of Children\u2019s Abilities\u2019 (PARCA) including both a parent-reported and parent-administered element and an assessment of verbal and non-verbal ability. The parent administered assessment included an odd-one-out task, design drawing task and puzzle task and the parent-reported assessment examined conceptual knowledge. Non-verbal ability was assessed with a 48-item vocabulary test and verbal ability with a grammar assessment which looked at length of sentences and correct use of both \u2018-est\u2019 words and \u2018but\u2019. The parent administered assessments have been validated in a subsampleSocio-economic status is a composite of maternal age at birth of eldest child, the mean score of maternal and paternal highest education level, as well as the respondent\u2019s (mother or father) occupation, administered by the Standard Occupational Classification 2000  when the child was two, which was the first age of contact.We calculated genome-wide polygenic scores, which are the SNP effect-size weighted sums of the number of trait-associated alleles. The SNP weights were derived from summary statistics for the largest GWA studies available for five psychiatric disorders at time of computation .The genome-wide polygenic scores were calculated using the software LDpredpsych \u00d7 GA) on cognitive outcome at age four, for five different psychiatric pathologies  separately (Eq. (28. Interaction terms for psychiatric genome-wide polygenic score (GPSpsych) with sex and socio-economic status as well as interaction terms for gestational age at birth (GA) with sex and socio-economic status were included to properly control for possible confounding effects29.Linear regression modelling was used to estimate the effect of the gene-environment interaction (GPSely (Eq. ). Prior psych \u00d7 GA on cognitive outcome. We therefore explored whether the estimate of the coefficient \u03b23 of the interaction term deviated significantly from zero. P-values lower than the significance level \u03b1\u2009=\u20090.05/5\u2009=\u20090.01 were considered significant to account for the family-wise error rate using the Bonferroni method, correcting for the five psychiatric pathologies explored. Calculations were implemented with \u201clm\u201d function in R (https://www.r-project.org/). Standard errors for the beta regression coefficients were estimated using the bootstrap technique with 10,000 bootstrap samples with random sampling of individuals with replacement.The aim of this analysis was to investigate the effect of the interaction term GPSAs a further exploration of our data, we looked at the extremes of the data undertaking a two-way analysis of variance (ANOVA) comparing mean cognition between the extreme quintiles of polygenic risk for individuals born at or below 34 weeks gestation and term-born (>\u2009=\u200937 weeks) individuals for the psychiatric pathologies that yielded significant results in our initial analysis. Similar analysis for the non-significant pathologies is presented in the Supplementary Information .2\\documFor completeness we also explored a possible association between cognition at four and genetic risk for each of the five psychiatric pathologies examined, details can be found in the Supplementary Analysis . The research was performed in compliance with the Declaration of Helsinki.Supplementary Information."}
+{"text": "We conducted formative research using in-depth interviews to identify preferences for and anticipated responses to receiving thank you notes and lay summaries of aggregate results among caregivers and adolescent participants of pragmatic pediatric studies conducted by the National Institute of Health-sponsored Pediatric Trials Network. We analyzed the data using qualitative thematic analysis. Nearly all participants said receiving a thank you note would make them feel valued, appreciated, and proud because they contributed to science. Similarly, nearly all participants said that receiving a lay summary of research results would make them aware of their role in improving the lives of children, feel like they are an active partner in research, and believe that researchers want to keep them informed. Participants also said that receiving a thank you note or lay summary may motivate them to participate in future research. Providing thank you notes as part of study participation should become a standard clinical trial practice, similar to the practice of providing lay summaries. \u2022Participants want to be thanked for their contributions to clinical research.\u2022Providing thank you notes and lay summaries demonstrate to caregivers and adolescent participants that researchers value and appreciate their contributions.\u2022Providing thank you notes and lay summaries may also increase caregivers\u2019 and adolescent participants\u2019 interest in participating in future research studies. Sharing lay summaries  of aggreWe engaged two stakeholder groups of PTN studies\u2014caregivers/parents and adolescent participants\u2014in formative research on their preferences for and anticipated reactions to receiving thank you notes and aggregated lay summaries of pediatric clinical research results of pragmatic studies. Findings from this study allow stakeholder data to guide PTN decisions about how, when, where, and what to provide in thank you notes and lay summaries.Here, we describe a subset of the study findings and focus on the perceived value of thank you notes and lay summaries as they are likely applicable to studies outside of PTN. Findings on the preferred content of thank you notes and lay summaries are presented elsewhere.2We conducted a qualitative descriptive study  with carDuring the one-on-one interviews, we asked participants to view several mock versions of thank you notes and lay summaries of PTN study results and describe their expectations of and anticipated reactions if they had received these materials as part of their PTN study participation. We also asked about a pre-identified list of potential reactions if not initially mentioned by the participant. We conducted the interviews by telephone, and they were audio-recorded with participant permission, transcribed, and analyzed using applied thematic analysis .Using NVivo 12 , two anaThe Duke University Health System Institutional Review Board (IRB) reviewed and approved the study, waiving informed consent for the caregiver interviews. For adolescent interviews, adolescents provided their oral informed assent and their caregivers provided parental permission.33.1We interviewed 27 participants (24 caregivers and 3 adolescents) of diverse race, ethnicity, education level, employment, geographic location, and disease state .3.2[Receiving a thank you note] would make me feel appreciated \u2026 like I made a difference in something. \u2014Adolescent, age 14[Receiving a thank you note] would give me a sense of purpose. You're not just a piece of data. You've got something else going on. You're involved. \u2014Caregiver, age 47Most participants said caregivers and adolescent participants should receive thank you notes because the notes demonstrate gratitude for their research contributions. Nearly all said receiving a thank you note would make them feel valued, appreciated, and proud because they contributed to science. Participants said:I think that [caregivers] should be thanked. It's hard on us being caregivers. We don't get to clock out. It's a daily struggle. It just feels good to know that there are people out there that support you. They feel for you when it comes to each individual situation.[Caregivers] can be thanked, and maybe know that your child's research is very significant. It's just imperative that [caregivers] feel like they have contributed to something. We are truly grateful that we could help someone else's child. To know that the study helped prevent another child from going through the different adverse effects, that really means a lot to us.Some caregivers expressed that their efforts caring for children with severe illnesses are often unrecognized, and a thank you note would be a source of encouragement. A 26-year-old caregiver said:As a parent of a child [with many health issues] \u2026 I think [receiving a thank you note] would encourage me to participate in more studies, to know that my kid helped someone find out information.Some also felt that providing a thank you note may promote altruism and interest in participating in future studies, including among adolescents. A 39-year-old caregiver explained:Participant preferences varied on the timing of providing thank you notes, and some suggested the length of the trial should be considered. For example, one thank you note can be provided at the end of shorter trials and two notes for longer trials\u2014one after enrollment and another at the end. Some participants cautioned on losing sentiment with providing too many thank you notes.3.3[Receiving a lay summary] would make me feel good. Because a lot of times, when you [participate in] these studies in the past, I don't know what came of that. I think [getting a lay summary] gives you more closure than anything. If you actually have it printed out in front of you, and [even though] you're not named individually, you personally know how you answered questions \u2026 this is what I contributed to. \u2014Caregiver, age 45[I would] definitely [take part in other research] because they are involving you in the study. They are keeping you in the loop basically. And next time, you're like, \u201cOkay, I'll enroll my child in other studies because they always keep me informed.\u201d \u2014Caregiver, age 31Most participants said caregivers should receive a lay summary of the research results, and about one-third said adolescents should. Participants stressed the importance of informing caregivers of the results, and some explained that caregivers can decide if and when to inform their child. Nearly all said receiving a lay summary would make them aware of their role in improving the lives of children, feel like they are an active partner in research, believe that researchers want to keep them informed, and motivated to participate in future research. Participants said:I'd be more likely [to take part in future research] if I knew the results of one study and was thanked. Knowing that the research team actually cared that you participated, and cared that you found out the results of the study, no matter how long it takes, [makes] you feel like your participation mattered.Most also expressed value in providing a lay summary years after their individual participation is over, acknowledging the long clinical trial timeline, and noted their expectation to receive individual-level results. A 37-year-old caregiver said:4Our findings provide new evidence suggesting that participants want to be thanked for their participation in research and value the acknowledgement. Our findings also contribute to existing evidence on the importance of lay research summaries by demonstrating the value and appreciation that participants place on receiving such information for pragmatic pediatric clinical research. Because of the anticipated participant response and ease of provision, we recommend that participant thank you notes become an expected study procedure in all clinical research similar to the provision of lay summaries.Participants\u2019 narratives also suggest that providing thank you notes and lay summaries may motivate future research participation. Research has shown that receiving a lay summary of research findings increases interest in participation in future research ; such daDr Corneli conceptualized and designed the study, designed the data collection instruments, drafted the initial manuscript, and reviewed and revised the manuscript.Dr. Zimmerman conceptualized and designed the study, and critically reviewed the manuscript for important intellectual content.Mr. Perry designed the study, coordinated and supervised data collection, collected data, carried out the analyses, and critically reviewed the manuscript for important intellectual content.Dr. Benjamin conceptualized the study, obtained funding, and critically reviewed the manuscript for important intellectual content.All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work.The authors have no conflicts to disclose.10.13039/100000071National Institute of Child Health and Human Development (NICHD) contract (HHSN275201000003I) for the Pediatric Trials Network (PI Danny Benjamin). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.This work was funded under the"}
+{"text": "This iatrogenic phenomenon, known little or not by many practitioners, responsible for significant dental and periodontal complications, both functional and aesthetic, is called \u201cWire Syndrome\u201d (WS). It is therefore considered an undesirable event of bonded orthodontic retainers, which must be differentiated from an orthodontic relapse. The objective was to perform, for the first time, a systematic review of the literature in order to define the prevalence of WS and to study its associated clinical characteristics. (2) Methods: A systematic review of the literature was performed following the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and recommendations using an electronic search strategy on four databases complemented by a manual search. All the prospective and retrospective clinical studies, including case reports and series, written in English or French, clearly mentioning the description, detection, or management of WS were included. Three independent blinding review authors were involved in study selection, data extraction, and bias assessment using the Mixed Methods Appraisal Tool (MMAT). (3) Results: Of 1891 results, 20 articles published between 2007 and 2021 fulfilled the inclusion criteria, with a globally high risk of bias since 16 articles were case report/series. The analysis of each article allowed the highlighting of WS through 13 categories, as follows: prevalence, apparition delay, patient characteristics, arch and tooth involved, families of movements, dental and periodontal consequences, type of wire, risk factors, etiologies, treatment, and preventive approach. (4) Conclusion: This systematic review of the literature elaborated a synthesis on WS, allowing general practitioners, periodontists, and orthodontists to understand this adverse event, to facilitate the diagnostic approach, and to underline preventive measures against WS. This review was registered in the International Prospective Register of Systematic Reviews . The long-term follow-up of orthodontic bonded retainers remains a challenge for orthodontists, but also for periodontists, as well as general practitioners. Whereas fixed retainer placement is a common procedure after orthodontic treatment, complications, which can be severe, can happen. Indeed, in 2007, Katsaros et al.  were theThe synthesis of clinical experience evoked by the authors cited above allows us to define and characterize Wire Syndrome (WS) as follows: Fixed orthodontic retainers can provoke aberrant, unexpected, unwanted, or unexplained tooth movement on teeth still bonded by a fixed retainer placed after orthodontic treatment, which could induce progressively iatrogenic dental and periodontal complications, functional and/or aesthetic, ranging from minor teeth displacement to teeth expulsion from the bone with loss of vitality. In the presence of severe WS, the retainer may become detached or fractured. WS is not a classic orthodontic relapse, and the position of the teeth does not correspond to any previous situation.However, neither general practitioners nor dental specialists, such as orthodontists and periodontists, are aware of the Wire Syndrome phenomenon. Concerning general practitioners, a lack of knowledge has been detected. A survey in eastern France showed that only 18.6% of general dentists were aware of the risks of adverse tooth movement associated with unintentionally active fixed retainers . HoweverTherefore, the aim of this study was to perform the first systematic review of the literature on Wire Syndrome (WS) in order to define the prevalence, to study its associated clinical characteristics, and, specifically, to facilitate the diagnostic approach of practitioners and to underline preventive and curative measures against WS.A systematic review of the literature (SRL) was performed, following as closely as possible the guidelines of Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and recommendations (reference). The protocol was registered in the International Prospective Register of Systematic Review (PROSPERO) (CRD42021269297).\u2013Participants (P): Patients with \u201cWire Syndrome\u201d (WS), i.e., dental movements described as aberrant, unexpected, unexplained, unwanted, or excessive;\u2013Interventions (I): Fixed orthodontic retainer bonded at the maxilla and/or mandible after orthodontic treatment;\u2013Comparisons (C): Patients not affected by \u201cWire Syndrome\u201d (only for studies including a control group);\u2013Outcomes (O): Define the prevalence of \u201cWire Syndrome\u201d and the characteristics associated with it.\u2013Study designs (S): All prospective and retrospective clinical studies, including case reports or series, written in English or French, clearly reporting the description, detection, or management of \u201cWire Syndrome\u201d or tooth movement described as aberrant, unexpected, unwanted, or unexplained in the presence of a bonded fixed retainer placed after orthodontic treatment were included, regardless of the length of the follow-up. In vitro studies, narrative reviews, author opinions, editorials, or commentaries were excluded.According to the question formulated using the \u201cPopulation, Intervention, Comparison, Outcome and Study Designs (PICOs) model\u201d: \u2756Electronic searchA search strategy, tailored to each database, combining keywords, Medical Subject Headings (MeSH) terms, and Boolean operators was performed without date restriction.The electronic search was conducted on four different databases on 1 September 2021 .\u2756Manual search\u2013From the bibliography of articles selected by the electronic search;\u2013\u25cbAmerican Journal of Orthodontics and Dentofacial Orthopedics;\u25cbEuropean Journal of Orthodontics;\u25cbJournal of Orthodontics;\u25cbJournal of Clinical Orthodontics;\u25cbOrthodontic & Craniofacial Research;\u25cbThe Angle Orthodontist;\u25cbRevue d\u2019Orthop\u00e9die Dento-Faciale;\u25cbL\u2019information Dentaire.From the search engine of a selection of orthodontic and dental journals:To complement the electronic searches, a manual search was conducted:https://www.zotero.org accessed on 1 September 2021). Two authors carried out the entire procedure independently. In the case of disagreement, a third author was interviewed, and a mutual discussion was conducted to reach a consensus.This research was conducted using the reference management software Zotero version 5.0.96.2 .Given the heterogeneity of the studies selected for this SRL, a tool that can evaluate the methodological quality of different types of studies within the same SRL was employed: The \u201cMixed Methods Appraisal Tool\u201d (MMAT)  .\u2756Single-stranded, round wire bonded on the canines only; diameter: 0.036 inch.\u2756Unknows strands with diameter: 0.0175 inch; 0.0215 inch; 0.0195 inch;Three strands with a diameter of 0.0155, 0.0195, or 0.0195 inches (heat treatment);Five strands with a diameter of 0.0215 inched (gold-plated);Six strands with a diameter of 0.0175 inches.Round, twisted, stainless steel wire:\u2756Six strands with a diameter of 0.018 inches.Round, coaxial, stainless steel wire:Most cases of WS are seen in the presence of round, twisted, stainless steel wires ,18,21,23Risk Factors: Predisposing factors of WS were investigated in three studies [\u2756p < 0.0001) [p = 0.029) [p = 0.049) [Patient-related factors: Different parameters were found to be significant in WS patients, such as lower facial level increase ( 0.0001) , vestibu= 0.029) , and pre= 0.049) . However= 0.049)  did not \u2756p = 0.03) [p < 0.01) [p = 0.270), inter-canine distance (p = 0.065), or change in incisor inclination (p = 0.151). Orthodontic treatment-related factors: Different parameters were found to be significant in WS patients, such as debonded at a young age ( = 0.03) , canine  = 0.03) , and abs < 0.01) . In cont < 0.01)  found no < 0.01)  showed n\u2756p \u2264 0.05) [p = 0.562) [p = 1.000) [Wire-related factors: No significant differences were found in patients with WS regarding debonded wire ( \u2264 0.05) , (p = 0.= 0.562)  and type= 1.000) . studies ,18,22:\u2756PEtiologies: Different etiological hypotheses were mentioned in the included studies, which can be grouped into three categories, as follows . After identifying 1891 articles, 20 articles were selected and analyzed, with a globally high risk of bias. Given the limited number of existing publications on WS and the relevant information found in the case reports and case series, these types of study designs were included. The description of WS is recent; the first publication on WS appeared in 2007 , followeRegarding the delay in apparition, WS appeared between 1 and 21 years after the placement of a retainer in the included studies. The majority of cases of WS appeared within the first five years after the placement of the retainer. This time interval should be considered with caution for several reasons. First of all, it is difficult to date the apparition of WS with precision, as retainer visits are intermittent. Moreover, it is difficult to detect early WS, and the patient often consults when complications are already severe . ConcernIn the presence of WS, the bonded retainer is intact in most cases but, in severe cases, the wire may become partially debonded or fractured due to important dental movement. In any situation, various and different clinical dental and periodontal signs can be found. The detection of one sign related to WS must immediately alert the practitioner to the possible presence of WS in order to stop the iatrogenic evolutive process and to start adapted therapy. Indeed, this syndrome is progressive and starts with minor dental and periodontal consequences until the loss of vitality and/or tooth expulsion. In addition, when treatment is not carried out ,21, clinWith regard to WS prevention, the most important preventive measure is the use of a bonded passive retainer. The use of a dental model to perfectly fit the retainer to the teeth before placement is recommended, as well as an indirect bonding protocol. Furthermore, special care should also be taken when the fixed retainer needs to be repaired. When a composite comes loose, small tooth movements may have already occurred and the wire is no longer a perfect fit. In this case, using an instrument to \u201cpush\u201d the wire to better fit the teeth results in an active wire that could potentially be responsible for subsequent WS. Therefore, a new passive wire should be bonded rather than repaired. Another strategy for preventing WS is double retention, which combines a fixed bonded retainer with a removable thermoplastic retainer ,5,8,18. Although there are explanatory hypotheses that could justify the risk factors mentioned by the authors, not all studies agreed. Indeed, WS seems to be due to a combination of different and multifactorial etiologies. In addition, it appears that the delay in apparition varies with etiology . Early WTM; CA Digital GmbH, Mettmann, Germany) versus round, 0.0175 inch (in), six-stranded, twisted, stainless steel wire retainers  and showed no significant difference between these two types of retainer after one year of placement. To reach a consensus on the preferred type of wire to use, randomized controlled studies must be conducted on a large sample size and over long observation periods.The type of wire seems to have an influence on the occurrence of WS. In the study of Padmos et al. , the mecFinally, some additional points should be made regarding this systematic review of the literature. First, only one study designed as a randomized controlled trial was included for analysis, and the risk of bias was considered high, as the majority of included studies were designed as case series/reports. Second, there was a lack of information in the included studies. For example, few data were provided on patient characteristics prior to orthodontic treatment , orthodontic biomechanics employed to treat the patient , bonding and retainer placement protocol, history of retainer failures , etc. Furthermore, the periodontal conditions, such as type of phenotype , tractioThis first review of the literature on Wire Syndrome (WS) included 20 articles published between 2007 and 2021, with a majority of case report/series leading to a globally high risk of bias. However, the analysis of the overall article provided an understanding of this adverse event associated with fixed orthodontic retainers, emphasized the importance of an early diagnosis, and highlighted preventive measures against WS for dental professionals worldwide, including general practitioners (GP), periodontists, and orthodontists. Indeed, the WS problem must involve all the dental health professions, including the general practitioners who will be able to refer, if necessary, the patient to a specialist practitioner; the continuity of the collaboration and the \u201cortho\u2013paro\u2013gp\u201d link will then be prolonged during the therapeutic time, thus guaranteeing optimal patient care. Further studies are needed to improve the knowledge about fixed orthodontic retainers, based on a large, well-documented sample and conducted over a very long observation period."}
+{"text": "The most common malignancy diagnosed in childhood is acute lymphoblastic leukemia (ALL). With significant advances in ALL treatment and excellent supportive care, overall survival is now up to 90%, depending on the risk group. As a result, the survivor population is growing significantly. At the same time, it is observed that survivors experience many health problems that substantially reduce their quality of life. Emerging evidence suggests that the increased susceptibility to multiple disorders related to accelerated aging in CCS may be attributed to chronic inflammation. Therefore, this study aimed to investigate whether asymptomatic survivors of ALL have a biological phenotype of accelerated cellular senescence early in life. For this purpose, a broad panel of 51 cytokines was tested. We show that survivors after ALL treatment have an inflammatory profile associated with premature cellular aging. Factors that increase the risk of immunological alterations include younger age at diagnosis, high-risk protocols, and radiation therapy.p < 0.05). Increased levels of pro-inflammatory cytokines, including the IL-1 family , IL-6 (p < 0.001), IL-17 (p < 0.001), IL-18 (p < 0.05), TNF\u03b1 (p < 0.01), IFN\u03b12 (p < 0.05), and IFN\u03b3 (p < 0.01), were found elevated in the entire study group, compared with the controls. Subjects treated previously according to the high-risk protocol had higher IL-18 levels than low- and intermediate-risk groups (p < 0.05). Elevated levels of IL-1ra, IL-6, IL-12 (p70), IL-17, LIF, M-CSF, CSF, and VEGF were found in ALL survivors treated before the age of 5, compared with subjects treated over 5 years of age (p < 0.05). Moreover, individuals who received radiotherapy presented elevated levels of both IL-18 (p < 0.05) and MIG (p < 0.05). In conclusion, we found that young asymptomatic survivors after ALL treatment demonstrated a biological profile of complex low-grade chronic inflammation.Childhood acute lymphoblastic leukemia (ALL) survivors are at higher risk of developing many late effects later in life. They experience multiple health problems that have significant public health implications, such as frailty, premature onset of lifestyle diseases, and second tumors. There is some evidence that chronic inflammation causes accelerated aging in childhood cancer survivors; however, the available data are very limited. The aim of the study was to evaluate the broad panel of cytokines among asymptomatic ALL survivors after anticancer treatment. The study included 56 subjects with a mean age of 16.11 \u00b1 3.98 years. The commercially available Bio-Plex Pro Human Cytokine Screening 48-Plex Panel Assay and Bio-Plex TGF-\u03b2 Assay were used for simultaneous determination of 48 cytokines and 3 isoforms of TGF-\u03b2. Among 51 tested cytokines, the levels of 33 were statistically significantly higher in ALL survivors than in the control group ( Acute lymphoblastic leukemia (ALL) is the most common malignancy in children. Currently, the overall long-term survival rate in children treated for ALL is approximately 80% due to advanced treatment and excellent supportive care ,2. Over One hypothesis holds that the differences in health status between childhood cancer survivors (CCS) and their equals result from premature cellular aging. Furthermore, emerging evidence suggests that the increased susceptibility to multiple disorders related to accelerated aging in CCS may be attributed to chronic inflammation . DespiteAnticancer therapy may lead to treatment-induced accelerated aging in several possible ways. They include cellular senescence, oxidative stress, telomere attrition, DNA damage, stem cell exhaustion, mitochondrial dysfunction, epigenetic alterations, etc. In general, cellular senescence is defined as the loss of the cells\u2019 ability to proliferate properly, resulting in an irreversible arrest of cell growth ,8. This In recent years, there has been an increase in the number of publications on the role of premature aging in patients with CCS; however, only few have examined the contribution of cytokines, and they have been limited to only few single proinflammatory cytokines  . MoreoveThe aim of this study was to investigate whether asymptomatic children and young adult survivors of acute lymphoblastic leukemia have a biological phenotype of accelerated cellular senescence early in life. For this purpose, a broad panel of 51 cytokines was tested.2). The waist-to-height ratio (WHR) was calculated by dividing waist circumference by height. Blood pressure was measured using a standardized sphygmomanometer. Hypertension (HT) was defined as a mean value of systolic blood pressure (SBP) and/or diastolic blood pressure (DBP) level\u2009\u2265 95th percentile adjusted for age, sex, and height. Echocardiography was performed to assess the heart\u2019s structures and fraction shortening and ejection fraction by a pediatric cardiology specialist. The study was conducted in accordance with the Declaration of Helsinki and was approved by the local ethics committee (Permission Number: R-I-002/328/2019).Fifty-six White survivors of acute lymphoblastic leukemia, without clinical features of any comorbid disease, were recruited for the study. They gave written informed consent prior to the enrollment. The characteristics of the survivors\u2019 cohort are presented in The commercially available Bio-Plex Pro Human Cytokine Screening 48-Plex Panel Assay  and Bio-Plex TGF-\u03b2 Assay  allowed the simultaneous determination of 48 cytokines, as well as 3 isoforms of TGF-\u03b2  in each well of a 96-well plate, respectively. The Bio-Plex assays were performed on serum specimens according to the manufacturers\u2019 instructions. The assay principle was based on the reaction with an antibody against a specific protein, which was covalently bound to fluorescently dyed magnetic beads, each with a distinct wavelength unique to the target protein. The bead-conjugated antibodies react with the sample containing the protein of interest. After the series of washes, biotinylated antibodies optional for different epitopes of the target proteins were added to the reaction. The final complex was made by adding a streptavidin\u2013phycoerythrin (SA-PE) conjugate. A dual-laser, flow-based microplate reader\u2014Bio-Plex 200 Reader \u2014detected the internal fluorescence of the individually dyed beads and the intensity of the signal on their surface. The obtained signal was expressed as median fluorescence intensity (MFI), analyzed, and presented as concentration (pg/mL) using the Bio-Plex Manager Software . The concentration of the analyte attached to the individual beads was proportional to the MFI of the phycoerythrin signal.2 test were used. For independent variables, the Mann\u2013Whitney U test or Student\u2019s t-test were used. We compared risk groups  using univariate analysis of variance (ANOVA), and Tukey\u2019s test was performed for post hoc analysis. Correlations were calculated by Spearman\u2019s test. A statistical significance was determined at 0.05.The statistical analysis was performed using GraphPad Prism version 9.3.1 . All values are expressed as a mean \u00b1 standard deviation (SD) or median (Me) and interquartile range (IQR) when appropriate. In the univariate analysis, Fisher\u2019s exact test and \u03c7p = 0.6) and median age of the study group did not differ from the control group\u201416.36 (13.32\u201319.27) years vs. 16.12 (14.16\u201317.65) years, p = 0.915.The characteristics of the study group are shown in p < 0.05). A comparison of the cytokine profile in the study and reference groups is presented in Among 51 tested cytokines, the levels of 33 were statistically significantly higher in ALL survivors than in the control group (p < 0.0001), IL-6 (p < 0.001), IL-17 (p < 0.001), IL-18 (p < 0.05), TNF\u03b1 (p < 0.01), IFN\u03b12 (p < 0.05), and IFN\u03b3 (p < 0.01)\u2014were found elevated in the entire study group [Increased levels of proinflammatory cytokines\u2014namely, the IL-1 family , as shown in To determine the effect of treatment intensity on cytokine levels, survivors were divided into three groups according to the treatment protocols used in childhood. As a result, subjects treated previously according to the high-risk protocol had only higher IL-18 levels, compared with low- and intermediate-risk groups (p < 0.05) , IL-17, LIF, M-CSF, CSF, and VEGF were found in ALL survivors treated before the age of 5, compared with subjects treated over 5 years of age ( < 0.05) .The potential impacts of some cytostatic agents  on serum cytokines and chemokines levels were further investigated; however, no effect was found.p = 0.037) and MIG (p = 0.012), as shown in One of the most harmful types of anticancer treatment, which is currently only targeted at carefully selected patients, is radiation therapy. In our cohort study, individuals who received radiotherapy had elevated levels of IL-18  such as Th1 , Th2 , and Th17 (IL-17A) in 87 asymptomatic ALL survivors. They showed a significantly higher concentration of IL-2, IL-10, and IL-17a, compared with controls . Our stuIn contrast, in a study by Sulicka-Grodzicka et al. of 50 young adults with CCS, higher levels of IL-6 were not confirmed. It is noteworthy that in addition to higher levels of C-reactive protein and fibrinogen, they found a shift toward memory and activated T cells, higher activation of cluster differentiation marker (CD) 38, and a decrease in naive T lymphocytes . MoreoveInterleukin 18 (IL-18) is a member of the interleukin 1 (IL-1) family expressed by a range of inflammatory cell types. In our study, a higher level of IL-18 was found in high-risk patients than in those who were treated according to intermediate- or low-risk protocols. In addition, its higher levels were found in the subset of patients who were treated with radiation therapy compared with those in whom it was not used. IL-18, together with IL-12, induces high levels of IFN\u03b3 production by T cells, and its expression correlates with disease activities of rheumatoid arthritis and Crohn\u2019s disease. Furthermore, in animal models, IL-18-deficient mice were more susceptible to bacterial infections than normal ones and showed uncontrolled disease progression. The assembly of the inflammasome in cells results in caspase-1 activation, followed by proteolysis and release of the cytokines IL-1b and IL-18 to induce pyroptotic cell death . All theFurther stratification of patients in this study was based on age at diagnosis. The rationale for this approach was the hypothesis that children treated at a younger age are more likely to develop chronic inflammation due to the immaturity of many organs and systems. As expected, high levels of several cytokines and growth factors among ALL survivors treated before the age of 5 were found. They included IL-1 receptor antagonist (IL-1Ra), IL-6, IL-12(p70), IL-17, leukemia inhibitor factor (LIF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), and vascular endothelial factor (VEGF).Unlike most cytokines of the IL-1 family, IL-1Ra has antagonistic effects on IL-1 \u03b1 and \u03b2, which have exquisite proinflammatory potency. It binds competitively to the same receptor as IL-1 but does not transmit a cellular signal, thus inhibiting IL-1-induced cellular changes . ContrarInterestingly, IL-12(p70) expression was also higher in a group of younger children at the time of diagnosis. Their bioactive form IL-12(p70) is produced mainly by macrophages, monocytes, neutrophils, and dendritic cells, and it primarily mediates Th1 cell differentiation and maintenance. Moreover, it indirectly exerts an anti-infective function by activating the cytolytic activity of natural killer (NK) cells and Th1 cells . On the Leukemia inhibitory factor (LIF) is a member of the IL-6 family and is expressed in almost all types of body tissues. LIF was first discovered as a cytokine that induces blast differentiation in myeloid leukemia; however, studies of LIF in other diseases, including cancer, indicate that it may potentially contribute to many other pathologies. The role of LIF appears to vary greatly depending on the tissue in which it is expressed. LIF is involved in tumor spread and metastasis, treatment resistance, and cachexia, and has also cancer biomarker potential . Since tMacrophages play a crucial role in inflammation and tissue regeneration, and their differentiation is mainly regulated by M-CSF derived from different tissues . ElevateIncreased levels of anti-inflammatory cytokines such as IL-4, IL-10, and IL-13 were also found in this study. Interleukin 4 is a major stimulant of Th2 cells and induces IgE production by B cells. Hence, it is involved in the development of allergy and is considered an important mediator of allergic inflammation . In turnMoreover, it is important to note that ALL survivors showed elevated levels of regulated on activation, normal T-cell expressed and secreted (RANTES), which is a member of the CC chemokine subfamily and plays a chemotactic role for monocytes, T cells, and eosinophils. It may have deleterious effects by recruiting immune cells that enhance inflammatory diseases such as arteriosclerosis, arthritis, nephritis, dermatitis, colitis, and many other disorders . There iAside from premature death, the most serious late effects of cancer treatment in children are second malignancies occurring later in life. The link between SASP factors and tumorigenesis has remained one of the most engaging scientific issues for years. There is ample evidence to support this linkage. It is emphasized that SASP promotes tumor cell proliferation, dissemination, angiogenesis, and invasiveness ,35,36. AINK4a and alternate reading frame protein product of the CDKN2A locus (ARF) [In addition to cellular senescence induced by SASP factors, many potential mechanisms lead to premature aging that should be noted. These include DNA damage, stem-cell depletion, mitochondrial dysfunction, oxidative stress, telomere attrition, and epigenetic changes. Much effort is currently being made in identifying a group of measurements of accelerated aging in cancer survivors. Researchers implemented the concept of biological age to estimate accelerated aging in children with cancer to better predict the aging process than chronological age . The risus (ARF) ,39,40.Finally, there are a few limitations to note. Firstly, single-center research should be interpreted in the context of the potential bias that may arise. Secondly, this pilot study was conducted on a relatively small group of individuals, which may affect the results. Finally, only cytokine levels as part of SASP were investigated, and no other parameters related to premature aging were analyzed. However, it is worth emphasizing that cellular senescence is a very complex process, and it is not possible to account for all factors and their interrelationships in a study performed on a small cohort of patients in one minor center.Strengths of the study include a homogenous group of acute lymphoblastic leukemia survivors, a relatively long follow-up time, and no ethnic diversity. To the best of our knowledge, this is the first study to address such a broad panel of inflammatory biomarkers among childhood acute lymphoblastic leukemia survivors.In conclusion, we demonstrated that ALL survivors present a biological profile of premature aging, manifested by increased secretion of multiple SASP biomarkers in the form of chronic inflammation. In addition, children treated for cancer at a younger age had higher levels of selected cytokines and growth factors than children treated at an older age, which may suggest a greater susceptibility of their healthy tissues to anticancer treatment administered in childhood. Furthermore, it is not yet clear how activation of cellular senescence mechanisms affects the development of different diseases. Additionally, in light of available studies indicating high morbidity among cancer survivors from multiple diseases, it seems reasonable to determine the exact pathogenesis of this condition. Thus, further longitudinal studies considering the contribution of a wide range of factors are needed.In conclusion, we demonstrated that ALL survivors present a biological profile of premature aging, manifested by increased secretion of multiple SASP biomarkers in the form of chronic inflammation. In addition, children treated for cancer at a younger age had higher levels of selected cytokines and growth factors than children treated at an older age, which may suggest a greater susceptibility of their healthy tissues to anticancer treatment administered in childhood. Furthermore, it is not yet clear how activation of cellular senescence mechanisms affects the development of different diseases. Additionally, in light of available studies indicating high morbidity among cancer survivors from multiple diseases, it seems reasonable to determine the exact pathogenesis of this condition. Thus, further longitudinal studies considering the contribution of a wide range of factors are needed."}
+{"text": "Impaired response to COVID-19 vaccination is of particular concern in immunosuppressed patients. To determine the best vaccination strategy for this vulnerable group we performed a single center, 1:1 randomized blinded clinical trial. Patients who failed to seroconvert upon two mRNA vaccinations (BNT162b2 or mRNA-1273) are randomized to receive either a third dose of the same mRNA or the vector vaccine ChAdOx1 nCoV-19. Primary endpoint is the difference in SARS-CoV-2 spike antibody seroconversion rate between vector and mRNA vaccinated patients four weeks after the third dose. Secondary outcomes include cellular immune responses. Seroconversion rates at week four are significantly higher in the mRNA  as compared to the vector vaccine group . SARS-CoV-2-specific T-cell responses are reduced but could be increased after a third dose of either vector or mRNA vaccine. In a multivariable logistic regression analysis, patient age and vaccine type are associated with seroconversion. No serious adverse event is attributed to COVID-19 booster vaccination. Efficacy and safety data underline the importance of a booster vaccination and support the use of a homologous mRNA booster vaccination in immunosuppressed patients.Trial registration: EudraCT No.: 2021-002693-10. Optimizing COVID-19 vaccination strategies for patients under immunosuppressive medication is of high importance. In this clinical trial including non-seroconverted immunosuppressed patients, a homologous mRNA booster vaccination resulted in higher seroconversion rate than a switch to a vector-based vaccine. Various types of vaccines have been approved by the European Medicines Agency (EMA), including vector vaccines, such as ChAdOx1 nCoV-19 (Oxford-AstraZeneca) or Ad26.COV2-S (Johnson&Johnson) and mRNA vaccines, such as BNT162b2 (Pfizer\u2013BioNTech) or mRNA-1273 (Moderna)7. Most recently, NVX-CoV2373 (Novavax) and VLA2001  have been approved by the EMA as a protein subunit vaccine and an inactivated whole-virus COVID-19 vaccine, respectively9. Multiple elements of the innate and adaptive immune system contribute to the vaccination response10. One way to assess the humoral immune response to different vaccines is to measure anti-SARS-CoV-2 antibodies against the receptor-binding domain (RBD). Immunocompromised individuals are less likely to mount an adequate immune response after primary vaccination. A significant number of patients do not seroconvert upon vaccination15, leaving them more susceptible to COVID-19 infections and subsequent severe disease courses16. Low antibody response rates have been observed in patients with immune-mediated inflammatory diseases, haemato-oncological malignancies, those following solid-organ transplantation, and patients undergoing hemodialysis21. Several studies have reported on the efficacy and safety of an additional booster vaccination in immunosuppressed patients. These mainly consist of the administration of a third mRNA vaccine in a homologous vaccination strategy26. Evolving evidence, however, suggests that a heterologous vaccination strategy might be more efficient in nonimmunocompromised healthy volunteers28. However, data on immunogenicity and safety of homologous versus heterologous booster vaccination strategy in patients who did not seroconvert are currently limited33. We, therefore, performed a blinded randomized controlled trial to address immunogenicity and safety of the third dose in non-seroconverted immunosuppressed patients, comparing mRNA and vector vaccines.The COVID-19 pandemic poses an unprecedented challenge to public health, and several mitigation strategies exist to combat this worldwide threat. Among such strategies, COVID-19 vaccination protects against a severe disease course and leads to accelerated viral clearanceSeventy-five patients under immunosuppressive therapy who had been immunized with two doses of an mRNA vaccine were screened for eligibility. Twenty-four patients were excluded due to the presence of detectable SARS-CoV-2-specific antibodies. Fifty-one non-seroconverted patients were randomized, of whom 25 were assigned to receive a vector and 26 to receive an mRNA vaccine as the third dose; five patients withdrew consent between the screening and the baseline visit Fig.\u00a0. Thus, ap\u2009=\u20090.006)  as compared to the mRNA group  Fig.\u00a0. Post ho8% as com+ peripheral B-cells and SARS-CoV-2 spike-specific memory B-cells were analyzed by flow cytometry. Patients without detectable CD19+ peripheral B-cells did not develop anti-RBD antibodies in either group  served as prepandemic control. Characteristics of HC and prepandemic patients can be found in Supplementary Tables\u00a0p\u2009=\u20090.0026) or the mRNA vaccine (p\u2009=\u20090.0396). No difference in SFCs/106 PBMCs was observed between patients who received the third vaccination with either an mRNA- or a vector-based vaccine , who were vaccinated three times with BNT162b2 and developed a humoral immune response, served as a positive control. Material from patients under immunosuppressive therapy collected before the COVID-19 outbreak  occurred during the four-week follow-up. The prevalence of systemic reactogenicity was similar in the vector and mRNA booster vaccine groups. 7/22 (32%) of vector-vaccinated patients developed arthralgia compared to 4/24 (17%) of patients with mRNA booster vaccination. Myalgia was reported in 7/22 (32%) vector-vaccinated patients compared to 9/24 (38%) mRNA-vaccinated patients. Fatigue was present in 11/22 (50%) vectors and in 10/24 (42%) mRNA-vaccinated patients. Local pain at the injection site was more frequent in mRNA  than in vector-vaccinated patients . Headache was reported in 8/24 (33%) of the mRNA-vaccinated patients compared to 9/22 (41%) of the vector-vaccinated patients.Duration of side effects was prolonged in the mRNA as compared to the vector-vaccinated group, especially for local reaction (mRNA: 1.46 versus vector 0.46 days per patient), myalgia , and headache  Fig.\u00a0.Fig. 5San\u2009=\u200936). None of the patients developed de novo anti-HLA antibodies.No thrombocytopenia or antibodies against platelet factor 4 (PF4) were observed after additional booster vaccination. None of the patients experienced an anaphylactoid reaction. In patients with previous organ transplantation, no acute transplant rejection was observed. Anti-HLA antibodies of patients with solid-organ transplantation were analyzed 12 weeks after the third vaccination with either a vector or an mRNA-based vaccine  than in the vector-vaccinated (heterologous) group. Overall, an additional COVID-19 vaccination resulted in humoral immune response in 41% of this initially vaccination-refractory patient population. Although the SARS-CoV-2-specific T-cell response was reduced in immunosuppressed patients as compared to HC, the cellular response could be increased after mRNA- and vector-based booster vaccination.36. While antibody levels have been determined at multiple time points during clinical routine before booster vaccination, it cannot be ruled out that individual patients had developed transient antibodies prior to study inclusion. Although case series and clinical trials have previously addressed the immunogenicity of a third vaccination in immunosuppressed patients, data comparing homologous versus heterologous vaccination strategies are currently limited33. A significant advantage was observed for the homologous booster dose in our study: the primary outcome showed a 44% higher seroconversion rate for mRNA (homologous) versus vector (heterologous) vaccination. In line with these data, anti-RBD antibody levels were significantly higher in patients who received an mRNA-based vaccination. To assess the stability of the humoral immune response, patients were invited to participate in an open-label extension study. Anti-RBD antibody levels were sustained over a period of 12 weeks, indicating a stable humoral immune response in immunosuppressed patients over at least 3 months.Patients without detectable anti-RBD antibody levels were included in our study, reflecting individuals at exceptionally high risk for severe COVID-19 disease courses. Although none of the patients seroconverted after primary vaccination, the heterogeneity of the patient cohort has to be considered since it has been reported that underlying disease-specific medication can contribute to vaccination response38. In the current trial, we excluded patients under rituximab treatment to ensure that absence of peripheral B-cells was not the main factor for an insufficient humoral immune response to COVID-19 vaccination. In addition, patients were stratified by the presence or absence of peripheral B-cells. Overall, the numbers of CD19+ peripheral B-cells did not correlate with anti-RBD antibody levels. However, when we analyzed SARS-CoV-2 spike-specific memory B-cells we observed a significant correlation with anti-RBD antibody levels, suggesting that the presence of anti-SARS-CoV-2-specific memory B-cells can be interpreted as predictive of antibody production40. ELISpot assays were performed using PBMCs to determine the in vivo effect of immunosuppressants on the development of circulating spike-specific T-cells. In line with previously published data on cellular responses in patients under immunosuppressive therapy42, we observed reduced T-cell responses in our patient cohort as compared to HC, suggesting a significant contribution of impaired T-cell function to the phenomenon of non-seroconversion after primary vaccination. A significant increase in SARS-CoV-2-specific T-cells was observed for both, mRNA- and vector-based booster vaccination, which was still diminished as compared to HC. In our study, T-cell responses to spike peptide pools from Wuhan and Omicron variants showed largely comparable results. This is in line with recent data reporting that the cellular response to SARS-CoV-2 variants is preserved in most infected and vaccinated individuals47. As T-cell responses to SARS-CoV-2 spike peptides could be influenced by pre-existing cross-reactive immunity against other human corona viruses49, we analyzed responses to the more variable S1 part containing the RBD and more conserved membrane-proximal S2 moiety of the spike protein separately. The results obtained revealed some variability between S1 and S2 responses in different groups, these, however, were not statistically significant. Although our data suggest that immunosuppressive therapy leads to impaired cellular immune responses, a bigger patient cohort would be needed to define the exact role of immunosuppressants. In addition, future in vitro assays should be performed to address the specific effects of different immunosuppressants on T-cell reactivation. Furthermore, we did not observe a significant correlation between SARS-CoV-2-specific T-cells and anti-RBD antibody levels, indicating more diverse roles of immunosuppressants in antibody production.In patients under B-cell-depleting therapy, such as rituximab, we and others have previously reported that seroconversion was impaired but a cellular immune response was preserved, supporting the importance of peripheral B-cells for seroconversion50. Common side effects  were observed, that were within the range reported in the approval studies6. While a longer duration of adverse events in patients boosted with a homologous mRNA vaccine than with the heterologous vector vaccine was observed in our patient cohort, this still lasted on average only 1.5 days.One of the biggest concerns of an additional booster vaccination in immunosuppressed patients relates to the risk of adverse reactions, which were documented by a patient diary within our study. No serious adverse event was recorded which could be related to booster vaccination. Reactogenicity was similar upon third vaccination as reported previously in heterologous vaccination regimens 51. To address this issue, we determined anti-HLA antibodies in solid-organ transplantation patients vaccinated either with vector or mRNA vaccine. In line with the most recent assessment report from the EMA and previous research53, no newly developed anti-HLA antibodies were detected 12 weeks post-vaccination.Concerns have been raised that cell culture-derived vector vaccines can induce HLA-sensitization in transplant candidates and recipients, which may negate future transplantations or activate low-level chronic anti-graft responsesOne limitation of the current study is the lack of placebo control, which was considered unethical. Despite early termination due to recruitment limitations, we were still able to demonstrate the statistical significance and benefit of homologous as compared to heterologous vaccination in immunosuppressed patients, which implies a robust difference between the two currently utilized strategies. In the current study, a heterogeneous population of immunosuppressed patients was included. Therefore, additional studies with larger patient cohorts will be needed to understand the effect of disease entity and immunosuppression on seroconversion and whether the enhanced humoral and cellular immune response in initially non-seroconverted patients also translates into clinical protection. Our data show that a humoral immune response can be mounted even in non-seroconverted patients who did not respond to primary vaccination and support a homologous vaccination strategy in immunosuppressed patients, also considering the acceptable safety profile.In this prospective blinded randomized controlled trial, adults (age\u2009\u2265\u200918 years) under immunosuppressive treatment without measurable SARS-CoV-2 spike protein-specific antibodies at least 4 weeks after their second COVID-19 vaccination were included. All patients had previously received two doses of an mRNA vaccine (BNT162b2 or mRNA-1273). Major exclusion criteria were known allergies to vaccines, previous infection with SARS-CoV-2, detectable anti-spike antibodies at the time of inclusion, or prior use of B-cell-depleting agents, such as rituximab. Healthy controls (HC) without immunosuppressive therapy as well as prepandemic immunosuppressed patients were recruited from the Vienna General Hospital and served as controls for the determination of SARS-CoV-2-specific T-cell responses. The detailed trial protocol can be found in the Supplementary Information. The trial was registered in the European Clinical Trials Database (EudraCT No.: 2021-002693-10) on the July 15, 2021.14, therefore patients were block-randomized in a 1:1 ratio based on the presence or absence of peripheral B-cells using a computerized algorithm (Randomizer). Patients were assigned to receive either a third dose of an mRNA vaccine (BNT162b2 or mRNA-1273 containing 100\u2009\u03bcg mRNA), corresponding to their primary vaccination compound) or a vector-based COVID-19 vaccine (ChAdOx1 nCoV-19).The presence of detectable B-cells is critical for seroconversion29 and is outlined in more detail in the Supplementary Information. In brief, four trial visits were performed. In addition, an optional open-label fifth visit was offered to the patients 12 weeks after vaccination. At screening, the eligibility of the patients was verified. An additional booster dose was applied at visit two (baseline), the cellular immune response was assessed at week one, the humoral immune response at week 4, and optionally at the extension visit at week 12 after the third vaccination. Safety was evaluated by a patient diary for the first week, then inquired at week 4.The study was designed as described previouslyIn this trial, laboratory assessors and patients were blinded to the type of vaccine used. This was done to ensure an objective assessment of vaccine reactogenicity by the patients. Blinding of vaccines was ensured by the Central Pharmacy of the Vienna General Hospital, where dose aliquots were prearranged in syringes without reference to the vaccine type used. The study procedures followed Good Clinical Practice guidelines and the Declaration of Helsinki. The trial protocol was approved by competent authorities and the ethics committee of the Medical University of Vienna (No.: 1583/2021). At inclusion, all patients and healthy controls provided their written informed consent. All trial visits were conducted monocentric in a tertiary hospital . The trial started on July 22, 2021, with the inclusion of the first patient. The last patient finalized the 4-week follow-up on October 8, 2021.Laboratory tests, including quantification of peripheral leukocytes, anti-SARS-CoV-2 antibody testing, determination of SARS-CoV-2-specific T-and B-cell responses, and anti-HLA antibodies are described in more detail in the Supplementary Information.The primary outcome of the study was the difference in antibody seroconversion rates between the two intervention groups (vector versus mRNA vaccine). According to the manufacturer\u2019s specification, seroconversion was defined as an anti-RBD antibody concentration of over 0.8 BAU/ml.Secondary endpoints included overall seroconversion rate and SARS-CoV-2 antibody levels at week 4 and cellular immune response before and 1 week following vaccination. Assessment of safety included incidence and severity of adverse events over 28 days. A paper-based patient diary was used to evaluate reactogenicity. The study sample size was targeted at 150 individuals, thus based on a Chi-squared test comparing the two groups, the minimal detectable difference was 21% at a power of 80%. During recruitment, routine access to the additional vaccination dose, as offered in our trial, was facilitated for high-risk patients in Austria, which substantially slowed our inclusion rates. To allow timely study completion, the decision was taken to stop inclusion prematurely at 75 enrolled patients, which would provide power to detect an effect size of at least 32% for our patient cohort. The observed effect size was ultimately larger, which eliminates considerations about a potential type II error, i.e., failure to reject the null hypothesis although it is actually false.\u03c4). For graphical presentation, in figures with a log-scale, not-detectable anti-RBD antibodies and cellular responses have been set to 0.1. For all analyses conducted, two-sided tests were used. Data collection was facilitated by Microsoft Excel 365. GraphPad Prism (version 9.1.0) and \u201cR\u201d version 4.0.3  were used for graphical presentation and statistical analysis. Following packages were utilized: \u201cggplot2\u201d, \u201cggbeeswarm\u201d, \u201ccorrplot\u201d, and \u201csjPlot\u201d for creating plots, \u201cpwr\u201d for power calculation, and \u201ctableone\u201d to create baseline tables.The analysis included all patients vaccinated with an additional dose. Differences in seroconversion rates were statistically compared by utilizing a Chi-squared test. Post hoc analyses, including a comparison of antibody levels, type of mRNA vaccine, antibody stability, as well cellular immunity, were performed by Kruskal\u2013Wallis- or Mann\u2013Whitney U test in unpaired groups or paired Wilcoxon test in paired groups. Factors associated with seroconversion rates were assessed by multivariable logistic regression analysis. Variables that might influence seroconversion were included in the model . Univariate associations between continuous variables were described via Kendall rank correlation coefficient (Further information on research design is available in the\u00a0Supplementary InformationReporting Summary"}
+{"text": "COVID-19 is associated with an increased mortality in hemodialysis patients. Therefore, achieving a long-lasting effective immune response to SARS-CoV-2 vaccines is essential. This study describes the humoral immune response in hemodialysis patients following three doses of mRNA vaccines against SARS-CoV-2, and explores the factors associated with a sustained immune response.We analyzed the monthly serological evolution of SARS-CoV-2 anti-S(RBD) antibodies for 1 year in 178 chronic hemodialysis patients who received three doses of SARS-CoV-2 mRNA vaccines. The primary outcome was sustained effective humoral response defined as anti-S(RBD) levels > 1,000 AU/ml after 4 months from the third dose. Multivariate logistic regression analyses were used to identify features associated with a sustained humoral immune response.After the initial two SARS-CoV-2 mRNA vaccine doses, 77.8% of patients showed an immediate effective humoral response, decreasing to 52.5% after 4 months. Antibody levels were significantly higher in COVID-exposed patients and HBV vaccine responders. After the third dose, 97% of patients showed an effective humoral response, and remained in 91.7% after 4 months. The mean monthly rate of antibody titer decline decreased from 33 \u00b1 14.5 to 25 \u00b1 16.7%. Multivariate regression analysis showed that previous exposure to COVID-19 and response to HBV vaccines were associated with an effective sustained humoral immune response.Immunization with SARS-CoV-2 mRNA vaccines elicits an effective immediate humoral immune response in hemodialysis patients, with a progressive waning in antibody levels. A third booster dose enhances the immune response with significantly higher antibody levels and more sustained humoral immune response. COVID-na\u00efve patients and patients without previous response to HBV vaccines are likely to benefit from receiving more booster doses to maintain an effective immune response. Patients on hemodialysis (HD) have an increased incidence of infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) compared to the general population, and when infected their mortality risk is higher \u20133. For tThere are no specific recommendations concerning the most adequate vaccine regimen for HD patients. Around 90% of HD patients without previous infection with SARS-CoV-2 develop an immediate humoral and cellular immune response after the administration of two doses of mRNA vaccines , 11. DesThere are no longitudinal studies concerning SARS-CoV-2 vaccine-induced immune responses for more than 4 weeks after receiving the third mRNA vaccine dose in patients on HD, which are needed to optimize clinical care and plan preventive strategies in this population.In this study, we describe the serological monthly changes against SARS-CoV-2 in HD patients after 1 year of follow-up from an initial immunization with two doses of mRNA vaccines. A third booster dose was administered after 5 months from the second dose, with continuous monthly serological assessment for 4 months following the third vaccine dose. We aimed to describe the factors associated with a sustained humoral response and identify the group of patients who are more likely to benefit from receiving a third booster dose.We designed an observational, longitudinal study to evaluate the immune response induced by the different mRNA vaccines against SARS-CoV-2 in patients attending two HD units: an in-center hospital dialysis unit and its affiliated satellite dialysis unit. The study comprised all HD patients who received vaccination against SARS-CoV-2 with a two-dose mRNA vaccine between December 28th 2020 and June 30th 2021, either BNT162b2 (BionTech/Pfizer) or mRNA-1273 (Moderna), given according to the manufacturer\u2019s recommendations. The type of vaccine was assigned according to the available vaccines at the local vaccination point or at our center.Patients were classified as having had a prior COVID-19 infection if there was clear medical documentation with a positive SARS-CoV-2 PCR swab or in presence of nucleocapsid-IgG specific antibodies in serological analyses before the administration of the first dose of the vaccine. Patients who had received the vaccination schedule before starting HD, patients with an unknown serological status for SARS-CoV-2 before vaccination and patients who either received an adenoviral vector-based vaccine or did not complete the full SARS-CoV-2 mRNA vaccine schedule were excluded from the study.The study was conducted according to the guidelines of the Declaration of Helsinki and received the approval of the Ethical Committee for Clinical Investigation of the Hospital Universitario Fundaci\u00f3n Alcorc\u00f3n. All patients signed an informed consent form and approved the use of their anonymized clinical information for medical research purposes.Patients received two initial doses of either mRNA vaccine (Pfizer or Moderna) separated by 4 weeks, followed by a third booster dose after 5 months. Serum samples for SARS-CoV-2 anti-spike 1 receptor binding domain [anti-S(RBD)] IgG antibody titers were obtained 2 months prior to the administration of the first dose of the vaccine as well as immediately before vaccination, immediately after the administration of the first and the second dose of vaccine, and thence in a monthly basis up to 4 months after the third booster dose, which would correspond to 10 months after the administration of the first dose . This seWe examined associations between anti-S(RBD) IgG antibodies and potential predictors such as demographic and clinical data, type of dialysis and type of the vaccine, previous exposure to COVID-19, history of effective humoral response to hepatitis B virus (HBV) vaccines, and treatment with immunosuppressants. Medical histories of study participants were extracted from their medical records.Antibody response was determined using the chemiluminescent microparticle immunoassay of IgG antibodies to SARS-CoV-2 in human serum  which provides quantitative measures of IgG antibodies specific for SARS-CoV-2 S1-RBD. Results are given as arbitrary units per milliliter (AU/ml), and were interpreted as positive according to the manufacturer\u2019s instructions, with a cutoff index value of > 50 AU/ml. The upper limit of quantification was 40,000 AU/ml. The humoral response was considered as effective when the value of SARS-CoV-2 anti-S(RBD) antibodies was > 1,000 AU/ml, which strongly correlates with a sustained neutralizing antibody response of 1/80 measured by plaque reduction neutralization titer , 19. TheDialysis vintage was defined as the time between the first HD session and the administration of the first dose of mRNA vaccine. Response to HBV vaccine was defined as an anti-HBs antibody titer > 10 IU/ml after four doses of Engerix-B or Fendrix. Immediate humoral response to SARS-CoV-2 vaccines was defined as an anti-S(RBD) antibody titer > 50 AU/ml 4 weeks after the second dose of the vaccine, and the same cutoff was used in every monthly serological assay. Effective humoral response was defined as an anti-S(RBD) antibody titer > 1,000 AU/ml, and sustained humoral response was defined as a persistent antibody titer > 1,000 AU/ml for more than 4 months.t-test, if normally distributed, or by the Mann-Whitney test if abnormally distributed. A Chi-square test was used for categorical variables. Multivariable logistic stepwise regression was used to determine the independent factors associated with an effective humoral response  4 months after the first two mRNA doses and 4 months after the third dose. The covariates of interest were selected based on the associations with serological response in univariate analyses, and any variables that were at the significance level p less than 0.10 in univariate analyses were included in these models. P-value of < 0.05 was considered statistically significant. Statistical analysis was performed with IBM SPSS Statistics for Windows, Version 22.0 .Demographic and clinical information are described for all patients included in the study. Data were presented as a number (percentage) for categorical variables, as mean \u00b1 standard deviation for continuous variables that were normally distributed, and as median  for continuous variables that were non-parametric. Continuous variables were first tested for normal distribution using Shapiro-Wilk, and then compared by One hundred and ninety-six patients on maintenance HD were screened for this study, 18 patients did not meet the inclusion criteria and were therefore excluded. Ultimately, 178 patients were eligible and were included in the study. p < 0.001). After receiving the third mRNA vaccine dose, the proportion of patients who seroconverted increased to 98.3%, and this rate remained stable for the following 4 months. Median antibody levels rose up to 26,037 AU/ml  and decreased to 9,180 AU/ml  after 4 months from the booster dose (p < 0.001).The evolution of IgG anti-S(RBD) antibody levels in the whole cohort throughout the study period is represented in p < 0.001].The proportion of patients with IgG anti-S(RBD) antibody levels over 1,000 AU/ml changed in a similar manner, from 77.8% of patients after receiving the first two doses, to 52.5% of patients 4 months after the second dose, rising up to 97% after receiving the third booster dose, and remaining in 91.7% of patients after 4 months from the third dose . Overalln = 33) and those who were COVID-19 na\u00efve at the time of vaccination (n = 145) (p = 0.04), and lower proportion of patients with online haemodiafiltration  in the group of patients with prior COVID-19 infection. Pre-vaccination levels of anti-S(RBD) IgG antibodies were elevated in 78.8% of COVID-exposed patients, with a median antibody titer of 1,123 AU/ml, of which 51.5% had antibody levels higher than 1,000 AU/ml before vaccination. On the other hand, in the COVID-na\u00efve group only two patients (1.4%) had positive anti-S(RBD) antibody levels that were slightly elevated (56 and 63 AU/ml) with negative IgG nucleocapsid antibodies and no history of COVID-19 infection, and were thus considered COVID-na\u00efve.We stratified the study population according to previous SARS-CoV-2 exposure into patients with prior COVID-19 infection before vaccination (n = 145) . There wp < 0.001), and remained so at every monthly assay. Serological changes in patients with previous COVID-19 exposure and in COVID-na\u00efve patients is represented in p < 0.001).The proportion of patients who achieved seroconversion was similar in both groups . Howeverp = 0.002), and these differences remained for the following 4 months after the initial two-dose vaccine. Nonetheless, after the third SARS-CoV-2 vaccine dose, the proportion of patients who had positive anti-S(RBD) antibody levels was matched in both groups  had received a full HBV vaccination regimen, of which 84 patients (68.9%) had an effective humoral response. h groups , but theh groups .p < 0.001)  antibodies of 33 \u00b1 14.5%. This decrease was significantly steeper in patients who were COVID-na\u00efve (36.5 \u00b1 11% per month) compared to patients with previous exposure to COVID-19 (17.5 \u00b1 6%) (< 0.001) .p < 0.001). Although the rate of antibody level decline remained significantly lower in patients with previous exposure to COVID-19 , COVID-na\u00efve patients also showed a more sustained humoral response after the third dose compared to the initial vaccination, with a monthly decrease in the rate of antibody level decline from 36.5 \u00b1 11 to 27.8 \u00b1 16% (p < 0.001). Also, patients who were responders to HBV vaccines showed a more sustained response after the third dose, with a decrease in the rate of antibody decline from 33.5 \u00b1 12.3 per month to 24.7 \u00b1 16% per month (p < 0.001) (The mean monthly rate of antibody titer decline decreased to 25 \u00b1 16.7% after the third dose (< 0.001) .We analyzed factors that were associated with an effective humoral response after the initial two-dose mRNA vaccination. Patients with previous exposure to COVID-19 , previous humoral response to HBV vaccines , patients who were < 70 years old  and patients who received mRNA-1273  were associated with an effective sustained humoral response after 4 months from vaccination in the univariate analysis. Patients with history of neoplasms were less likely to maintain an effective humoral response . In the multivariable linear regression model, both previous exposure to COVID-19 infection and previous humoral response to HBV vaccines maintained statistical significance. Details are presented in In the univariate analysis to determine the factors associated with a sustained humoral response after 4 months from receiving the third dose of mRNA vaccine, we found an association with response to HBV vaccines , and a tendency for a less likely effective seroconversion in patients receiving immunosuppressants . After adjusting for age, dialysis vintage, immunosuppression, and type of vaccine administered, the humoral response to HBV vaccines remained as an independent factor associated with an effective sustained humoral response after 1 year from the initial vaccination. All patients with prior exposure to COVID-19 developed an effective humoral response, and therefore could not be included as a factor in the multivariate analysis .Throughout the year following SARS-CoV-2 vaccination, there were 23 vaccinated patients (12.9%) who were diagnosed with SARS-CoV-2 infection, of which 3 cases (1.7%) were after receiving the first two doses, and the remaining cases (11.2%) were diagnosed after receiving the third dose of vaccination. Twenty-one patients (91.3% of the infected patients) remained asymptomatic. There were only two cases (1.1%) of severe COVID-19 with acute respiratory failure requiring hospital admission and high flow oxygen therapy among vaccinated patients included in this study, both of which had an inadequate humoral response to the vaccine. The first patient was an 85-year-old male patient who became infected 3 months after receiving the first two mRNA vaccine doses, having reached a peak SARS-CoV-2 anti-S(RBD) antibody titer of 1,007 AU/ml in the first month after vaccination, and decreasing to 510 AU/ml in the second month, just before infection. The second patient was a 72-year-old female patient under immunosuppression with tocilizumab due to severe rheumatoid arthritis. She never responded to the initial two doses of mRNA vaccine nor to the booster dose (peak SARS-CoV-2 anti-S-RBD antibodies 1 AU/ml) and was infected 3 months after receiving the third dose. Neither patient had a history of COVID-19 infection before vaccination, and both patients died.In this serological study we examined the monthly changes in SARS-CoV-2 anti-S(RBD) IgG levels for 1 year after vaccination. We observed that patients who were COVID-na\u00efve and those who had not previously responded to HBV vaccines benefited the most from a third mRNA vaccine dose against SARS-CoV-2, since it provided them with an effective humoral response , both immediately after vaccination and also sustained in time for at least 4 months after receiving the booster dose, whereas the first two doses had been unable to elicit such a sustained response.Several studies have determined the factors that are associated with an immediate humoral response few weeks after vaccination, including serum albumin levels, lymphocytes, dialysis vintage, intravenous iron dose, immunosuppressant treatment, previous infection with COVID-19, and a positive response to HBV vaccines \u201322. In op = 0.009). In this way, 4 months after receiving the booster dose, there were no significant differences in the protective humoral response between COVID-na\u00efve patients and those who were previously infected with COVID-19 .Na\u00efve patients for COVID-19 infection are the ones who benefit the most from vaccination and especially from the administration of a third dose . In our During the study period, the accumulated incidence in our area was around 20,500 cases per 100,000 population , predomip < 0.001). Therefore, this group of patients not only benefits from the first two vaccine doses but also from the booster effect of a third dose, although to a lesser extent than in the COVID-na\u00efve population.HD patients who have had a COVID-19 infection have less protection against reinfection than the general population . HoweverPrevious studies have compared the humoral response after the second and third doses. One study analyzed the humoral response after the third dose of the vaccine, showing a slight increase in the proportion of patients who achieve seroconversion, but with a very intense humoral response, reaching a 36-fold increase in median antibody titers after the booster dose , and anoIt has been previously described that there is an association between humoral responses after HBV and SARS-CoV-2 vaccines in HD , 32, 33.It should be noted that the most used vaccine in our patients was mRNA-1273, 77.5% of the patients for the first two doses and 91.7% for the third dose (with no significant differences between the different groups of patients). It has been described that the type of vaccine acts as an independent factor for response to vaccination and that mRNA-1273 is the one that obtains the best results, perhaps due to its higher dose of mRNA .We believe that in the future, the rate of antibodies will continue to decline and a significant percentage of patients will probably lose protection in the mid-term. Therefore, monitoring the levels of antibodies in the HD population should be a priority in order to propose new immunizations when necessary, as other authors have suggested .Consequently, we could revaccinate when SARS-CoV-2 anti-S(RBS) IgG levels drop below a certain threshold, in a similar way to how revaccination of HBV is recommended when anti-HBs antibodies drop below 10 mIU/mL. We suggest that monitoring of anti-S(RBD) IgG levels should be particularly frequent in patients who did not respond to HBV vaccines, in COVID-na\u00efve patients and in those with lower antibody levels.Our study has a number of limitations. First, the rate of neutralizing antibodies was not determined, which according to other authors can decrease progressively after the first two doses and can rise very significantly after the third dose. However, other studies have found a significant correlation between anti-S(RBD) antibodies and the neutralizing antibody response, and in our study we found that the changes in anti-S(RBD) IgG antibody levels resemble those described in neutralizing antibodies , 19. AnoIn conclusion, as a consequence of receiving a third dose of SARS-CoV-2 mRNA vaccine, HD patients achieve a better and more sustained humoral immune response over time, which probably translates into lower morbidity and mortality rates in SARS CoV-2 infection. Patients who are most likely to benefit from the booster dose are COVID-na\u00efve patients and patients who were non-responders to HBV vaccines.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Medical Research Ethical Committee (CEIm), Hospital Universitario Fundaci\u00f3n Alcorc\u00f3n. The patients/participants provided their written informed consent to participate in this study.EG-V and AS: conceptualization, writing\u2014original draft preparation, and writing\u2014review and editing. EG-V: methodology and project administration. EG-V, AS, and ML-P: validation. AS: formal analysis. JA-S: investigation. EG-V and EG: resources. EG-V and ML-P: data curation. JA-S, AC, CC-C, and GF-J: visualization. All authors have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Lung transplant recipients (LuTX) represent a vulnerable population for severe acute respiratory syndrome coronavirus 2 (SARS\u2010CoV\u20102). Even though many vaccines are already developed, more clinical data need to support effective immunological response in immunocompromised patients.Stable LuTX recipients with no medical history of coronavirus disease (COVID\u201019) were enrolled. Currently available messenger RNA (mRNA)  and non\u2010mRNA  vaccines were given according to availability, boosters were all mRNA\u2010based. SARS\u2010CoV\u20102 Spike1 immunoglobulin G (IgG) antibody titer was evaluated before and 2 weeks after second and third dose. Difference between mRNA versus non\u2010mRNA vaccines was assessed.p\u2009=\u2009.002). 6/23 (26%) patients received two doses of mRNA vaccine developed COVID\u201019 after the second injection in an average of 178 days, half of them recovered, half of them died in intensive care unit (ICU). 3/6 (50%) patients with two doses mRNA and recovered from COVID\u201019 had significantly higher level of antibody (average 20847.3\u2009U/ml) than without infection. After the booster vaccine, 1/24 (4%) developed infection.Forty\u2010one patients  received two doses of SARS\u2010CoV\u20102 vaccines: 23 of mRNA, 18 of non\u2010mRNA, and 24/41 (58%) received a third dose. Median 92 months passed since transplantation, and serum level of tacrolimus was median 5.5\u2009ng/ml. Positive serology was found in 37% of all patients after the second dose, 86% had mRNA vaccine. After the third dose, 29% became positive who had no antibody before. Significantly higher level of antibody was found after the second mRNA than non\u2010mRNA vaccines (2.2 vs. 1568.8\u2009U/ml, respectively, Immunosuppression therapy may induce a weaker SARS\u2010CoV\u20102 response in LuTX recipients; therefore, third dose is a priority in transplanted patients. The highest antibody level was measured recovering from COVID after two doses. Our data confirm that booster mRNA vaccine could increase antibody levels, even if immunization was started with non\u2010mRNA vaccine. Serological findings following SARS CoV 2 vaccines in lung transplant recipients As LuTX patients receive high doses of immunosuppressant medications, they are at very highest risk of developing severe coronavirus disease 2019 (COVID\u201019) and increased mortality.Transplant recipients were excluded from recent SARS\u2010CoV\u20102 vaccine trials, so there are insufficient data about efficacy, durability, or safety in this patient subpopulation. Our study aims to assess specific immunological response to different types of vaccinations and determine the proportion of seroconverted patients and outcomes in Hungarian LuTX patients.22.1Before 2015 our patients underwent lung transplantation in Vienna. The first lung transplantation procedure was performed in Budapest, Hungary on December 12,\u00a0 2015. The Department of Pulmonology, Semmelweis University is the only center for LuTx posttransplant care in Hungary. Our transplant activity is 20 cases per year. Our center uses the immunosuppression protocol of Medical University of Vienna. The induction therapy was antithymocyte globulin (ATG) of patients who underwent lung transplantation before 2008. From 2009 monoclonal antibody targeting CD52  30\u2009mg intravenously was the induction therapy followed by lower maintenance immunosuppression.2.2LuTX patients in the study period between January and December 2021 included 148 individuals. Patients were recruited at least 1 year after the surgery, if they were in stable functional condition, and if no rejection nor infection was present. Those who had immunoglobulin deficiency were excluded. In Hungary, according to actual regulations individuals who were infected with SARS\u2010CoV\u20102 within the last 3\u20136 months were exempt from vaccinations, including 30 LuTX patients.Out of the potentially eligible 118 patients, 41 volunteered for baseline SARS\u2010CoV\u20102 IgG and IgM measurements at study entry to help determine baseline SARS\u2010CoV\u20102 serostatus for the analysis of vaccine efficacy and were approved by local ethical committee (TUKEB\u2010IV/861\u20101/2021/EKU).At the time of the study BNT162b2, mRNA\u20101273, ChAdOx1, BBIBP\u2010CorV, and Gam\u2010COVID\u2010Vac vaccines were approved in Hungary. As there was no recommendation which vaccine should LuTX patients receive, all were vaccinated by the offered and available vaccines at centrally assigned vaccination points. SARS\u2010CoV\u20102 Spike1 IgG antibody (Roche\u00ae) titer was measured 2 weeks after the second dose of the respective vaccine and if available, after the third dose at their regular control (2\u20136 weeks postvaccination). Positive serology was considered >0.8\u2009U/ml serum level of anti\u2010SARS\u2010CoV\u20102\u2010Spike1.2.3t test after testing for normality using Kolmogorov\u2013Smirnov test, while \u03c72 test was applied for analyzing categorical data. A p value <.05 was defined as statistically significant.Statistical analysis was performed using Graph Pad software . Data are expressed as mean\u2009\u00b1\u2009standard deviation (SD) or median. Differences between groups for parametric data were evaluated with Student's 3N\u2009=\u200920). The most common non\u2010mRNA vaccine was ChAdOx1 (N\u2009=\u200916). Twenty\u2010four of 41 (58%) patients got third dose after the second one on average 177\u2009\u00b1\u200942 days later, all third doses were mRNA\u2010based. No serious adverse events other than general lethargy and local skin reaction were recorded neither after the second nor the third vaccination.Forty\u2010one LuTX recipients were enrolled into the analysis. Baseline characteristics are summarized in Table n\u2009=\u200918) recipients received ATG as induction therapy and 23 alemtuzumab. Each patient received tacrolimus and prednisolon. The median dosage of prednisolone was 5\u2009mg/day. The median tacrolimus serum level was 5.5\u2009ng/ml (IQR: 2.5\u201311.2\u2009ng/ml) before the first vaccination.Eighteen (Eleven out of 41 patients (26%) were treated with tacrolimus+everolimus+prednisolone. The total serum level of tacrolimus and everolimus was median 6.6\u2009ng/ml (IQR: 3.6\u201311.2\u2009ng/ml). Each patient except seven cases received mycophenolate\u2010mofetil, median 1500\u2009g/day (IQR: 500\u20132000\u2009g/day). Under the vaccination period, the immunosuppressive treatment did not change.N\u2009=\u200926; 64%), 3/41 patients (7%) had low\u2010positive antibody level (<10\u2009U/ml) and six patients (15%) developed >1000\u2009U/ml antibody titer 2 weeks after second vaccine. Thirteen out of 23 (57%) mRNA\u2010vaccinated patients became seropositive after two shots. Eighteen out of 41 patients received two doses of non\u2010mRNA vaccine; positive serology was found only in two cases (11%). A significant difference was found between the response of mRNA versus non\u2010mRNA vaccines , and the highest immune responses  were found in patients vaccinated with two doses of BNT162b2.Immune response for SARS\u2010CoV\u20102\u2010Spike1 was not measurable in most cases after the second dose  still did not develop any immune response neither after the second nor the third vaccination. However, seven patients (29%) had positive antibody after the third dose who had none before and in these patients the average antibody titer was 2435\u2009U/ml. Five of them received ChAdOx1, two of them BNT162b2 vaccines.p\u2009=\u2009.05). Only one of them was asymptomatic and recovered at home, while the other five required hospitalization. Two patients had moderate disease with 10%\u201320% involvement of the lungs, after a short time of hospitalization they recovered with no functional loss and high antibody titer  was measured thereafter. Three out of six patients had severe illness and needed intensive care, where they died soon, and after their second vaccination 183, 186, and 216 days have passed, respectively. Figure Six patients developed SARS\u2010CoV\u20102 infection after the second vaccination in an average of 178 days, all of them received BNT162b2. Three patients had no detectable antibody, while the other three had 140, 160, and 1346\u2009U/ml antibody levels respectively after two doses of vaccination. Significantly higher antibody levels were detected after recovering from infection  than after two doses of vaccines  while 57% of the patients became seropositive after two doses of mRNA vaccines. Among all patients vaccinated three times, positive antibody response increased from 17% to 46%, with a corresponding increase in neutralizing capacity after the third dose. The highest individual antibody level (>25000\u2009U/ml) was measured after two doses of mRNA vaccines, and then recovering from SARS\u2010CoV\u20102 infection. Infection could be life\u2010threatening in transplant recipients. In our patient population mortality was high (50%) among infected recipients, even though two or three doses of vaccines. No serious side effects of vaccination were observed. In the most recent recommendation, third dose is theoretically suggested, but that was not yet available in many countries.N\u2009=\u200948); however, 85% of the patients had high antibody response after COVID\u201019.Several studies exists who studied the effectiveness of the booster vaccines in heart\u2010 transplanted patients,The low antibody response rate is alarming but not unexpected in LuTX recipients. Previous experiences with Influenza vaccine show a lower rate of immune response in solid organ recipients, however, vaccination demonstrated reduced influenza\u2010related lower respiratory tract disease and hospitalization despite low antibody response.Other concerns regarding the SARS\u2010CoV\u20102 vaccines include the lack of long\u2010term safety data, potential reduction in efficacy in immunocompromised patients, unknown durability of the immune response, and potential for vaccine\u2010associated allograft rejection. Despite the low proportion of seroconversion observed in our patients, SARS\u2010CoV\u20102 vaccines provide potential benefit and decreases the risk stabling transplant recipients.We know our study limitations with the low number of the cases, and vaccine efficacy cannot be defined obviously by the presence or absence of antibodies, which may be deceptive. We should also note that in lines with positive antibody response there is still no universal cutoff value. However, we observed low antibody response in patients vaccinated with non\u2010mRNA vaccines, none of them get COVID\u201019. Additionally to neutralizing antibodies T helper1 CD4\u2009+\u2009and CD8\u2009+\u2009T cell response might contribute to immunity against SARS\u2010CoV\u20102. Further data are needed to evaluate B\u2010 and T\u2010cellular responses after SARS\u2010CoV\u20102 vaccination and disease.The emerging studies on SARS\u2010CoV\u20102 vaccines indicate that they are safe, and our experience with different SARS\u2010CoV\u20102 vaccines proved to be safe in lung transplant recipients. Currently, the third booster dose of SARS\u2010CoV\u20102 vaccine is the most appropriate way to increase the immune response among lung transplant recipients. The durability of this immune response should be evaluated in the need of additional boosters in the future. However, we still have insufficient amounts of data using non\u2010mRNA\u2010based vaccines among transplant recipients, there is promising result about switching to a different type of vaccine  in nonresponders thereby offering a new strategy to increase immune response. Vaccination is the most promising way to tackle the pandemic, and third or more doses of vaccine are becoming a chance to avoid serious COVID\u201019 complications.All authors have read and approved the final manuscript. All authors performed the research. Enik\u0151 B\u00e1rczi, Anik\u00f3 Boh\u00e1cs, and Veronika M\u00fcller designed the research study, analyzed the data, and wrote the paper.The authors declare no conflicts of interest."}
+{"text": "Overall, comprehensive analysis of a small cohort showed that co-administration of SARS-CoV-2 vaccine and IO is not associated with increased irAEs. Nevertheless, the detection of autoantibodies post anti-SARS-CoV-2 vaccination warrants further investigation (NCT03702309).Despite more than 2\u2009years having elapsed since the onset of SARS-CoV-2 pandemic, a level of hesitation around increased SARS-CoV-2 vaccine toxicity in cancer patients receiving immunotherapy (IO) remains. This hesitation stems from the idea that IO agents could elicit an overwhelming immune stimulation post vaccination and therefore increase the risk of vaccine-related toxicity. The aim of our study was to explore serological responses to SARS-CoV-2 vaccination in patients treated with IO and describe the level of immune stimulation using parameters such as blood cytokines, autoantibody levels and immune related adverse events (irAEs) post vaccination. Fifty-one evaluable patients were enrolled in this longitudinal study. Absolute levels and neutralization potential of anti-SARS-CoV-2 antibodies were not significantly different in the IO group compared to non-IO. Chemotherapy adversely affected seroconversion when compared to IO and/or targeted treatment. Following vaccination, the prevalence of grade \u22652 irAEs in patients treated with IO was not higher than the usual reported IO toxicity. We report, for the first time, that anti-SARS-CoV-2 vaccination, elicited the generation of five autoantibodies. The significantly increased autoantibodies were IgM autoantibodies against beta-2 glycoprotein ( For more than 2\u2009years, the world has faced tremendous public health, economic, and ethical challenges due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic . Cancer The development of vaccines preventing severe disease, hospitalization and death from COVID-19 has marked a significant achievement , 6. DespCancer patients have been prioritized for receipt of initial vaccination series and boosters around the globe. Despite this, there is ongoing concern as to whether vaccine efficacy is impacted by increased immune vulnerability in the setting of malignancy or secondary to immune-modulating therapies (such as immuno-oncology (IO) agents). The heterogeneous nature of neoplastic diseases and diverse therapies applied has further complicated efforts to interrogate this relationship.Emerging evidence suggests that the use of certain anti-cancer therapies during SARS-CoV-2 vaccination may alter immune correlates of protection. In some cancer patients, including a high proportion of those receiving cytotoxic or B-cell depleting therapies, lower rates of seropositivity and T-cell responses have been reported after two doses of mRNA vaccine \u201315. VaccIn the present study, we comprehensively explored peripheral immune responses to SARS-CoV-2 vaccination in IO-treated cancer patients. We also described the immune-mediated toxicities in IO-treated patients who received anti-SARS-CoV-2 vaccination and lastly, we conducted an in-depth analysis of auto-antibody generation following vaccination.Immune Response to COVID-19 Vaccine in immunotherapy (IO) and non-IO Treated Cancer Patients  between March 2021 and July 2022. Patients were allowed to have any of the approved vaccines in Canada at the time of study recruitment, such as Pfizer (BNT162b2), Moderna (mRNA-1273), AstraZeneca [AZD1222 (ChAdOx1)] or their approved combinations.Patients with a diagnosis of solid malignancy, treated at Princess Margaret Cancer Centre, University Health Network (PM-UHN), with either a standard of care (SOC) therapy or within a clinical trial were recruited. Blood samples from patients enroled were banked under the study oC. After processing, serum samples were aliquoted and stored in liquid nitrogen vapor phase for long term storage until time of downstream analysis.Sampling at 6 time points per patient were undertaken: at baseline (BASE), between 1st and 2nd vaccine doses (PRE2D), at 1\u2009week (1\u2009W), 1\u2009month (1\u2009M), 4\u20136\u2009months (4\u20136\u2009M) and 10\u201312\u2009months (10\u201312\u2009M) after the 2nd dose, as per Fig. Patient demographics, cancer diagnosis, past medical history, treatment details, concomitant medications, and immune related adverse events (irAEs) of \u2265grade 2 severity by Common Terminology Criteria for Adverse events Version 5.0 (CTCAE 5.0) were collected prospectively and entered into a Medidata Rave database. This de-identified database is password protected and encrypted, to be compliant with PM-UHN standards for all investigator-initiated studies.The most updated Charlson Co-morbidity Index (CCI) was used to describe patients\u2019 overall health status . CCI is This study was conducted with the approval of UHN Oncology Research Ethics Board (CAPCR/UHN REB #: 21\u20135797) and all patients signed informed consent. Participants who were concurrently treated in an interventional study with experimental treatment, were allowed to take part in this observational study (VIVACIOUS), according to their respective study protocols; or were not specifically disallowed to do so, if protocols were silent on this matter. The laboratory exploratory analysis was performed by scientific staff who was blinded to patients\u2019 identity.Serum samples from patients (20\u2009\u00b5L) were analyzed using Elecsys\u00ae Anti-SARS-CoV-2 S assay on the cobas e411 analyzer (Roche Diagnostics) for the quantitative detection of total antibodies (IgM/IgG/IgA) to the SARS-CoV-2 spike (S) protein receptor binding domain (RBD). This assay is a double-antigen sandwich electrochemiluminescence immunoassay, which uses streptavidin-coated microparticles to separate bound from unbound substances prior to applying a voltage to the electrode. Application of a voltage to the electrode then induces chemiluminescent emission which is measured by a photomultiplier. Samples were run in singleton. The measuring range of this assay is 0.40\u2013250\u2009U/mL (up to 2500\u2009U/mL with on-board 1:10 dilution), with a concentration of \u22650.80\u2009U/mL considered positive. Serum antibody levels against SARS-CoV-2 S protein are the result of immune response to either natural infection or vaccination but cannot distinguish between the two .The GenScript SARS-CoV-2 surrogate Virus Neutralization Test (sVNT) Kit was used to detect circulating neutralizing antibodies against SARS-CoV-2 that block the interaction between the viral S glycoprotein RBD with the angiotensin converting enzyme-2 (ACE2) cell surface receptor. The interaction between RBD and ACE2 leads to viral endocytosis into the host cells and subsequent viral replication. This assay detects any antibodies in serum and/or plasma that neutralize the RBD-ACE2 interaction.oC (30\u2009min) with respective positive and negative controls. Samples were then loaded on plate wells and incubated at 37 oC for 15\u2009min. Absorbance was measured at 450\u2009nm. A result of \u226530% is considered positive for the detection of SARS-CoV-2 neutralizing antibodies . Similarly, the neutralizing antibody level at 1\u2009M was 94.5%  for the IO group and 92.7%  for the non-IO group  and neutralization levels (97.7 vs 89.1 p\u2009=\u20090.003) compared to patients who received only two vaccine doses  after the second dose. Most cytokines  were undetectable for most patients. Overall, there were no statistically significant differences between either the baseline and the intra-dose measurement, nor the baseline and post 1\u2009week measurement Fig. . Figure Patients treated with IO were prospectively followed-up for irAEs of more than grade 2 severity, from the date of first vaccine dose to 90\u2009days following the 4th dose. Two out of 35 (5.7%) patients experienced immune related toxicity after 1st dose and before the 2nd vaccine dose, 8/34 (23.5%) after the 2nd dose and before the 3rd, 2/29 (7%) following the 3rd dose and 2/13 (15.3%) after the 4th dose Fig. . Most ofp\u2009<\u20090.001) and also IgM antibodies against: Human beta-2 glycoprotein 1 , Myeloperoxidase (MPO) , Nucleosome , and against Short Palate, Lung and Nasal epithelium carcinoma associated protein 2 (SPLUNC2)   between IgM anti-MPO autoantibody and IL-2 cytokine levels post-vaccination was found  following vaccination, as defined by a significant increase of their mean log-transformed fluorescent intensity (mean Log2 MFI). These autoantibodies were an IgG antibody against Myosin heavy-chain 6 (MYH6)  Fig. . There w01) Fig. . In viewund Fig. .Fig. 4AuWe conducted the VIVACIOUS study in a Canadian tertiary cancer centre during the height of initial SARS-CoV-2 pandemic waves. We aimed to understand multi-dimensional longitudinal immune correlates after anti-SARS-CoV-2 vaccination in solid cancer patients treated with immunotherapies and to compare these with patients receiving non-IO-based treatments. At that time, there was considerable vaccine hesitancy for some patients receiving cancer immunotherapies owing to emerging data on potentially autoimmune-related complications linked to mRNA vaccines (such as myocarditis). Thus, we further endeavoured to undertake an in-depth analysis of immunological and clinical parameters that could provide insights into this risk. Unlike other large series, we did this by adding multiple timepoint blood collections through the initial series and the booster vaccines doses and cytokine/autoantibody assays to assess this risk in a multi-dimensional manner.In performing this analysis, we found that IO-treated cancer patients were largely indistinguishable from non-IO-treated cancer patients with respect to absolute levels and neutralization capability of anti-RBD antibody generation after primary series anti-SARS-CoV-2 vaccination. Although others have reported reduced rates of seroconversion after vaccination amongst patients with haematologic malignancies, we confirm that patients with solid cancer receiving immunotherapy generate robust antibody responses. Both quantitative and functional testing of anti-SaRS-CoV-2 antibodies following vaccination seem to be largely unaffected by cancer immunotherapy, providing surrogate evidence that B cell function remains intact during treatment with immunotherapy. This finding is in keeping with what is reported in the literature so far , 33, 34 et al. showed that third and fourth vaccine doses can restore waning antibody titres in solid cancer patients [et al. recently reported that 3 vaccination doses are imperative for achieving neutralizing antibodies that offer protection against the Omicron variant in patients with solid malignancies [As with healthy subjects, we further observed that serological responses wane over the 4\u20136\u2009months following the second vaccine dose which highlights the importance of implementing booster vaccinations , 8. A repatients . We notepatients , 37. Intgnancies . In our Although most of our participants generated robust antibody responses after initial primary vaccine series, we identified several patients with low seroconversion. Interestingly, all of these patients were male and 3/4 of them had a head and neck primary. In the literature, there is conflicting data as to whether patient sex or primary tumor type have an impact on seroconversion after vaccination. At least one publication reported higher antibody titres amongst female solid cancer patients, an observation also made in our cohort of patients . In our We next examined whether cytokine production differed after vaccination in IO-treated and non-IO-treated patients. Given the broad immunomodulatory impacts of vaccination and immunotherapies, we were somewhat surprised to find no significant, lasting differences after vaccination in any of the cytokines studied. A moderate positive correlation was observed between IL-2 and anti-MPO autoantibody generation and interestingly, a recent case report on cytokine release syndrome following anti-SARS-CoV-2 vaccination in a patient receiving ICI was accompanied by an increase in IL-2 .Having examined the impact of IO therapies on vaccine efficacy, we further explored whether vaccine administration might affect immunotherapy-related toxicities. Isolated thrombocytopenia (without thrombosis) has been described with mRNA-based COVID-19 vaccines  and a paLastly, we observed an increase of five antibodies against self-antigens after vaccination, an effect that was not treatment dependent. Human beta-2 glycoprotein 1, a critical regulator of complement and coagulation systems, is also a target of anti-phospholipid antibodies implicated in the antiphospholipid syndrome . In our Intriguingly, we observed an elevation of autoantibody against alpha-cardiac myosin heavy chain (MYH6), a component of the contractile system in cardiac muscle . MYH6 haOur study has various limitations, such as the small number of patients included and the lack of characterising immune responses at a cellular level by interrogating IL-2/IFN-\u03b3-expressing T lymphocyte responses and functionality. Moreover, for our neutralization test, we considered 30% as the threshold for positivity, although we note that more recent evidence has suggested that higher neutralization levels are required for adequate protection against infection . In desiIn conclusion, we have found that administration of anti-SARS-CoV-2 vaccines can be safely delivered in patients undergoing cancer immunotherapy, without elevating the risk of immune related adverse events. The absolute and neutralizing levels of anti-RBD antibodies are decreasing with time and repeat booster vaccination is required to achieve desirable levels of immunity. The observation that anti-SARS-CoV-2 vaccination de-represses generation of five autoantibodies, a cardiac myosin amongst them, warrants further investigation.Supplementary Tables and Figures"}
+{"text": "At multivariate analysis, only treatment with MMF  and active neoplasia  were independent predictors of seroconversion failure. These findings suggest that MMF dose reduction or suspension may be required to optimize vaccine response in these patients.Vaccines are the most effective means to prevent the potentially deadly effects of SARS-CoV-2 infection, but not all vaccinated individuals gain the same degree of protection. Patients undergoing chronic immunosuppressive therapy due to autoimmune diseases or liver transplants, for example, may show impaired anti-SARS-CoV-2 antibody response after vaccination. We performed a prospective observational study with parallel arms, aiming to (a) evaluate seroconversion after anti-SARS-CoV-2 mRNA vaccine administration in different subgroups of patients receiving immunosuppressive treatment for rheumatological or autoimmune diseases or to prevent organ rejection after liver transplantation and (b) identify negative predictors of IgG anti-SARS-CoV-2 development. Out of 437 eligible patients, 183 individuals were enrolled at the Rheumatology and Hepatology Tertiary Units of \u201cMaggiore della Carit\u00e0\u201d University Hospital in Novara: of those, 52 were healthy subjects, while among the remaining 131 patients, 30 had a diagnosis of spondyloarthritis, 25 had autoimmune hepatitis, 10 were liver transplantation recipients, 23 suffered from connective tissue diseases (including 10 cases that overlapped with other diseases), 40 were treated for rheumatoid arthritis, and 5 had vasculitis. Moreover, all patients were receiving chronic immunosuppressive therapy. The immunogenicity of mRNA COVID-19 vaccines was evaluated by measuring IgG anti-SARS-CoV-2 antibody titers before vaccination and after 10, 30, and 90 days since the first dose administration. Of the selected cohort of patients, 24.0% did not develop any detectable anti-SARS-CoV-2 IgG after a complete mRNA-based two doses primary vaccination cycle. At univariate analysis, independent predictors of an absent antibody response to vaccine were a history of liver transplantation (OR 11.5, 95% CI 2.5\u201353.7,  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a coronavirus responsible for coronavirus disease 2019 (COVID-19), a very heterogeneous disease ranging from a nearly asymptomatic state to a life-threatening condition with high in-hospital mortality, with the highest toll among the elderly or those with chronic diseases and cancer ,4. COVIDSince 2020, more than 50 vaccines against COVID-19 have been developed and tested in phase II and III trials ,9,10,11.Patients affected by chronic immune-mediated inflammatory diseases have high rates of morbidity and mortality due to infections that rely on immunodeficiency, related to either the autoimmune diseases themselves or to the immunosuppressive drugs used to treat these illnesses . Thus, tTo date, few data are available on the efficacy of anti-COVID-19 vaccination in patients with inflammatory autoimmune diseases or in transplanted patients receiving immunosuppressive treatments. To fill this gap, we performed a prospective observational study to assess seroconversion rates after vaccine administration in different subgroups of patients undergoing chronic immunosuppressive treatment due to autoimmune diseases or liver transplantation.We performed a prospective observational study (acronym RIVALSA) with parallel arms including healthy subjects and autoimmune/liver-transplanted patients receiving immunosuppressive drugs with planned COVID-19 vaccination between March and July 2021. Inclusion and exclusion criteria are reported in The study protocol was approved by the local ethical committee (CE 72/21), and the study was conducted in strict accordance with the Declaration of Helsinki. Among all the patients attending either the Rheumatology or Hepatology Unit of \u201cMaggiore della Carit\u00e0\u201d University Hospital in Novara , eligible subjects (patients receiving immunosuppressive drugs to treat autoimmune diseases or to prevent organ rejection after liver transplantation) were asked to participate in the study; from the 437 subjects screened, 131 patients were included in the study together with 52 healthy subjects selected among the healthcare professionals of these units and/or their relatives. (1)Assessment of the IgG anti-SARS-CoV-2 titer in response to vaccination in patients who completed the primary vaccination cycle receiving immunosuppressive therapy due to inflammatory autoimmune diseases or organ rejection prevention after liver transplantation and proportion of the failure of IgG seroconversion relative to healthy subjects in patients who completed the primary vaccination cycle, 90 days after the first vaccine dose;(2)Identification of predictors of the absence of an IgG anti-SARS-CoV-2 seroconversion 90 days after the first vaccine dose in patients who completed the primary vaccination cycle.The predefined endpoints of the cohort study were as follows:All individuals enrolled in the study underwent blood sampling before anti-SARS-CoV-2 vaccination (t0) and after 10, 30, and 90 days from the first vaccination dose . Blood samples were collected using EDTA as an anticoagulant, and blood fractions were immediately separated via centrifugation and stored at \u221280 \u00b0C until the time of analysis.IgG anti-spike protein antibodies were determined by performing an ELISA test, using a CE-IVD commercial kit (EUROIMMUN Medizinische Labordiagnostika AG Anti-SARS-CoV-2 QuantiVac ELISA (IgG), L\u00fcbeck, Germany) ,26, foll2 test or Fisher\u2019s exact test when appropriate, while continuous variables were compared with the Mann-Whitney U test. Odds ratios (ORs) with confidence intervals (CIs) were calculated at univariate analysis. A set of variables associated with seroconversion failure at univariate analysis were used to build multivariate stepwise logistic regression models. The statistical significance threshold was set at 0.05 (two-tailed). Statistical analyses were performed with Statistica for Windows release 12  and MedCalc\u00ae Statistical Software version 20.014 .The relevant clinical and experimental data of each subject  were collected from clinical records or during an ad hoc interview and stored in a dedicated database (REDCap). Categorical variables were expressed as frequencies (percentage), while the measures of central tendency and dispersion of continuous variables were, respectively, expressed as medians and interquartile range (IQR). Categorical variables were compared with Pearson\u2019s \u03c7Among the 131 patients, 69.5% were women with a median age of 58 years (IQR 49\u201367). Healthy controls had a median age of 32 years (IQR 29\u201345) and were mainly women (65.4%). Most of them were healthcare professionals and had no comorbidities.The baseline population features (patients and controls) are shown in Comparing the two groups, we observed a significant difference in the median antibody titers between healthy subjects and patients at 10, 30, and 90 days since the first vaccine dose .This difference was also confirmed after the exclusion of those subjects with baseline antibody positivity for any time with the exception of the assessment at 90 days after vaccination .p = 0.0010).Interestingly, 50 out of the 50 healthy individuals with complete follow-up (100%) developed anti-SARS-CoV-2 IgG antibodies; by contrast, only 100 out of 119 patients with complete follow-up (84.0%) showed a detectable antibody response to vaccination at 90 days  was built. Treatment with MMF and active cancer as comorbidity were independent predictors of the absence of vaccine response in this model  nor with b-DMARDs  was associated with the absence of IgG anti-spike antibody detection. These results are even more interesting when compared with the existing literature about DMARDs and immune response to vaccines. Indeed, previous studies assessing antirheumatic drug effects on vaccine immunogenicity were mainly focused on seasonal influenza and pneumococcal vaccines. These showed reduced humoral responses in methotrexate-, abatacept-, and rituximab-treated patients and a minimal or absent response in patients treated with anti-cytokine drugs. Such data thus raise the question about the effectiveness of anti-SARS-CoV-2 vaccination in DMARD-treated patients and foster new studies aimed to evaluate immunomodulatory drug effect on COVID-19 vaccination in rheumatologic patients ,33,34,35+ T cells in a third of patients treated with methotrexate [With the aim to evaluate the immunogenicity of a single dose BNT162b2 mRNA vaccine, Bugatti et al., studied 140 chronic inflammatory arthritis patients and observed that anti-cytokine drugs had a low impact on vaccination (>80% response), while the treatment with methotrexate and/or glucocorticoids had a greater effect in reducing the IgG seroconversion, even after methotrexate withholding before and after vaccination . In a laotrexate . On the otrexate , a resulInterestingly, studying a cohort of 404 rheumatic patients, Ruddy et al., showed that anti-TNF-\u03b1 therapy did not influence antibody response (100%), while only 82% of patients receiving glucocorticoids and 73% receiving MMF had IgG seroconversion . In our Notably, MMF and calcineurin inhibitors impaired immunogenicity to vaccination in our population as well. Indeed, the present study provided evidence of a perdurable absence of antibody response up to 3 months after the first dose administration and the following second one, suggesting a definitive absence of seroconversion. Such interesting results are in agreement not only with those of Furer et al., on autoimmune inflammatory rheumatic patients  but alsoIn univariate analysis, being affected by SLE, undergoing liver transplantation, and being treated with MMF or calcineurin inhibitors were all predictors of the lack of response to vaccination. When all of these predictors were analyzed together in a comprehensive multivariate model, only MMF treatment remained associated with an increased risk of vaccination failure. Such observation is of great interest, as MMF is a common treatment used in either SLE or liver transplantation and may account for the principal lack of immunogenicity observed in these patients. The observed impaired humoral response to mRNA-based vaccines in patients treated with MMF and calcineurin inhibitors could be explained by their pharmacological mechanism of action. Both classes of drugs, in fact, are known to impair T-cell proliferation and activation processes, especially by suppressing follicular T helper cells, thus reducing B-cell-mediated antibody production ,46,47,48As an additional point of interest, we observed that the concomitant diagnosis of neoplasia was a negative predictor of antibody response in all the analyses. This result is not surprising if we consider that, due to their therapeutic regimen, these patients show an increased risk to develop severe COVID-19 manifestations. To date, oncological patients have been generally excluded from anti-SARS-CoV-2 efficacy clinical trials, and studies assessing vaccine immunogenicity in these individuals are still few. The emerging literature on this topic highlights that seroconversion in oncological patients is significantly reduced compared with a healthy population ,50,51.Recently, Mitchell et al.  observedDue to the ongoing pandemic situation, and according to recent lines of evidence highlighting that vaccine efficacy decreases over time ,54, manyAs the pandemic situation evolved, different SARS-CoV-2 variants emerged, acquiring new mutations that could account for immune escape properties. Such lines of evidence foster studies aimed to investigate the ability of vaccine-elicited immunity to neutralize these new threats. According to the \u201cIstituto Superiore di Sanit\u00e0\u201d , at the time of the present study, the prevalent variant in Italy was the lineage B.1.1.7 (Alpha variant). Different research groups investigated the ability of post-immunization sera to neutralize circulating variants, highlighting that the Alpha variant is unlikely to escape neutralization through both BNT162b2- and mRNA-1273-elicited antibodies and to increase reinfection risk ,60,61,62The present study has some limitations. First of all, the sample size was relatively small, and the population of patients was heterogeneous in terms of diseases, limiting the possibility to identify predictors of vaccination failure with smaller effects than those evidenced due to eventual biases. Additionally, control cohort heterogeneity could, to some extent, limit the possibility to elaborate conclusions stronger than those proposed. Furthermore, we focused only on people receiving mRNA-based vaccines, as only few of our patients received adenoviral vaccines, thus precluding the evaluation of this kind of vaccine immunogenicity. In addition, the chosen assay detected only IgG and not IgM or IgA against SARS-CoV-2 spike protein. Therefore, we cannot exclude that other antibodies targeted to other viral antigens may be present. Moreover, we did not evaluate T-cell-mediated response; thus, a deeper investigation of the cell-mediated response to viral infection would be necessary to better elucidate the role of MMF in vaccination failure. Due to the local COVID-19 infection recording protocol, not establishing a routine identification of viral variants, it was not possible to draw any conclusion about the possible role of the timely prevalent variants of concern in determining vaccination failure. Furthermore, the adopted study setting and the healthcare data availability did not allow a deeper investigation of vaccine efficacy in protecting against symptomatic infection. Finally, the monocentric design of the study may have led to some bias due to the clinical practice of our center.In this prospective, observational study, we showed that a limited but relevant proportion of patients receiving immunosuppressive therapy for inflammatory autoimmune diseases or prevention of solid organ rejection after liver transplantation did not develop detectable anti-SARS-CoV-2 IgG three months after a complete, two-dose mRNA vaccine schedule. The principal predictors of the lack of seroconversion were ongoing treatment with MMF and the compresence of active neoplasia as comorbidity. Individuals under treatment with calcineurin inhibitors or those affected by SLE or undergoing liver transplantation also had a high risk of a failure of vaccine response, but the risk may be biased by concomitant MMF treatment. Our study suggests that MMF treatment modification, its temporary suspension, or a different vaccine regimen may be required in these populations to enhance immune responses."}
+{"text": "Patients with end-stage renal disease (ESRD) requiring hemodialysis (HD) constitute a high-risk group in terms of susceptibility to and severity of infection. High mortality rates have been observed in this population group with SARS-CoV-2 infection, and thus strategies to halt viral transmission are deemed crucial. Vaccination with an mRNA-based vaccine has been shown to decrease the risk of severe disease and substantially reduce mortality, both in healthy individuals and ESRD patients. The cellular and humoral immune responses elicited in HD patients, however, are diminished when compared to healthy controls, with the waning immunity that ensues placing them at high risk for reinfection. Several variables have been found to be associated with this suboptimal response to vaccination, with immunosuppression, older age, and underlying comorbidities playing a central role. Lately, the effects of booster dose administration have been studied in HD patients, with results indicating an enhanced immune response capable of inducing a protective immune profile in this group.The outbreak of SARS-CoV-2 has raised considerable concern about the detrimental effects it can induce in public health, with the interest of the scientific community being focused on the development of preventive and therapeutic approaches. Patients with end-stage renal disease (ESRD) are amongst vulnerable populations for critical illness owing to the presence of other comorbidities, their defective immune system, and their inability of self-isolation. To date, vaccination constitutes the most promising method to manage viral dispersion. Therefore, it is particularly important to investigate the effectiveness of available vaccines against SARS-CoV-2 in this risk group. Here, we summarize initial experience regarding the humoral and cellular immune responses elicited in dialysis patients after completion of the recommended vaccination regimen, as well as after booster dose administration, with one of the two mRNA vaccines, namely, BNT162b2 and mRNA-1273. In conclusion, a significantly diminished and delayed immune pattern was observed in ESRD patients compared to healthy population, with a peak in antibody titers occurring 3\u20135 weeks after the second dose. A booster dose significantly augmented the immune response in dialysis patients with either mRNA-based vaccine. Variables adversely correlating with the weak immunogenicity observed in dialysis patients include immunosuppressive therapy, older age, comorbidities, longer time in hemodialysis treatment, and higher body mass index. On the contrary, previous COVID-19 infection and administration of the mRNA-1273 vaccine are deemed to induce a more favorable immune response. Further investigation is needed to thoroughly understand the efficacy of mRNA-based vaccines in hemodialysis patients and define predictive factors that can influence it. The COVID-19 pandemic poses a considerable threat to public health , with caHowever, severe concerns have been raised about the immunization status and safety of dialysis patients after vaccination due to their distinct clinical condition . End staThe aim of this review is to investigate the cellular and humoral immune response patterns induced in dialysis patients after administration of the BNT162b2 or mRNA-1273 vaccines.The adequate laboratory examination of humoral response levels plays a highly significant role in assessing vaccination efficacy. Serological protocols relying on the quantification of specific anti-viral antibodies are considered as reliable biomarkers . As immuOverall, case-control studies have demonstrated a defect in the humoral immunity elicited by HDPs compared to healthy participants after two doses of the mRNA vaccine, regardless of the diagnostic test used ,22,23,24In conclusion, evidence from analyzed vaccine studies evaluating both the cellular and humoral responses indicates a significantly lower and delayed humoral response in HDPs after vaccination, compared to healthy individuals, that reached a peak in antibody titers 3\u20135 weeks after the second dose. Markedly impaired anti-BNT162b2 humoral responses were also observed by case-control studies evaluating only the antibody status of vaccinated patients. More specifically, according to Rincon-Arevalo et al. , a delayThe suboptimal humoral response after a two-dose vaccine regimen and the need for continuous and robust antibody responses in dialysis patients necessitates modifications and/or enhancements to the primary vaccination regimen. Although vaccination with two doses of an mRNA-based vaccine prevents severe SARS-CoV-2 infection to a significant extent in dialysis patients, the waning humoral response that ensues about 4 months after the last dose increases the risk of reinfection without excluding the possibility of severe disease . A boostAlthough antibodies are considered to play a significant preventive role against COVID-19 infection, T-cell effects also complement this protective purpose, with the BNT162b2 vaccine eliciting robust CD8+ and CD4+ RBD-specific T-cell responses . A promip < 0.0001). In addition to IFN\u03b3 responses, interleukin (IL)-2 levels also increased between the second and the third vaccine dose. Consistent findings were demonstrated by Strengert et al. [In line with humoral immunogenicity, the T-cell-mediated IFN\u03b3-response was significantly diminished in HDPs after completion of the recommended vaccination regimen, regardless of the days elapsed from the last injection. Schrezenmeier et al.  and Stumt et al. , althougt et al. . Nonethet et al.  recomment et al.  suggestet et al. . Inconsit et al. . Duni ett et al.  also stuGenerally, a considerable lack of information on cellular immunity in HDPs following completion of any type of available COVID-19 vaccine scheme was identified in the literature, highlighting the urgent need for further investigation. p = 0.96). Additionally, there was a statistically significant decline in the T-cell response between 3 weeks and 3 months after the third dose (p < 0.001). This observation needs to be interpreted cautiously, since it may reflect normal T-cell physiology rather than failure of the third dose to achieve a sustained cellular response. Becker et al. [p = 0.564). When comparing the T-cell response after the third and after the fourth dose, however, a statistically significant difference was observed (p < 0.0005). The cellular response after a booster vaccine dose has been less extensively analyzed compared to the vaccine-elicited humoral response, but results of associated studies provide insight into the potential protective role of T-cell-mediated immune responses against SARS-CoV-2 infection among dialysis patients. Melin et al.  analyzedr et al.  assessedThe results of the above studies highlight the need to conduct more research regarding the effect of booster dose administration on the T-cell response, since published results, although promising, are scarce, and thus definite conclusions are hard to make. Among the factors associated with poor immune responses after vaccination completion in HDPs, the presence of an immunosuppressive regimen was the most reported ,23,24,55Other predictors of the observed weak immunogenicity among dialysis patients are associated with the clinical characteristics of the analyzed population. Patients with kidney failure are characterized by decreased lymphocyte count, systemic inflammation, malnutrition, and a defective clearance of uremic toxins, with all these parameters suggested to affect vaccination outcomes . The ureHowever, reviewed studies indicate that increasing the viral exposure of this vulnerable cohort may circumvent their immune dysfunctions, as previous COVID-19 infection was associated with favorable antibody responses ,21. In aAn optimal approach to strengthen the immune response to SARS-CoV-2 vaccination in dialysis patients requires adherence to a vaccination regimen. This could be accomplished via a meaningful interaction between the healthcare provider and the patient and should aim to reinforce the positive outcomes following vaccination. Results from published studies support the administration of an mRNA-based vaccine in dialysis patients, both as part of a primary vaccine series and also during booster dose administration. Vaccines with a different mechanism of action have been shown to confer lower cellular and humoral immune response rates when compared to an mRNA-based vaccine, although it needs to be noted that studies contemplating a non-mRNA-based vaccine regimen in dialysis patients are limited. Regarding the optimal mRNA-vaccine that should be used, studies have found that the mRNA-1273 vaccine is associated with higher antibody titers when compared to the BNT162b2 vaccine, although antibody neutralization capacity and cellular responses between these two vaccines have not been studied extensively, making conclusions about the most appropriate mRNA-based vaccine in dialysis patients problematic ,62. As bESRD patients in HD treatment are shown to develop a promising but impaired humoral and cellular immune response, compared to the healthy population, after completion of the vaccination regimen with the available mRNA anti-SARS-CoV-2 vaccines, BNT162b2 and mRNA-1273. Taking into consideration the risk factors associated with this vulnerable group, adaptation of vaccination protocols should be a health priority. Booster dose administration with mRNA-based vaccines seems to augment both the humoral and cellular responses in DPs, and implementation of this strategy may result in sustained immunological responses in this high-risk group. Additionally, further research regarding the lifespan and the effectiveness of observed responses are urgently needed, with great attention to patients receiving immunosuppressive therapy, older individuals, and those with comorbidities, conditions deemed to adversely contribute to immunization outcomes."}
+{"text": "Further, we demonstrate a decline in antibodies over six months in responders and controls, and question if second vaccination non-responders benefit from of a third vaccination.Here we confirm lower humoral and cellular responses in hematological patients compared with controls and highlight the response in risk-groups such as CAR-T-cell recipients. The main risk factor for a poor humoral response was anti-CD20 therapy. CD19p = 0.001). Risk factors for a poor serological response were age (<65a), history of anti-CD20 therapy within the year preceding vaccination, CD19+ B-cells < 110/\u00b5L, and CD4+ T-cells > 310/\u00b5L. The magnitude of T-cell response was higher in patients <65a and with CD19+ B-cells < 110/\u00b5L. Patients and healthy controls demonstrated a significant decrease in SARS-CoV-2 S antibody levels over the period of six months (p < 0.001). A third vaccination demonstrated a strong serological response in patients who had responded to the previous doses (p < 0.001). The third vaccination yielded seroconversion in three out of 19 patients in those without serological response. We conclude that both humoral and cellular responses after SARS-CoV-2 immunization are impaired in patients with hematological malignancies. A third vaccination enhanced B-cell response in patients who previously responded to the second vaccination but may be of limited benefit in patients without prior seroconversion.Here we analyzed SARS-CoV-2-specific antibodies and T-cell responses after two coronavirus disease 2019 vaccinations over a six-month period in patients with hematological malignancies and assessed the effect of a third vaccination in a subgroup. Sixty-six patients and 66 healthy controls were included. After two vaccinations seroconversion was seen in 52% and a T-cell-specific response in 59% of patients compared with 100% in controls ( The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus (SARS-CoV-2), poses a major challenge to the health care systems around the world ,4.Concerns regarding the serological response to vaccines in patients with hematological malignancies are based either on the direct impact of the underlying disease or the administered therapy ,6,7,8,9.In addition, hematological patients are at an increased risk of breakthrough infection, especially in terms of accelerated waning of antibody levels over time and increasing immune-evasion capabilities of emergent variants ,15. A thIn the present study, we aim to evaluate serological and T-cell responses after two doses of mRNA COVID-19 vaccination and describe the efficacy of a third vaccine dose in patients not responding to the first two immunizations.The study is part of a prospective cohort study performed at the Medical University of Vienna, Vienna, Austria, \u201cCharacterization of the responsiveness after mRNA SARS-CoV-2 vaccination in patients with immunodeficiency or immunosuppressive therapy\u201d; Eudra CT Nr. 2021-000291-11. Ethical approval for this study was granted by the local ethics committee of the Medical University of Vienna, Austria (EK Nr. 1073/2021).Patients with hematologic malignancies older than 18 years, who were treated at the Clinical Division of Hematology and Hemostaseology, were enrolled in the study. Further, 66 age- and sex-matched healthy controls (HCs) were included. All patients and HCs were vaccinated as participants of the Austrian vaccination program. Antibodies against the SARS-CoV-2 receptor-binding domain and the nucleocapsid protein were determined before vaccination as well as 10\u201320 days after the first, 12\u201328 days, and 5\u20136 months after the second vaccination. T-cell responses were evaluated in 32 patients 12\u201328 days after their second vaccination and in 10 patients 28 days after their third vaccination. At each visit, medication history, disease activity, leukocyte subtypes , the possibility of meanwhile SARS-CoV-2-infection , and vaccination associated adverse events were reported. \u00ae Anti-SARS-CoV-2 assay [\u00ae e801 analyzer  at the Department of Laboratory Medicine, the Medical University of Vienna (certified acc. to ISO 9001:2015 and accredited acc. to ISO 15189:2012).Humoral immunity was quantified by assessing antibodies to the receptor-binding domain of the viral spike protein using the Elecsys anti-SARS-CoV-2 S immunoassay . The ran-2 assay . Antibod5 cells per well were incubated with SARS-CoV-2 peptides , AIM-V medium , or PHA  in 96-well plates coated with 1.5 \u03bcg anti-IFN-\u03b3  for 24 h. After washing, spots were developed with 0.1 \u03bcg biotin-conjugated anti-IFN-\u03b3 , streptavidin-coupled alkaline phosphatase , and 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium (Sigma). Spots were counted using a Bio-Sys Bioreader 5000 Pro-S/BR177  and Bioreader software generation 10. T-cell responses were considered positive when mean spot counts were at least threefold higher than the mean spot counts of the unstimulated wells.Cellular immunity was assessed by a T-cell stimulation assay. PepMix\u2122 SARS-CoV-2 peptide pools were purchased from JPT . The pools comprised 15-mer peptides overlapping by 11 amino acids (aa) covering the entire sequences of the SARS-CoV-2 spike protein. The spike peptides were split into two sub-pools, S1 (aa 1-643) and S2 (aa 633-1273). Peptides were dissolved in dimethyl sulfoxide and diluted in an AIM-V medium for use in the ELISpot assays. For ex vivo ELISpot assays, PBMCs were thawed. A total of 1\u20132 \u00d7 10The statistical analysis was performed using SPSS Statistics 27 , and figures were produced in the R packages \u201cggplot2\u201d, \u201cggpubr\u201d, and \u201cviridis\u201d. According to the distribution, continuous variables are represented as the median with 25% quartile (Q1) and 75% quartile (Q3). Differences in unpaired groups were assessed using the Wilcoxon rank-sum test. For multiple testing, Bonferroni correction was applied when indicated. Categorical variables are represented as numbers and rates in percent. Differences in categorical variables of unpaired groups were compared using Fisher\u2019s exact test. The association of relevant variables with seroconversion was described with univariate and a multivariable logistic regression analysis adjusting for age and sex.Overall, 66 patients suffering from hematologic malignant diseases, as well as 66 healthy controls (HCs), were included in the study. The median age of our cohort was 62  years, and 26 (39%) were female. The median age of the HCs was 50 , and 26 (39%) were female. Patient\u2019s baseline demographics are highlighted in p < 0.001). Additionally, SARS-CoV-2 S antibody levels were significantly lower in patients  compared to HCs .At the baseline, two patients and three healthy controls had detectable antibodies in the nucleocapsid-based chemiluminescence assay. After the first mRNA SARS-CoV-2 vaccination fewer patients  showed antibody response in comparison to healthy controls . The hematological malignancies associated with the lowest response were aggressive non-Hodgkin lymphomas (NHL) , followed by indolent NHL , CLL , multiple myeloma , and myeloid diseases . One patient with Langerhans cell histiocytosis did seroconvert. One patient each with ALL and Hodgkin\u2019s lymphoma did not seroconvert. Several variables such as age < and >65a, sex, CD8+ T-cells < 280/\u00b5L and CD19+ B-cells < 110/\u00b5L, anti-CD20 therapy, and chemotherapy were analysed for association with the rate of seroconversion. The presence of Anti-CD20 therapy within the last year , the number of CD19+ B-cells < 110/\u00b5L , and the number of CD4+ T-cells < 310/\u00b5L  were associated with lower rates of seroconversion. Age > 65a correlated with an increased rate of seroconversion  (After the second mRNA SARS-CoV-2 vaccination seroconversion was evident in 52% (34/66) of the patients and in 100% (66/66) of the HCs (= 0.046) . Multiva= 0.046) .p = 0.1). It was highest in patients who received allogeneic stem cell transplantation , followed by a patient who received autologous stem cell transplantation . Six patients received anti-CD19 CAR T-cells, of which only one patient (17%) showed seroconversion after four weeks.Obinutuzumab was exclusively administered in patients <65a  and patients <65a suffered numerically more often from aggressive NHL, associated with the poorest seroconversion rate . The overall response rate in bone marrow transplant recipients was similar to the other patients in this cohort . Antibody titers were lower in patients aged <65a , history of anti-CD-20 therapy within the last 12 months , CD19+ B-cells < 110/\u00b5L , and CD4+ T-cells < 310/\u00b5L   in comparison to HCs  .+ B-cells (p < 0.001). CD4+ T-cell levels correlated with the seroconversion rate of the different hematological malignancies (p = 0.037). Additionally, CD4+ T-cell levels were lowest in patients with aggressive NHL , followed by indolent NHL  and CLL . CD4+ T-cell levels did not correlate with the history of anti-CD20 therapy within the last 12 months (p = 0.14).The history of anti-CD-20 therapy within the last 12 months correlated with lower CD19SARS-CoV-2 S antibody levels were reassessed 202 days  after the second vaccination in 53 patients and 58 HCs. For further analysis, the patients were divided into two groups in dependence of seroconversion four weeks after the second vaccination . p < 0.001; HCs four weeks after vaccination, . Interestingly, 10 of 32 (31%) of the 2VR demonstrated a delayed increase of SARS-CoV-2 S antibodies between the four-week visit and the six-month visit, compared to 7 of 58 (12%) of the HCs. Further, SARS-CoV-2 S antibodies of patients responding to the second vaccination (2VR) did differ significantly compared to HCs six months after the second vaccination .SARS-CoV-2 S antibodies of second vaccination responders and HCs significantly decreased over the course of six months after the second vaccination . The third vaccination was performed in 33 patients  at a median of 176  days after the second vaccination. The vaccination was performed in 17 patients (51.5%) with BNT162b2, eight patients (224.24%) with mRNA-1273, and in eight patients (24.24%) with AZD1222. Only 3 of the 19 2VNR experienced seroconversion after the third vaccination (16%). The 2VR demonstrated a significant increase in SARS-CoV-2 S antibody level . A logistic regression model did not find an association for sex, anti-CD20 therapy within the last year, CD8+ T-cells < 280/\u00b5L or CD19+ B-cells < 110/\u00b5L, or frequency of T-cell responses  compared to patients >65a , and CD19+ B-cells < 110/\u00b5L , compared to patients with CD19+ B-cells within the normal range  at 4 weeks after the second vaccine dose. After the third dose, T-cell responses were reassessed in 10 of 22 patients. Five of those ten patients had already mounted T-cell-specific responses after the second vaccination, which increased to 7 out of 10 patients after the third vaccination (SARS-CoV-2-specific T-cell responses were determined in 32 (49%) patients after the second vaccination and 16 HCs to assess if vaccination induced a cellular immune response. Interestingly, 19 out of 32 patients (59%) demonstrated a T-cell response compared to 100% (16/16) of the HCs  are significantly correlated with anti-SARS-CoV-2 S IgG levels after the second vaccination. CD4+ T-cell levels were particularly low in patients with aggressive NHL, the disease with the lowest humoral response. An analysis of T-cell responses in humans with COVID-19 disease and unexposed individuals demonstrated that virus-specific CD4+ T-cells are present in 100% of convalescent patients and that CD4+ T cell levels correlated with the magnitude of anti-SARS-CoV-2 S IgG [+ T-cell responses guide the adaptive immune response to the second vaccination [We demonstrated that levels of CD4-2 S IgG . This iscination .Another risk factor for seroconversion we found was age <65a. This is somewhat in contrast to previous literature, but the uneven distribution of obinutuzumab/rituximab therapy and the numerical higher number of aggressive lymphomas in patients aged <65a might contribute to this finding .+ T-cells was shown [+ B-cells within the normal range. We did not find a gender-associated difference in the SARS-CoV-2-specific T-cells response [In addition to the antibody response to mRNA SARS-CoV-2 vaccination, T-cell responses contributed to vaccine efficacy in healthy controls ,25,27. Fas shown . In our response . Furtherresponse .A special subgroup in our cohort comprises six anti-CD19 CAR T-cells recipients. Only 1 of 6 patients demonstrated seroconversion. Given the mechanism of action with T-cells eliminating CD19-expressing malignant and normal cells, this finding is not surprising .Another aim of this study was to assess long-term responses after mRNA SARS-CoV-2 vaccinations in hematological patients. In both HCs and patients, the visit after six months demonstrated a significant reduction of anti-SARS-CoV-2 S IgG levels. However, patients responding to the second vaccination more often demonstrated a delayed increase of SARS-CoV-2 S antibodies in comparison to HCs.Previously, the lack of immunogenicity of mRNA SARS-CoV-2 vaccination has led to evaluations of third vaccine immunization in different COVID-19 high-risk cohorts. Recent reports of transplant recipients demonstrated that a third dose of mRNA vaccine had substantially higher immunogenicity compared to the placebo ,30. FirsWe report several limitations. In this cohort, hematological patients were older than HCs, as HCs consisted mainly of healthcare professionals working at our center. Hence, the demonstrated difference between patients and HCs could be overestimated, as older age was shown to be a major factor for weaker serological responses . Further+ 19 B-cell and CD4+ T-cell levels were shown to be additional predictive markers for seroconversion. T-cell responses were reported in 59% of the patients and were higher in patients with low CD19+ B-cell counts. Whether the preserved T-cell response in patients with anti-CD20 therapy and low CD19+ B cell counts contribute to some degree of protection requires further evaluation. Over a period of six months, vaccine responders and HCs demonstrated a significant decline in antibodies. In those patients, the third vaccination was demonstrated to be effective in booster B-cell response. In vaccine non-responders, a third vaccination yielded seroconversion in a minority of the patients, suggesting a limited benefit from a third vaccination in terms of seroconversion in this group.In conclusion, our findings demonstrate that the full immunization schedule was able to induce anti-SARS-CoV-2 S IgG responses in 52% of patients. B-cell depleting therapy was the major factor for a poor serological response. CD"}
+{"text": "The antibody response decreased 12 weeks after the primary vaccination. After the booster, humoral and cellular responses significantly improved, with comparable seroconversion rates between the heterologous and homologous groups. Positive sVNT against the wild type occurred in >90% of LT patients, with only 12.3% positive against the Omicron variant. Conclusions: ChAdOx1/BNT162b2 evoked a significantly higher immunological response than ChAdOx1/ChAdOx1 in LT recipients. The booster strategy substantially induced robust immunity against wild type in most patients but was less effective against the Omicron strain.Background: Heterologous prime-boost vaccination potentially augments the immune response against SARS-CoV-2 in liver transplant (LT) recipients. We investigated immunogenicity induced by different primary prime-boost vaccination protocols and the subsequent response to the booster vaccine among LT recipients. Methods: LT recipients, who received primary immunisation with ChAdOx1/ChAdOx1 or ChAdOx1/BNT162b2, were administered the third dose of mRNA-1273 three months following the primary vaccination. Blood samples were collected before and after primary vaccination and post-booster. The levels of receptor binding domain antibody (anti-RBD) and neutralising antibody (sVNT) and spike-specific T-cell responses were assessed. Results: Among the 89 LT recipients, patients receiving ChAdOx1/BNT162b2 had significantly higher anti-RBD titres, sVNT, and cellular response after primary vaccination than those receiving ChAdOx1/ChAdOx1 ( The rapid spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has become a global public health concern and has been declared a pandemic since December 2019 ,4. PatieAlthough considered safe and effective in minimising disease severity and mortality, several reports have revealed inadequate immune responses in LT recipients after a standard two-dose SARS-CoV-2 vaccination, with serological response rates ranging from 47\u201379% ,9,10. HoTo explore these issues among LT recipients, we conducted this study with three main aims: (1) to evaluate SARS-CoV-2 specific humoral and cellular immune responses after primary vaccination with heterologous versus homologous prime-boost protocols, (2) to explore immune dynamics after primary immunisation, and (3) to assess the subsequent response to the booster vaccine following different primary vaccine series.This prospective, longitudinal observational study was conducted between June 2021 and February 2022 at a single liver transplant centre . SARS-CoV-2 na\u00efve LT recipients aged \u2265 18 years were enrolled in the study. All LT patients were immunised with either homologous [ChAdOx1 /ChAdOx1] or heterologous [ChAdOx1/BNT162b2 ] as their primary vaccine protocol. Vaccination schemes were determined by the national policies of the Ministry of Health of Thailand according to the availability of vaccines. All participants received an additional dose of mRNA-1273  three months following the standard two-dose vaccine series. The exclusion criteria were LT patients with a history of confirmed SARS-CoV-2 infection, prior immunisation with any SARS-CoV-2 vaccine, acute cellular rejection, or pregnancy. Blood samples were collected immediately before the first vaccination, four weeks after the second vaccination, twelve weeks after the second vaccination, and four weeks after the booster vaccine.Demographic, clinical, and biochemical data were obtained from the electronic medical records at the time of the first vaccination. All LT recipients were screened for active respiratory tract infection and SARS-CoV-2 exposure by questionnaire at every visit. Adverse events after vaccination were assessed using a questionnaire and a phone interview.\u00ae anti-SARS-CoV-2 S using Cobas e411 immunoassay analysers . The sensitivity and specificity of this assay were 93.9% and 99.6%, respectively  (p = 0.32)  U/mL vs. 152.1 (13.6\u2013678.8) U/mL, (= 0.02)] . However = 0.32) .p = 0.09, 43.8% reduction rate), whereas the median anti-RBD IgG in patients receiving ChAdOx1/BNT162b2 significantly decreased to 638.5 (142.5\u20131245.5) U/mL . Nevertheless, the SARS-CoV-2 spike total antibodies remained significantly higher in the heterologous prime-boost group than in the homologous prime-boost group (p < 0.001).Twelve weeks after the primary vaccination, a decline in anti-RBD antibodies was observed in both primary vaccine platforms . The medp < 0.001). Seroconversion occurred in 81.3% of LT recipients receiving ChAdOx1/ChAdOx1/mRNA-1273 and in 94.7% of those receiving ChAdOx1/BNT162b2/mRNA-1273. Although the additional vaccine yielded strikingly high antibody levels, no significant difference was observed in the anti-RBD titre and seroconversion rate between the two groups after the booster  (The booster vaccine significantly improved immune response . The medctively) .p = 0.025). Owing to the high seroconversion rate after booster vaccination, we could not identify significant predictors of the immune response following the third vaccine.Potential variables as predictors of poor humoral response after primary vaccination were evaluated using logistic regression analysis . In univp = 0.01] (p < 0.001)  was investigated at four weeks following primary immunisation and four weeks after the booster vaccination. Following primary immunisation, participants receiving ChAdOx1/BNT162b2 had significantly higher median sVNT levels than those receiving ChAdOx1/ChAdOx1  . ChAdOx1< 0.001) .p = 0.06) (p = 0.04). Notably, the sVNT level demonstrated a significant correlation with the anti-RBG titre after primary vaccination  and post-booster .After an additional dose of mRNA-1273, the median sVNT level to wild type increased to 97.6% (96.8\u201397.8%) in patients vaccinated with ChAdOx1/BNT162b2 and 97.3% (69.2\u201397.6%) in those immunised with ChAdOx1/ChAdOx1; the difference was not statistically significant ( = 0.06) . HoweverNeutralising antibodies to the Omicron variant were assessed in blood samples after booster vaccination. Notably, almost all LT patients had a substantially low level of sVNT to the Omicron strain . Only 10p > 0.05) (6 PBMC cells of LT patients receiving the heterologous primary vaccine protocol was significantly higher than those receiving the homologous protocol (p = 0.02) or sVNT  and spike-specific T-cell responses.The T-cell response was evaluated in 58 randomly selected LT patients: 41 in the ChAdOx1/BNT162b2 group and 17 in the ChAdOx1/ChAdOx1 group. The baseline clinical and laboratory characteristics were comparable between the two groups ( > 0.05) . The resprotocol . A signiOverall, most patients experienced minor adverse events after injection of the primary vaccination and booster . Both vaIn this prospective study, we evaluated the immunogenicity and dynamics of SARS-CoV-2 specific immune response as well as the subsequent benefit of the booster among LT recipients who received homologous versus heterologous primary vaccination. Following primary immunisation, the heterologous protocol (ChAdOx1/BNT162b2) induced a more robust immune response than that with the homologous protocol (ChAdOx1/ChAdOx1). The antibody level eventually decreased three months later, regardless of the vaccine platform. The additional third dose significantly improved humoral and cellular immune responses in both the primary vaccine groups. Most LT recipients had strong immunity against the wild-type strains. Nevertheless, the neutralising activity against Omicron variants was substantially attenuated in both groups, even after booster vaccination.n = 11). In addition, Mendizabal et al. reported that heterologous adenoviral vector/mRNA improved the humoral response in Argentinian LT patients [At the beginning of the pandemic, the mRNA vaccine was unavailable in most low- and middle-income countries. In Thailand, inactivated and adenoviral-vectored vaccines were the first vaccines to be inoculated in the general population during the outbreak before the arrival of mRNA vaccines . The usepatients . In our patients ,21. We aWe also evaluated cellular immune responses, which may contribute to immune longevity . Our finNotably, the decrease in total anti-spike antibodies was observed in both vaccine protocols three months after the primary vaccination. Immune waning may diminish the protective effect against SARS-CoV-2, especially in patients in the homologous group, wherein less than half of those demonstrated detectable antibodies. Antibody kinetics were consistent with the results observed in a recent study in LT recipients .Regarding the diminished immune response in LT recipients after primary immunisation ,24, the The emergence of variants of concern, for example the Omicron variant (B.1/B.1.1.529), has raised concerns about vaccine efficacy. With highly prevalent mutations in the spike protein resulting in the potential to escape the host immune response, it is anticipated that a markedly low humoral immune response against the Omicron strain will be observed . DespiteThe strength of our study is that it was a longitudinal prospective study that provided comprehensive information regarding SARS-CoV-2-specific immune response, including anti-spike antibodies, neutralising activities, and T-cell responses, from baseline to post-booster in LT recipients. We investigated different types of primary vaccine regimens based on previous studies conducted in the Western region. Therefore, our results could be helpful in settings where the mRNA vaccine was not the predominant platform for primary immunisation or experienced vaccine supply issues, especially in developing countries.Our study had some limitations. First, although we present clinical evidence in a real-world setting, the study was conducted in a single transplant centre, precluding generalisation to a broader population of LT recipients. Second, the study was limited by the absence of randomisation. We observed a relatively lower number of participants in the homologous vaccine group than in the heterologous vaccine regimen. However, the baseline characteristics were comparable between patients immunised with the two different vaccine platforms. Third, our study included only LT patients receiving homologous primary vaccination with ChAdOx1/ChAdOx1 and thus does not reflect whether ChAdOx1/BNT162b is superior to homologous BNT162b/BNT162b protocol. Lastly, there were no comparative data among healthy populations for immune responses. In a state of immunosuppression, weaker immune responses were expected among LT recipients in several studies ,8,9. DesPrimary vaccination with the heterologous regimen induced better SARS-CoV-2 specific immune response among LT patients than the homologous protocol. Waning immunity was observed after the primary immunisation, regardless of the vaccine regimen. The booster strategy produced a more robust immune reaction in most LT recipients. Nevertheless, protective efficacy against emerging virulent strains and immune sustainability after booster vaccination should be assessed in future studies."}
+{"text": "Effective SARS-CoV-2 vaccination in patients receiving treatment with B-cell depleting agents is challenging. Information on vaccination responses in these patients are a valuable tool to develop efficient vaccination regimens.In this single-center retrospective observational study, we report the humoral and cellular response in 34 patients receiving anti-CD20 antibody treatment for renal immune disease.After base immunization with SARS-CoV-2-vaccines, 92.0% developed a cellular, 32.4% a humoral response. Humoral immunity correlated with B-cell counts and the timespan between anti-CD20 antibody treatment and vaccination. All patients with\u2009>\u200921/\u00b5l B-cells, or\u2009>\u2009197\u00a0days after treatment showed humoral response.Adequate timing of SARS-CoV-2-vaccinations after anti-CD20 antibody treatment and CD19 measurements are crucial to generate immunity. Awaiting partial B-cell recovery by postponing regularly scheduled anti-CD20 treatment should be considered in patients with stable immune disease.Trial registration: This study has been retrospectively registered in the German Clinical Trials Register (DRKS00027049) on 29/10/2021.The online version contains supplementary material available at 10.1186/s12879-022-07722-7. Successful vaccination of risk groups is essential to reduce morbidity and mortality in the COVID-19 (coronavirus disease 2019) pandemic. Vaccine-induced immunity relies on antibody-driven , and T-cell mediated (cellular) immune response .Effective vaccination of immunocompromised patients is especially challenging as many immunosuppressive agents impair the response to vaccines, increasing the risk for severe COVID-19, prolonged infection, and viral evolution , 5.Several immune-mediated and hematologic diseases are treated with monoclonal anti-CD20 antibodies. These drugs deplete circulating B-cells, leading to impaired maturation of memory B-cells and reduced numbers of antibody-producing plasma cells. ConsequChronic kidney disease is a further independent risk factor known to impair vaccination responses . Hence, Here, we report the humoral and cellular immune response to mRNA- and vector-based SARS-CoV-2 vaccines  in patients receiving the monoclonal anti-CD20 antibody Rituximab for renal autoimmune disease. Our results help to optimize vaccination strategies by taking prior therapies and cellular parameters into account.In this single-center retrospective observational study, we report data from the University of Freiburg Medical Center, Germany. Inclusion criteria were age\u2009\u2265\u200918\u00a0years, current treatment with the anti-CD20 antibody Rituximab for renal autoimmune disease, and completed base-immunization with either mRNA-based , or vector-based  SARS-CoV-2 vaccines. In vaccination regimens requiring two doses for base immunization, the second dose was given between 2 and 12\u00a0weeks after the first vaccination. Exclusion criteria were prior SARS-CoV-2 infections, and administration of antimetabolite drugs  within two months before vaccination.Demographic data, past medical history, treatment details and laboratory findings  were extracted from electronic patient records of outpatient visits between April and September 2021. B cell counts were measured in between 5\u00a0weeks prior, to 12\u00a0weeks after vaccination by flow cytometry , and\u2009\u2265\u2009200 mIU/ml (for IFN-\u03b3 negative control\u2009>\u2009100 mIU/ml), using a double-cut-off strategy integrating the result of background stimulation according to in-house standard procedure  received mRNA vaccines (BNT162b2: n\u2009=\u200925 [73.5%]), mRNA-1273: n\u2009=\u20091 [2.9%]). Six patients (17.6%) received vector-based vaccines . Two patients (5.9%) received a first dose of the vector-based vaccine ChAdOx1, followed by a mRNA-based vaccine .Humoral response was detected in 11 (32.4%) patients. Cellular response was detected in 23 (92%) patients. One individual had neither response, while an isolated response was observed in another individual. Kidney function, proteinuria, or hematuria, as well as timespans between first and second vaccinations and the timepoint of testing after vaccination were not altered between response groups (Table Kidney function, proteinuria and hematuria did not correlate with SARS-CoV-2-IgGs or IFN-\u03b3-release (Additional file B-cell counts at vaccination correlated with the time from last anti-CD20-antibody treatment to vaccination and were significantly higher in patients with a humoral response Table A. SigmoiPatients receiving additional corticosteroids are overrepresented in humoral and cellular non-responder groups Table . A subgrFinally, there were no significant differences in humoral, or cellular response in patients receiving different vaccination regimens. However, in line with previous reports , we obseAnti-CD20 treatment impairs humoral response to vaccinations. Data on SARS-CoV-2 vaccines is still limited, but previous studies have shown negative effects on antibody production after the BNT162b2 vaccine in patients receiving B-cell depleting agents , 14.This study confirms impaired humoral response to SARS-CoV-2 vaccinations in patients receiving anti-CD20 treatment for renal autoimmune diseases. We show that the humoral response strongly correlates with B-cell counts and the interval between last anti-CD20 antibody administration and vaccination. Both, time from Rituximab to humoral response and CD19 counts necessary for vaccination response, are similar to results reported in parallel studies \u201320. ThisOur study shows that the cellular response to SARS-CoV-2 vaccines is independent of CD19 depletion, confirming other recent reports , 21. TheIn line with other recent studies \u201324, we oWe do not observe reduced kidney function to be a statistically significant risk factor for impaired vaccination response. However, parallel studies report reduced response to mRNA SARS-CoV-2 vaccines in both subjects with chronic and end stage renal disease , 26.This study has several limitations, the major being the small study population, the retrospective character, and the lack of a standardized follow-up. The multitude of anti-CD20 antibody treatment regimens further complicate the interpretation. However, our study provides valuable information on how to optimize the humoral vaccination response, and how to reduce the risk for COVID-19 in an especially vulnerable patient cohort.Based on our data, awaiting partial B-cell recovery, and thus postponing regularly scheduled anti-CD20 treatment, should be considered in patients with stable renal disease. We and others  observedAdditional file 1. Supplemental Figure S1, Supplemental Tables S1\u2013S5, Supplemental Methods."}
+{"text": "Although mRNA-based vaccines against SARS-CoV-2 induce a robust immune response and prevent infections and hospitalizations, there are limited data on the antibody response in individuals with humoral immunodeficiency. The aim of this study was to evaluate the humoral immune response after two vaccine doses with BNT162b2 or mRNA-1273 in patients with humoral immunodeficiency disease.This cross-sectional study assessed 39 individuals with hypogammaglobulinemia under immunoglobulin replacement therapy. IgG anti-SARS-CoV-2 spike protein antibodies (anti-S) were measured 4 weeks to 4 months after two doses of an mRNA vaccine against SARS-CoV-2. The proportion of patients, who developed a humoral immune response to the spike protein were evaluated and compared to 19 healthy controls.After vaccination with two vaccine doses, 26/39 patients (66.7%) with humoral immunodeficiency disease and all healthy controls developed anti-S. In subjects with baseline IgG <3 g/l, only 1/5 (20%) showed a humoral immune response. 10 out of 26 with CVID (38.5%) and 7/9 under immunosuppressive drugs (77.8%) developed no immune response (13 subjects with no response) compared to 0/19 in healthy controls. Subgroup analysis in patients without immunosuppressive drugs revealed lower anti-S in patients with moderate to severe humoral immunodeficiency disease: baseline IgG <3 g/l: 12.0 AU/ml (95%CI 12.0\u2013125.0), baseline IgG 3\u20135 g/l: 99.9 AU/ml (95%CI 14.4\u2013400.0), baseline IgG >5 g/l: 151.5 AU/ml (95%CI 109.0\u2013400.0), healthy controls 250.0 AU/ml (95%CI 209.0\u2013358.0), p = 0.007.In most patients with mild to moderate humoral immunodeficiency we found only slightly lower anti-S antibodies compared with healthy controls after two vaccine doses with BNT162b2 and mRNA-1273. However, in patients with a decreased baseline IgG below 3 g/l and/or under immunosuppressive drugs, we found severely impaired humoral immune responses. Following the global spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccinations have been introduced since the end of 2020 to control the pandemic. In particular, mRNA-based vaccines are considered highly effective in prevention of infection and hospitalization, even against variants of concern ,2. To daWe studied the humoral immune response against the S1/S2 spike protein of SARS-CoV-2 in patients with humoral immunodeficiency disease. Subjects with a primary or secondary hypogammaglobulinemia  and treated by intravenous (IVIG) or subcutaneous immunoglobulin (SCIG) replacement therapy in our outpatient clinic were included if they had received two mRNA Covid-19 vaccine doses  or mRNA-1273 ) 4\u20136 weeks apart between January and June 2021. CVID was defined with the following characteristics: significant reduced total IgG, reduced IgA or IgM values, poor vaccine response, recurrent infections and absent T-cell impairment . As the All data on disease, treatment, and vaccinations were collected by the attending physicians based on the medical records. All individual vaccination dates and the vaccines administered were obtained from the original vaccination records in each study participant. Healthy volunteers were recruited among the private and professional surroundings of the investigators without performing a matching procedure. Single serum samples for analysis of vaccine antibodies were collected 2 weeks to 4 months after the second Covid-19 vaccination in all study participants and healthy controls.Blood samples were run on an Abbott ARCHITECT i2000 instrument using the Abbott SARS-CoV-2 nucleoprotein assay and the DiaSorin LIAISON\u00ae SARS-CoV-2 S1/S2 IgG assay following the manufacturer\u2019s instructions. The Abbott assay is a chemiluminescent microparticle immunoassay for qualitative detection of IgG in human serum or plasma against the SARS-CoV-2 nucleoprotein. The Liaison SARS-CoV-2 S1/S2 is a chemiluminescence assay, that uses paramagnetic microparticles coated with S1 and S2 fragments of the viral surface spike protein (positive cut-off of >12 AU/ml). According to the manufacturer\u2019s information, the Liaison SARS-CoV-2 S1/S2 IgG assay of >15 AU/ml reached a plaque-reduction neutralization test (PRNT) titer of 1:40 in 17/18 patients and a PRNT titer of >1:160 if SARS-CoV-2 S1/S2 IgG of 80 AU/ml were analysed . PresencThis study was approved by the local ethics committee . All subjects included signed informed consent.Statistical analysis were performed using Graphpad Prism 9 . Patient characteristics are summarized with descriptive statistics. Values are median and interquartile ranges (IQR) for continuous variables, categorical variables reported as n (%). The 95% confidence intervals (95%CI) from the proportions of patients with positive anti-S values in a specific group were calculated as Wilson/Brown interval. Anti-S titers were compared by Kruskal-Wallis or Mann-Whitney test, the median 95%CI was calculated for all groups.47 patients with a humoral immunodeficiency treated by immunoglobulin replacement therapy and vaccinated twice with one of the Covid-19 mRNA vaccines were identified in a single center. Eight subjects were excluded . Of the 39 patients included, 28 were female (71.8%) compared to 15/19 healthy controls (79.0%). The median age was 61 years  in the study group and 53 years  in the control group . CharactAfter two vaccine doses, we found detectable anti-S levels in 26/39 subjects (66.7% [95%CI 51.0\u201379.4%]) with a humoral immunodeficiency disease, and in all healthy controls . In CVID, the proportion of patients with presence of anti-S was 16/26 (61.5% [95%CI 42.5\u201377.6%]), and thus lower than in non-CVID patients  and in the healthy control group , p = 0.002. All individuals with a humoral non-response to the vaccines (n = 13) were either under immunosuppressive drugs (including anti-CD20 therapy in the past)  and/or had a diagnosis of CVID . Two patients treated by immunosuppressive treatment showed a low anti-S response . Median In a post-hoc analysis, we obtained anti-S levels from 22 patients after the third vaccination . A third26/39 of our patients with humoral immunodeficiency showed a detectable immune response against SARS-CoV-2 after mRNA vaccination. After the second Covid-19 vaccine, the majority of individuals with a mild to moderate IgG deficiency had a specific anti-S concentration that was slightly below the level of healthy controls. Even in most patients with CVID an immune response was detected after a second dose of the Covid-19 mRNA vaccine. Our findings are in line with recent studies, demonstrating an increase of anti-S levels in most individuals with humoral immunodeficiency without immunosuppressive drugs after two Covid-19 vaccinations \u201322. In aTherefore, anti-S antibody determination might be useful to detect non-responders after completion of a two Covid-19 vaccination schedule in patients at risk, to enable a timely third vaccination. In patients under immunosuppressive drugs or humoral immunodeficiency, a full vaccination schedule should include three Covid-19 mRNA vaccinations followed by a booster vaccine . If a deOur study has limitations: Although both mRNA vaccines seem to be effective in a majority of patients with humoral immunodeficiencies, we have no long-time data since the observational period was very short. Blood samples were taken over a wide time interval (4 weeks to 4 months) in both study groups. Since antibody titers decrease over time, it is therefore likely, that the measured antibody levels underestimate the peak levels in these patients. Furthermore, it has to be assumed that patients with a humoral immunodeficiency have a faster decline of the IgG-anti-S titer than healthy controls. In addition, it is unclear whether the anti-S antibodies we measured are neutralizing in nature. We cannot make any statement about the T-cell response, which certainly plays a major role. Specific T-cell responses seem to be defective in about one third of CVID patients . Lastly,Although individuals with humoral immunodeficiencies have impaired responses to vaccines, BNT162b2 and mRNA-1273 mRNA vaccines produce humoral immune responses in the majority of these patients. Most individuals with mild to moderate humoral immunodeficiency disease without immunosuppressive drugs did not have significantly reduced anti-S values compared to healthy individuals. Treatment with immunosuppressive drugs and a diagnosis of CVID are risk factors for reduced or even absent humoral response. In these individuals, a full vaccination schedule should include three Covid-19 mRNA vaccinations as primary series followed by a booster vaccine.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file."}
+{"text": "Corona mortis, can lead to death in the worst-case scenario if injured. Despite being well-known, exact anthropometric data are lacking. The purpose of this study was to determine diameters of the regular obturator artery, the Corona mortis and the inferior epigastric artery. In addition, the level of origin of the Corona mortis was quantified. The obturator artery and its norm variants were dissected bilaterally in 75 specimens  and measured using two different methods. The Corona mortis was present in 36 of the 150 hemipelves (24%), presenting in one third of all cases bilaterally. Its level of origin measured from the commencement of the inferior epigastric artery was subject to high variability (4.4\u201328.3\u00a0mm). The mean diameters of the Corona mortis  and the regular obturator artery  were similar for both methods. There were no significant sex nor side differences. The diameter of the inferior epigastric artery was significantly smaller distal to the origin of the Corona mortis. The high incidence, non-predictable level of origin of the Corona mortis and its size similar to the regular obturator artery support its clinical relevance even to date. Clinicians should always be aware of an additional arterial vessel close to the pelvic brim.An enlarged anastomosis connecting the vascular territory of the external iliac and the obturator artery may replace most or all of the latter. This relatively common vascular variation, known as  Corona mortis, what means crown of death. The first author, who described the course of this replaced obturator artery was the anatomist Haller . While alive, all individuals bequested their post mortem bodies for their use in medical education and research. In addition to their informed consent, institutional approval was obtained from the Ethics Committee of the Medical University of Vienna . All specimens were embalmed by perfusion and immersion with a diluted solution of formalin (1.6%) and phenol (4%). Bodies with malignant tumours or vascular surgical interventions in the pelvic and femoral regions were excluded.Corona mortis was determined overall, and regarding sex and body side. As generally accepted . To minimize the probability of a methodological error, two different methods of measurements were performed. With the first one, the diameter of the vessels was determined without any compression. This method was named \u201cunfolded\u201d , IBM SPSS Statistics 26 , and GraphPad Prism 9.3.1 . To prove a possible correlation between the incidence of a \u03b1\u2009=\u20090.05. In the text, mean values\u2009\u00b1\u2009standard deviations are given.The significance level was set at Corona mortis was observed in 27 individuals (36.0%). It was present in 16 of the 38 males (42.1%) and in 11 of the 37 females (29.7%). In relation to the pelvic halves, a Corona mortis occurred in 36 of 150 cases (24%). Thereby it was present in 19 male (25%) and 17 female hemipelves (23%).In the 27 individuals with a Corona mortis, it was present on only one side in 18 cases (66.7%). In the remaining nine individuals (33.3%) the Corona mortis was observed bilaterally. This was twice as much the case in females than in males . In males, the unilateral cases were present in nine left and only four right sides. In females, the unilateral cases were more balanced (two left vs. three right sides). For detailed information, please refer to Table A Corona mortis with sex (p\u2009=\u20090.771) or with body side (p\u2009=\u20090.444).There was no correlation between the occurrence of a Corona mortis were coincidentally present in only two left male hemipelves   distal to the commencement of the inferior epigastric artery from the external iliac artery. Although it originated more distally in women and on the left side on average, there was no difference between sexes (p\u2009=\u20090.210) nor sides (p\u2009>\u20090.999). In the four cases with Corona mortis origin from the external iliac artery, this was highly variable, ranging from 15.0 to 27.7\u00a0mm proximal to the commencement of the inferior epigastric artery. Due to the small number of cases, no further statistical analyses were conducted.The 9%) Fig.\u00a0. In only9%) Fig.\u00a0. The levCorona mortis was 2.5\u2009\u00b1\u20090.5\u00a0mm (\u201cunfolded\u201d), and 2.1\u2009\u00b1\u20090.4\u00a0mm (\u201cHillen\u201d). The maximum diameter of the Corona mortis was 3.7\u00a0mm (\u201cunfolded\u201d) and 3.1\u00a0mm (\u201cHillen\u201d). However, these values were measured in two different male individuals. The minimum diameter was determined in one female individual with 1.3\u00a0mm (\u201cunfolded\u201d) and 0.9\u00a0mm (\u201cHillen\u201d). In cases with bilateral Coronae mortis the diameters were mostly the same on both sides and showed differences of 0.0\u00a0mm to at most 0.6\u00a0mm (both measurements methods). Only in one bilateral case, the difference amounts 1.2\u00a0mm (\u201cunfolded\u201d). For further details regarding sex and body side, please refer to Fig.\u00a0Corona mortis, neither for the method \u201cunfolded\u201d (p\u2009=\u20090.947) nor for the method \u201cHillen\u201d (p\u2009=\u20090.880). There were also no statistically significant differences in the diameters of the Corona mortis between sexes or body sides for both methods.The diameter of the regular obturator artery averaged 2.4\u2009\u00b1\u20090.6\u00a0mm (\u201cunfolded\u201d) and 2.0\u2009\u00b1\u20090.5\u00a0mm (\u201cHillen\u201d). This vessel originated in 79 cases from the ventral and in 35 cases from the dorsal trunk of the internal iliac artery. In two cases, it was impossible to identify a clear trunk-formation of the branches of the internal iliac artery. The average diameter of the Corona mortis was 3.4\u2009\u00b1\u20090.7\u00a0mm (\u201cunfolded\u201d) and 2.8\u2009\u00b1\u20090.5\u00a0mm (\u201cHillen\u201d). Distally to the commencement of the Corona mortis it was reduced to 2.8\u2009\u00b1\u20090.5\u00a0mm (\u201cunfolded\u201d) and 2.3\u2009\u00b1\u20090.4\u00a0mm (\u201cHillen\u201d), respectively  as well as for the method \u201cHillen\u201d (p\u2009<\u20090.001).The average diameter of the inferior epigastric artery proximally to the origin of the Corona mortis measured with the method \u201cHillen\u201d were significantly smaller than those measured with the method \u201cunfolded\u201d (p\u2009<\u20090.001). The mean difference was \u2212\u00a00.4\u2009\u00b1\u20090.2\u00a0mm .Diameters of the regular obturator artery and the \u00a0mm Fig.\u00a0d. HoweveCorona mortis, the incidence was calculated from 32 former anatomical, surgical, and radiological studies including 8.257 hemipelves  are also based on case numbers far below 100. Among others, Japanese authors observed by trend a lower occurrence of a Corona mortis , and 2.1\u2009\u00b1\u20090.4\u00a0mm (\u201cHillen\u201d). It was slightly higher in male than in female bodies, but was almost equally concerning the side of the body. The mean diameters measured by other authors ranged from 1.63 to 4.0\u00a0mm (Hong et al. In the present study, the mean diameter of the Corona mortis but also the diameter of the regular obturator artery was measured for comparison. The difference between their two mean diameters was only 0.1\u00a0mm for both measurements methods. Therefore, the Coronae mortis observed here are likely to have almost completely replaced the non-existent regular obturator artery. In practical terms, this means that injury to the Corona mortis may cause a similar amount of blood loss as an injury to the regular obturator artery.In this study, for the first time, not only the diameter of the Corona mortis. As expected, there was a significant reduction in diameter after commencement of the Corona mortis. On the other hand, this means that in the case of a Corona mortis, the inferior epigastric artery in its area of origin, is considerably stronger than usual. In practice, this means that in the case of a Corona mortis, not only is there an increased risk of bleeding from it. Injury to the inferior epigastric artery proximal to the origin of the Corona mortis may also result into higher amount of blood loss than in cases with a regular obturator artery. In the case of arterial occlusive disease, a Corona mortis may also become an important link between the circulation territories of the external and internal iliac arteries. People with this vascular variant may be better able to compensate for occlusions in this area and may not become symptomatic or may become symptomatic later.This study was also the first to compare the diameter of the inferior epigastric artery proximal and distal to the origin of the To minimize the probability of a systematic error, the measurement of diameters was accomplished in two different ways. The determined values of both methods were statistically significantly different from each other but the measured values correlated highly between the two methods. The results of both methods are affected by different factors. The measurement described by Hillen  may be iCorona mortis. However, it was quantified for the first time that the level of origin of the Corona mortis is highly variable in relation to the origin of the inferior epigastric artery. In addition, it was demonstrated for the first time that the diameter of a Corona mortis is nearly identical to that of a regular obturator artery. It was also shown that the diameter of the inferior epigastric artery decreased significantly after the commencement of the Corona mortis. All these results support the clinical relevance of this common vascular variant: It can be expected in about a quarter of all hemipelves. One cannot predict at which level the Corona mortis originates from the inferior epigastric artery or the external iliac artery. Therefore, clinicians should always be aware of the possibility of an additional arterial vessel in this area, which can lead to severe arterial bleeding in the event of an injury.The current study confirms previous data on the incidence of a"}
+{"text": "In bi- and monolingual children, nonword repetition tasks (NWRTs) differentiate between typically developing (TD) and children with Developmental Language Disorder (DLD). Language specificity is a crucial factor in nonword construction especially for multilingual children. While language-specific nonwords seem less artificial than non-specific nonwords, the application of language-specific phonemes may be less suitable for bilingual children who are exposed to the target language less than monolingual peers. This study evaluates the concurrent and predictive value of a novel, computerized NWRT implemented in the MuLiMi web-platform and its potential in the discrimination of bilingual children with and without DLD, investigating the role of nonwords\u2019 language specificity. Thirty-seven children  with at least one Italian-speaking parent, living and attending kindergartens in Germany were tested with the MuLiMi NWRT and German standardized language tests. Caregivers and kindergarten teachers filled in questionnaires. Fourteen of the children were re-tested after 8\u201312\u2009months. The results suggest that the new test\u2019s concurrent and discriminative validity are good. Analysis of variance revealed highly significant differences between children with and without (an objective risk of) phonological disorders and a significant interaction between nonword specificity and risk group. Significant correlations of initial scores with follow-up scores collected after 8\u201312\u2009months were also found, as well as correlations with improvements in language abilities. In conclusion, although both language-specific and language-non-specific nonword repetition can support DLD risk identification in bilingual children, language-specific stimuli appear to be particularly sensitive indicators. This is interpreted as confirming DLD children\u2019s reduced sensitivity to frequent, familiar characteristics of the linguistic stimuli. The test\u2019s discriminative and concurrent validity showed to be robust to various potentially influencing factors like patterns of language exposure. The adequate identification of Developmental Language Disorder (DLD) in bilingual children has been described as a serious diagnostic challenge  with conNWRTs are considered increasingly relevant for the identification of DLD in bilingual children as children\u2019s phonological processing abilities can be assessed directly and\u2014at least compared to other tasks\u2014relatively independently of their prior vocabulary knowledge that might be reduced compared to their monolingual peers due to variations in language input and exposure. Being less subject to the influence of language experience in the language of assessment , they woThe extant literature provides controversial evidence as to which type of NWs  yield the optimal discriminative power for differentiating between weak language performance due to underexposure and actual language impairment in bilingual children. No effects of language experience and dominance were found on the repetition performance of bilingual TD and DLD children with either Arabic, European-Portuguese, or Turkish as their L1 (family or heritage language) who used the French and the German versions of the LITMUS NWRT \u2014not distThe number of conclusions to be drawn from previous research is limited, especially because findings on the relationships and interactions between language experience and repetition performance on language-specific NWs are mixed. In this study we compare NWs that take into account the characteristics of both languages and NWs that are relatively free (or freer) from such language-specific attributes in order to establish which NWs are more useful (discriminant) for the language assessment of bilingual children.Indeed, a prominent issue concerns the need or usefulness of administering NWRTs in the L1, in the L2 or in both languages for the diagnosis of DLD. This heterogeneity in findings may be explained by the great variability in methodological approaches that were applied in these studies, such as differences in the construction of the NWRTs, the characteristics of the NWs, the targeted language of assessment (the children\u2019s L2 or L1) and the children\u2019s age and patterns of language dominance.via questionnaires) has proven to be a sound method  language-specific (LS) items that take children\u2019s language experience/dominance into account and (2) language-non-specific (LNS) items that are intended to assess children\u2019s language abilities regardless of their language background. In order to avoid circular procedures, we will address diagnostic accuracy, taking into account information deriving from alternative tests of phonological ability and information collected through parental and kindergarten teachers\u2019 questionnaires, including active use of language in addition to passive exposure. Finally, the goal of this study is to assess the validity of the \u201cMultiMind Nonword Repetition Task,\u201d which is part of a web-platform .The differences emerging between the groups of children with and without an objective risk of having DLD  based on pre-existing diagnoses and/or standardized language tests scores .The correlations between NWRT scores at T1 and follow-up (T2) scores .In order to identify the type or the types of NWs that have the best discriminative power we pose the following research questions (RQs):RQ1: Is MuLiMi NWRT repetition accuracy valid and reliable in the identification of DLD  as assessed by children\u2019s risk status based on German standardized test scores? If yes, is performance in repeating LS items or LNS items more discriminant? And are accuracy scores on the (various types of) NWRT in accordance with parental questionnaires and kindergarten teachers\u2019 subjective ratings?RQ2: Are there any advantages in using a bilingual NWRT over a monolingual one?RQ3: Is children\u2019s language dominance correlated with children\u2019s NW repetition accuracy and does language experience (dominance and exposure) influence improvement in NW repetition accuracy?RQ4: Does NW repetition accuracy at T1 predict improvement in overall language performance?M\u2009=\u200959.05, SD\u2009=\u20098.56) participated in this study. For the purpose of this study, two participants were excluded because of contradictory information in their parental questionnaires concerning whether or not they had an official diagnosis of DLD. At the time of testing, out of the 37 children, seven had already been diagnosed with DLD by German Speech and Language Therapists and were receiving treatment in Germany, 13 were considered TD, and 17 had neither been diagnosed nor treated for DLD but were identified as being at-risk for language impairment because they scored below a t-score of 40 in at least one of the standardized tests applied in this study (see section \u201cGerman Standardized Tests\u201d). Due to the phonological nature of the NWRT, a more specific type of \u201cphonological risk\u201d was identified for appropriate comparisons. This group comprised children who scored below a t-score of 35 in the German standardized NWRT (Mottier-test). According to this criterion, 10 children did, and 27 children did not, present with phonological risk. When participating in this study, all children were living in Germany. They all had at least one native Italian-speaking parent and were exposed to Italian\u2014even though to varying degrees\u2014on a daily basis. Participants with two Italian-speaking parents had been exposed to the German language for at least 2\u2009years. While eight of the children were attending a German kindergarten, 31 were enrolled in a bilingual Italian-German kindergarten program. A subset of the children  participated in a follow-up study (T2) that occurred 8\u201314\u2009months after the first evaluation (T1) repeating both standardized and experimental tests. Participant recruitment took place through either kindergartens or Speech and Language Therapy clinics. The study was approved by the institutes\u2019 Ethical Committees and all parents signed informed consent according to the Declaration of Helsinki.Thirty-nine simultaneous or early-sequential bilingual Italian-German speaking children aged from 3;10 to 6;1\u2009years  and an additional experimental protocol constituting a novel, web-based set of screening tasks for the assessment of risk of DLD . The Italian-German NWRT that is reported in this study is part of this experimental battery. Furthermore, caregivers were asked to complete a language background questionnaire providing information about their children\u2019s language experience and exposure patterns and an extensive questionnaire on pregnancy, general and language development. Finally, kindergarten teachers were asked to complete a short survey on the children\u2019s language skills across linguistic areas.n\u2009=\u20096; the other set is specific with respect to German ones, LS German, n\u2009=\u20096), recorded by expert native speakers of the respective language, and one set of LNS NWs . To make sure that the LS NWs complied with the phonotactic constraints of the respective language, phonemes and consonant clusters specific to each language were identified and used  or incorrect . As noted before, variations in accent or in the phonetic realization of single phonemes depending on language-specific features  were considered acceptable as long as the phoneme was clearly identifiable. The raw scores obtained in the MuLiMi NWRT reflect the number of NWs that were correctly repeated by each child  in the garbage can]. By contrast, an utterance such as \u201cWeil ist traurig\u201d with the verb in second position is considered level 3. A level is considered mastered if the child produces at least three utterances corresponding to that level throughout the entire test. As for Subject-verb-agreement, scores are calculated by first identifying all utterances that contain a subject and a verb and in a next step counting all the utterances with correct subject-verb agreement. Finally, the ratio between the number of occasions for subject-verb-agreement and the correctly realized instances is calculated. For instance, a common, correct response to the question elicitation \u201cWas passiert hier?\u201d [What is happening (in this picture)?] is \u201cDie (Kinder) spielen Fu\u00dfball.\u201d [They are playing football.] By comparison an utterance such as \u201cSpielen Fu\u00dfball\u201d is not included in the analysis as it is missing the subject. The maximum score is 1.0, children\u2019s performance again is represented on a four-point scale.Children\u2019s morpho-syntactic abilities in German were assessed using the elicited production task of the Linguistische Sprachstandserhebung Deutsch als Zweitsprache  was used to assess children\u2019s language background. Depending on the families\u2019 preference, the questionnaire was provided either in German or in Italian. The questionnaire included 34 questions concerning what language(s) children hear and speak on a daily basis. Children\u2019s main caregivers were asked to use a seven-point scale using a combination of frequency adverbs and a percentage scale to estimate the proportion of their children\u2019s Italian compared to German exposure in different contexts (one example for language input in the home: \u201cWhat language(s) does the child hear from his/her mother\u201d (1) only German; 100% German, 0% Italian, (2) predominantly German, hardly any Italian; 90% German, 10% Italian, (3) mostly German, sometimes Italian; 75% German, 25% Italian, (4) the same amount of German and Italian; 50% German, 50% Italian, (5) sometimes German, mostly Italian; 25% German, 75% Italian, (6) hardly any German, predominantly Italian; 10% German, 90% Italian, and (7) only Italian; 0% German, 100% Italian). Based on this information, an individual input  the child hears on a daily basis) and output  the child speaks on a daily basis) score were calculated for each language. The values obtained reflect the children\u2019s current language experiences at the time of their participation in this study.A further questionnaire was completed by the child participant\u2019s kindergarten teacher concerning the presence of difficulties in the domains of phonology, lexicon, morphosyntax and pragmatics. Each domain was judged according to a four-level scale .via the Mozilla Firefox web browser. For a subset of children (n\u2009=\u200914), follow-up (T2) data was collected between 8 and 14\u2009months after the initial evaluation (T1). Parents and teachers\u2019 questionnaires were filled individually on printed forms and returned to the researchers after a few days.Each child was tested individually in a quiet room by well-trained researchers either in one of the kindergartens or one of the Speech and Language Therapy clinics where they were recruited. In the few cases where this was not possible due to COVID-19 contact restrictions, children were tested in a quiet room in their homes. The test battery was administered in two separate sessions lasting between 40 and 50\u2009min each. The NWRT was carried out using a Lenovo laptop, model YOGA 720-15IKB under the Windows 10 Pro operating system. The online screening platform MuLiMi was accessed Children were assigned to one of three groups  based on the information about a pre-existing diagnosis of DLD (DLD group) and whether they had obtained a t-score below 40 in at least one of the standardized tests applied in the study\u2019s test protocol but without a pre-existing diagnosis of DLD (at-risk group). A further, more specific criterion for classifying children at-risk for phonological problems was applied dividing the group with respect to a cut-off of 35  for the t-score in the Mottier test .A variable expressing linguistic dominance was created based on parental reports, estimating the average number of hours each child is exposed to/actively speaks Italian during a regular week. Then, the number of hours was converted into a percentage in order to express the ratio between children\u2019s weekly language input and output in Italian vs. German. A compound score for language dominance in which the input and output scores of both languages were merged was created based on the following equation:Based on the resulting score, children\u2019s language dominance was assigned. The variable displaying dominance ranges from \u22121 (German dominant) to 1 . Children with a score of \u22121 to \u22120.16 were considered German dominant; \u22120.15 to 0.15 balanced; 0.16 to 1 Italian dominant.t-scores). Point-biserial correlations were computed for the association with dichotomous variables , whereas Spearman\u2019s correlations were used in the case of three- or four-levels variables . In all other cases Pearson\u2019s correlations were computed, under the condition that the variables\u2019 distributions satisfied all requirements for such analyses. Nonparametric tests were used for the comparisons of small groups  or for non-continuous variables. Repeated-measures ANOVAS were performed on continuous, close-to-normally distributed variables (the scores obtained on various subsets of the NWRT) with Specificity (LS vs. LNS) and (after excluding LNS NWs) Language  as within-subject factors and Phonological risk (with vs. without) as a between-subjects factor. The effects of dominance as a covariate were also assessed. Nonverbal intelligence and age were added as covariates  whenever they had been found to correlate with the variables under exam.Data were analyzed by means of IBM SPSS Statistics v.26. The association between children\u2019s NW repetition performance and their linguistic profile was assessed through partial correlation analyses, controlling for children\u2019s language dominance, age (in months), and nonverbal intelligence . While the TD children leaned more towards the German-dominant end of the continuum compared to the DLD children who as a group were rather dominant in Italian, the children in the at-risk group represented a greater variety of experience patterns. Even though the difference in dominance patterns did not reach significance across the three groups , pairwise comparisons showed a significant difference in language dominance between the TD and the DLD group , whereas for the TD vs. at-risk group and the at-risk vs. DLD group no significant differences emerged (sp\u2009>\u20090.05).2\u03c7(2)\u2009=\u200911.232, p\u2009=\u20090.004, with a mean rank of 26.46 for TD, 16.69 for at-risk and 10.50 for DLD children]. A Mann\u2013Whitney-U-Test confirmed significant differences for TD vs. at-risk children  and TD vs. DLD children  but not for at-risk vs. DLD children (p\u2009>\u20090.05).In both the CPM and the Mottier-test, the TD children scored highest, followed by at-risk and then DLD children (see t(28)\u2009=\u20093.590, p\u2009=\u20090.001], at-risk vs. DLD children  and TD vs. DLD children .Performance (raw scores) in the Mottier-test was normally distributed in the sample. Running pairwise comparisons, significant differences emerged in the Mottier-test raw scores for TD vs. at-risk children . However, there was a significant interaction effect between NW Specificity and children\u2019s Phonological risk status . Post-hoc tests showed that the NW repetition performance was significantly different for children with and without Phonological risk for both types of NWs . The repetition of LS NWs was significantly better than repetition of LNS NWs for the children without risk , whereas no significant difference emerged for the children with Phonological risk, who even performed (non-significantly) better with LNS than with LS NWs , but not for nonverbal intelligence and language dominance (sp\u2009>\u20090.05). No significant interaction between NW Specificity and children\u2019s language dominance (p\u2009>\u20090.05) emerged either. Furthermore, the covariates did not show any significant interaction with Phonological risk.Furthermore, a repeated-measures ANOVA with NW Specificity (LS vs. LNS) as within-subject factors and Phonological risk (with vs. without) as a between-subjects factor and dominance, nonverbal intelligence and age as covariates was performed. There was no significant main effect for Specificity (M\u2009=\u20093.00 for both types) but not on number of phonemes  and this could have affected accuracy, Length in phonemes was set as a covariate in a repeated measures ANOVA with Phonological risk as a within-subject factor (subjects in this case were the NWs) and NW Specificity (LS and LNS) as a between-subject factor. The results showed that the effect of Phonological risk remained significant: F\u2009=\u20096.965, p\u2009=\u20090.017, \u03b72\u2009=\u20090.279 as well as the interaction between Phonological risk and Specificity: F\u2009=\u20098.954, p\u2009=\u20090.008, \u03b72\u2009=\u20090.332. Also the effect of Length almost reached significance, F\u2009=\u20094.372, p\u2009=\u20090.051, \u03b72\u2009=\u20090.195, but its interaction with Phonological risk did not, p\u2009=\u20090.327. When repeating the analysis with the three-level variable Clinical status as a within-subject factor , Specificity as a between-subject factor  and Length as covariate , the results clearly showed (see sp\u2009\u2264\u20090.001), the discriminative power  of LS turned out to be significantly higher (covarying for Length) than that of LNS for the comparisons between DLD and the other two groups, ps\u2009\u2264\u20090.036, and higher for LNS than LS for the comparison between TD and at-risk children (p\u2009=\u20090.025).Since LS and LNS nonwords were matched on number of syllables \u2009=\u20098.028, p\u2009=\u20090.008] and a significant effect of Phonological risk status , but no significant interaction effect between Language and Phonological risk status (p\u2009>\u20090.05). Among the covariates, only age , but neither nonverbal intelligence, nor language dominance showed a significant effect on repetition performance. Moreover, a highly significant interaction emerged between age and language \u2009=\u200919.385, p\u2009<\u20090.001), due to performance on LS-German NWs being rather stable at different ages, but performance on LS-Italian NWs increasing with age.A second repeated-measures ANOVA on LS items with Language  as within-subjects factor and Phonological risk (with vs. without) as between-subject factor, covarying for age, nonverbal intelligence, and language dominance, yielded a significant main effect of Language [ps\u2009\u2264\u20090.001). A cut-off of 12.5 would allow for a sensitivity of 0.800 and a specificity of 0.815 in identifying phonological risk, and a sensitivity of 0.857 and a specificity of 0.767 in identifying DLD. When comparing LS and LNS NWs according to their sensitivity and specificity in identifying children with phonological risk and with DLD, respectively, it can be seen that LS produce better ROC curves (AUC\u2009=\u20090.933 and 0.931) than LNS NWs . Use of LS NWs alone would allow reaching a specificity (with unchanged sensitivity) of 0.900 for DLD identification and 0.963 for phonological risk identification . All ROC curves are presented in In order to confirm the discriminative power of the NWRT, an exploratory analysis of diagnostic accuracy  was performed with respect to the identification of the presence of phonological risk , as well as of DLD (as opposed to TD) in the clinical status classification, in spite of the small sample size. The ROC curves produced by the total NWRT score were very encouraging, with AUC (area under the curve) values as high as 0.917 and 0.898, respectively  and the compound scores LS-total  and LNS-total  and with the NWRT total score . These results remained stable when controlling for age and nonverbal intelligence. Children\u2019s scores obtained in the Mottier-test correlated higher  than with the repetition of LS-German NWs. The comparison of correlation coefficients for LS total and LNS total with Mottier raw scores did not yield fully significant results . For the two categorical variables verb placement and subject-verb agreement based on the LiSe-DaZ German standardized test, Spearman correlations revealed significant associations with NW repetition performance across subcategories and in the total score  except for the association between verb placement in the LiSe-DaZ and LNS-total . All the NWRT scales were also significantly associated with raw scores in the PPVT German standardized vocabulary test . This pattern was substantially maintained when controlling for age and nonverbal intelligence . Repetition performance across all NWRT scales further correlated with the German CLT compound (noun and verb comprehension) score , whereas the Italian CLT compound score was significantly associated only with repetition performance of LS-Italian  and LNS-total  but not with LS-German  nonwords, and thus not significantly associated with the NWRT total score. This pattern of results remained substantially stable when controlling for age and language dominance, but when adding nonverbal intelligence as a control variable no significant associations were found with the CLT Italian compound score .Performance across all NWRT scales was significantly associated with several standardized measures. Most importantly, the raw scores in the Mottier-test were significantly associated with the percentage of correctly repeated LS NWs (n\u2009=\u200936) or output (n\u2009=\u200935) in the two languages correlate with any of the NWRT scales .For one of the children conflicting information between the parental questionnaire and teachers\u2019 reports emerged regarding his/her amount of Italian output. This participant was thus excluded from the analyses on the Italian output variable. No significant correlations emerged between children\u2019s language dominance scores and their performance on any of the subcategories of NWs, nor did children\u2019s language input . Particularly teachers\u2019 judgment of children\u2019s productive phonology skills correlated significantly with children\u2019s NWRT scores across all scales  except for LS German .Teachers\u2019 global evaluation of children\u2019s language performance correlated significantly with children\u2019s clinical/risk status , risk score  and family global input score . Furthermore, children\u2019s performance in all NWRT scales was significantly associated with the scores in the parental questionnaire with the exception of the QUIR-DC FIGS (see t-scores).One of the children had to be excluded from the analysis concerning parental questionnaires since her/his parents never returned the QUIR-DC questionnaire. Children\u2019s clinical/risk status was significantly associated with the responses of their parents in the QUIR-DC general score  and for LNS-total . Language dominance was not significantly associated with improvement in NWRT scores from T1 to T2 . However, an interesting, negative association was found between the amount of Italian output at T1 and the improvement of LS-German scores  as assessed by children\u2019s risk status based on German standardized test scores? If yes, is performance in repeating LS items or LNS items more discriminant? And are accuracy scores on the (various types of) NWRT in accordance with parental questionnaires and kindergarten teachers\u2019 subjective ratings?t-scores). The same results emerge from the correlations between the children\u2019s scores on the NWRT and their position within the classifications expressing clinical status or phonological risk. They further highlight that LS NWs are more discriminant than LNS NWs, especially when the goal is to discriminate between TD and both at-risk and DLD children, or children with phonological risk. ROC curves  confirm that the NWRT, and especially LS NWs, may allow the identification of children with either phonological risk or DLD with very good sensitivity and specificity figures.Reliability of the MuLiMi NWRT was assessed both in terms of inter-rater reliability and of internal consistency. Both indexes were found to be satisfactory. For what concerns validity, children\u2019s performance across all NWRT scales was significantly associated with a wide range of standardized test results. The significant correlations between children\u2019s performance on the MuLiMi nonwords and their scores on further standardized language tests indicate that the task can support the identification of bilingual children\u2019s risk of DLD, irrespective of children\u2019s nonverbal intelligence  is positively associated with most language tests  influence improvement in NW repetition accuracy?Similar to what has been found in previous studies e.g., , TD chilEven though dominance differed significantly between the TD and DLD group , no significant association between children\u2019s language dominance and their repetition performance in any of the nonword subcategories was found. Moreover, a measure of German input quality in children\u2019s homes collected through parents\u2019 questionnaires did not correlate significantly with children\u2019s nonword performance in any of the scales cf. . NonetheThe absence of any influence of language experience on nonword repetition may also suggest that the bilingual children included in this study had already acquired sufficient experience with both of the languages to perform equally well (or badly) on German and Italian language-specific nonwords cf.  and thusRQ4: Does NW repetition accuracy at T1 predict improvement in overall language performance?Overall, the significant associations between nonword repetition performance at T1 and T2 across subcategories suggests that the MuLiMi NWRT scores are sufficiently stable across time. Since several months passed between T1 and T2 and since some of the children received Speech and Language Therapy treatment during this period, these correlations cannot be considered as proper measures of test\u2013retest reliability.Notably, significant associations of children\u2019s nonword repetition accuracy at T1 and their improvement of NWRT scores were found for LNS nonwords but not LS nonword repetition performance. Since performance on LNS at T1 was generally lower than performance on LS nonwords, it is likely that this difference indicates a larger space for improvement for the former than for the latter. Nonetheless, a negative correlation of LNS nonwords at T1 with improvement on the same measure was found. Also, we found greater discriminative power for LS compared to LNS items with respect to phonological risk status. This could indicate that a low performance on LNS items (compared to low performance on LS items) is less likely to reflect a specific impairment of phonological skills. In other terms, there is a greater probability for the repetition of LNS items to improve with time, possibly regardless of clinical intervention.The main limitation of the study, due to the difficulty of finding children with DLD belonging to this particular language combination and to limited access to kindergartens and clinical structures during COVID-19-related restrictions, is a relatively small sample size and unequal number of TD children and children with (risk of) DLD. Moreover, the children included in this study do not sufficiently represent the great heterogeneity of language experience scenarios in the bilingual population. Participants were predominantly early sequential or simultaneous bilinguals who most likely had already had sufficient exposure in both languages to perform equally well on LS-German and LS-Italian nonwords. This might be due to the recruitment method with the help of  bilingual kindergartens where children receive dual language input. It was therefore not possible to examine how later onset of exposure and acquisition of a second language would influence performance on LS vs. LNS nonwords. The fact that all DLD children were Italian-dominant is also remarkable. This might be a random effect due to small sample size for the DLD group, but it may also suggest that insufficient language exposure to the societal language can be a possible confound in diagnosis, or that it could interact with neurobiological risk factors making the language impairment in the L2 more evident  and thus enhancing the probability of early diagnosis and treatment. Other possible explanations may call socio-cultural factors into play, the analysis of which is beyond the scope of the present study. These issues could be addressed in future studies where a greater heterogeneity of  acquisition patterns is represented in the sample, including simultaneous, early as well as later sequential bilinguals. It would also be interesting to include monolingual participants in order to examine how repetition is affected by the presentation of nonwords whose characteristics go against the phonotactic constraints of their native language.Furthermore, even if whole word scoring is sufficient to discriminate DLD from TD children, in how far in-depth analyses on syllable- & phoneme-level as well as error analyses could provide additional, useful information will need to be addressed in future studies.A more general methodological issue and characteristic of most studies on DLD in bilingual population is the circularity between classification and verification. In fact, the classification of children into risk groups is largely based on the results of standard test procedures whose informative value and application with multilingual children is limited. This limitation applies not only to the test procedures with monolingual norms  but also to tests with norms for the bilingual population, which (like the LiSe-DaZ) normally refer to a large variety of L1s and to certain bilingual acquisition scenarios only. In this perspective, it would be useful if future studies could incorporate phonological and articulatory tests in both the L1 and the L2.Finally, the effect of \u201cneutral\u201d intonation in the presentation of LNS nonwords needs to be investigated in more detail since this type of presentation while avoiding language-specific prosodic features on the one hand, on the other hand makes the stimuli sound rather unnatural, a characteristic that might influence repetition performance.Summarizing the results of the study with respect to the research questions that were initially raised, it can be concluded that children\u2019s repetition accuracy in the MuLiMi NWRT differs according to their risk status , confirming the task\u2019s discriminative validity, with a stronger capacity of LS than LNS nonword repetition accuracy to discriminate between children with and without a phonological risk (RQ1). Furthermore, MuLiMi nonword repetition performance is associated with German standardized test scores, confirming the task\u2019s concurrent validity (RQ1). Both teachers\u2019 subjective ratings of children\u2019s language performance and parental reports about children\u2019s language development are largely associated with NW repetition accuracy (RQ1). For bilingual children, the bilingual MuLiMi NWRT is more informative than the monolingual Mottier-test (RQ2). Neither nonword specificity nor children\u2019s language dominance had an impact on NWRT (RQ3), but improvement in NW repetition performance from T1 to T2 did show an effect of language use (RQ4). Preliminarily investigating the MuLiMi NWRT\u2019s predictive validity, children\u2019s repetition performance at T1 correlated significantly with repetition performance at T2 (pointing to stability of the measure) and with improvement (RQ4) which for certain nonword subscales was significantly associated with children\u2019s language use (RQ3).Thus, in line with previous research e.g., , a combiDue to the independence of repetition performance from language dominance, a combination of LS and LNS items provides a satisfactory solution for the identification of language disorders despite the great variety of language experience patterns present in the bilingual population. The present results also suggest that the task is sufficiently robust with respect to the possible impact of nonverbal intelligence and age (within the kindergarten and preschool age range) variations, beyond different language dominance patterns.For clinical purposes of DLD risk identification and in order to plan and design appropriate intervention strategies, LS nonword repetition performance is the most suitable measure. Repetition of LNS nonwords seems to be more informative with respect to possible improvement in performance, probably due to less specific, more exposure-related impairments. It is thus suggested that a comprehensive assessment should comprise both LS as well as LNS items: the reduction of the items to a relatively small amount of nonwords very carefully selected from the initial list so as to maximize item distinctiveness while preserving validity and reliability allows for relatively fast but highly reliable and informative testing outcomes.The raw data supporting the conclusions of this article are available on the Zenodo repository, DOI: 10.5281/zenodo.6519135. Personal data concerning participant children will be made available upon request to the corresponding author. Access will be granted to named individuals in accordance with ethical procedures upon completion of a formal data sharing and collaboration agreement, and approval by the ethics committee and legal authority.The studies involving human participants were reviewed and approved by Ethics Committee of the Scientific Institute Eugenio Medea, scientific section of the association \u201cLa Nostra Famiglia,\u201d via Don Luigi Monza 20, Bosisio Parini (LC) 23842, Italy on 17 June 2019 (no. 43/19); Ethics Committee of the Catholic University Eichst\u00e4tt-Ingolstadt, Ostenstra\u00dfe 26, 85072 Eichst\u00e4tt, Germany, on 21 January 2020 (no. 009-19). Written informed consent to participate in this study was provided by the participants\u2019 legal guardian/next of kin.ME and TB contributed to the conception of the study, definition of the experimental materials for German and Italian, performed data collection, processing and analyses for the study, and contributed to the interpretation of results. ML took care of the conception and definition of the experimental design, assisted in the definition of experimental materials, participated in the interpretation of results, performed statistical analyses, and supervised the whole study. All authors contributed to writing the manuscript, read, and approved the submitted version.This project has received funding from the European Union\u2019s Horizon 2020 program for research and innovation under the Marie Sk\u0142odowska Curie Grant Agreement No. 765556  and by the Italian Ministry of Health, Grant RC2021 to ML. The APC was funded by the IRCCS E. Medea, Bosisio Parini, Italy.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "The study of neurotransmitters and stress hormones allowsthe determinationof indicators of the current stress load in the body. These speciesalso create a proper strategy of stress protection. Nowadays, stressis a general factor that affects the population, and it may causea wide range of serious disorders. Abnormalities in the level of neurohormones,caused by chronic psychological stress, can occur in, for instance,corporate employees, health care workers, shift workers, policemen,or firefighters. Here we present a new nanomaterials-based sensorstechnology development for the determination of neurohormones. Wefocus on fluorescent sensors/biosensors that utilize nanomaterials,such as quantum dots or carbon nanomaterials. Nanomaterials, owingto their diversity in size and shape, have been attracting increasingattention in sensing or bioimaging. They possess unique properties,such as fluorescent, electronic, or photoluminescent features. Inthis Review, we summarize new trends in adopting nanomaterials forapplications in fluorescent sensors for neurohormone monitoring. Neurohormones, which are themain regulators of the stress response, are physiologically activesubstances produced by the nervous system.8 As very often the first symptoms of stress-induced diseases  areunderestimated, the development of biosensors that would enable themonitoring of parameters related to exposure to stressful conditionsis extremely important. Knowledge about the level of neurohormonesis an essential factor in modern medicine; however, there is no availablemethod for accurate, sensitive, fast, and direct analysis that wouldallow monitoring the concentration of these species.Nowadays, stress is ageneral factor affecting the population,and it may cause a wide range of serious disorders. Chronic stresscan have a large pathophysiological impact on neuroendocrine12 The employment of nanomaterials (NMs) in biosensors permits theuse of many new signal transduction technologies in their manufacturing.Nanomaterials, e.g., nanoparticles (NPs), nanotubes, nanofibers (NFs),nanorods, or quantum dots (QDs), have large possible application inthe construction of biosensors.13 Thesematerials possess a wide range of different properties, and thereforethey are included in functional materials , which can be bound to the biological molecules and usedin biosensors to determine or amplify different signals.14 The unique optical properties of NMs, especiallyquantum dots, make the NMs attractive fluorophores that can be usedboth in vitro and in vivo in various biological studies, where traditionalfluorescent labels based on organic molecules do not provide long-termstability or high enough intensity or where a simultaneous detectionof many signals is needed.15 In sensors,the signal detection is based on the registering of a change in oneof the physical properties  of sensing materials induced by the interaction withthe analyte. The main advantages of using the nanoparticles, e.g.,changing their optical properties, such as emission color, intensity,polarization, or emission kinetics, can be used as the principle inoptical sensors systems.13 The use of carbon-and cadmium-based nanomaterials for the construction of sensors requiresadjusting their properties  adequately to the needs thatarise. Moreover, to obtain specificity of NMs in their sensing action,the surface modification, called functionalization, must be appliedfirst.17 There are several methods for detectingan analyte with NPs, e.g., emission quenching from NPs, increase inNPs\u2019 emission due to passivation of the NPs\u2019 surfaceby the analyte, and nonradiative F\u00f6rster resonance energy transfer(FRET).18 Nanostructured materials withgreat potential for the easy identification of individual chemicalcompounds with minimized sample volumes and their treatment are particularlypromising in the process of biosensors development. Such materialsinclude, e.g., carbon nanotubes (CNTs), graphene, and quantum dots,which very often are combined in optical sensor/biosensor detectionsystems. Optical biosensors represent a common type of biosensors,where the detection is based on the interaction of the optical fieldwith a biorecognition element.19 Thesetools are stable owing to their resistance to radio interference andother electromagnetic waves. It is worth mentioning that they usuallyshow high sensitivity, while simultaneously measuring without causingcontamination of the sample with the reaction product.20The evolutionof biosensors was driven by the need for faster andmore versatile analytical methods for application in important areasincluding clinical diagnostics, food analysis, environmental monitoring,and industry analysis in complex sample matrices , with minimal sample pretreatment. Nowadays, an increasing interestin combining nanotechnology with biosensor construction is observed.Here, an overview of the recent developmentsin the field of nanomaterials-basedoptical sensors and biosensors that showed suitable detection limitsto determine neurohormones in biological fluids is presented 1.222 The most commonly useddetection methods in optical sensors are based on fluorescence andabsorption phenomena.Many various detection methods  can be utilized in the design of sensors and biosensors.One of the most accurate and at the same time versatile methods isthe optical one. The operating principle of sensors using this methodis based on several optical phenomena which can be divided into twogroups\u2013luminescent  and nonluminescent .2.123 The quantity describing the ability of a substance to absorblight of a specific wavelength is absorbance. According to the well-knownLambert\u2013Beer\u2019s law, it is possible to determine analyteconcentration based on absorbance measurements. The absorbance magnitude(A) can be expressed as the logarithm of the ratioof the incident light intensity (I0) andthe light transmitted to the detector (I).25\u20131 M\u20131), l is the optical path length (in cm), and c is the concentration of the analyte (in M).Absorption is a phenomenonin which the photon energy of incident electromagnetic radiation isabsorbed partially or in total by the examined analyte. As a result,the valence electrons of the atom are excited to higher energy levels,and subsequently absorbed energy may be transformed into heat or emittedas other electromagnetic radiation .26 Its length may be extended by incorporating,e.g., optical mirrors.27In absorptionspectroscopy, a light source of a given wavelength or selected spectrumis positioned opposite to a sensor. The key element in absorbancemeasurements is the selection of an optical path length that willprovide an adequate signal-to-noise ratio. Depending on the designof the sensor and the nature of the performed measurements, the opticalpath may follow either perpendicular or parallel to the fluidic channel.2.228 Thisradiation has a longer wavelength than the excitation one. The photonenergy (E) is expressed as the product of Planck\u2019sconstant (h) and the frequency of the wave (\u03bd),which can be presented as the ratio of the speed of light (c) and the wavelength (\u03bb).The phenomenon in whichelectromagnetic radiation is emitted as a result of exposure to radiationof a different, shorter wavelength is called fluorescence. A fluorophoreor a fluorescent dye absorbs the energy of incident photons and isexcited to a higher energy level as a result of radiation-free transitions.Over a short period of time, called the excited state lifetime, avibrational relaxation of the fluorophore occurs. As a result, itsenergy is decreased, and eventually the electrons return to theirvalence band radiating photons.ex) and emission (\u03bbem) radiation is known as the Stokes shift and can range from 10 to150 nm. The fluorophore can be excited numerous times, and the fluorescentemission can be obtained repeatedly before it is no longer able tofluoresce. This process is also known as photobleaching.29 The efficiency of the fluorescence process isdetermined by the quantum yield of the fluorophore and is a ratioof the emitted to the absorbed number of photons.23 Most commonly, fluorescence measurements are performedutilizing radiation in the range from 250 to 750 nm.28 Selected common fluorescent labels along with their excitationand emission wavelengths are presented in 30The wavelength difference between theexcitation , and the photodetector that recordsthe light intensity and produces a measurable value.Novel optical-basedbiosensors utilize relatively small-sized lightsources that are based on semiconductor compounds (mainly from theIII-V and II-VI groups). The most popular light sources are semiconductorlasers, superluminescence diodes, and light-emitting diodes (LEDs).Light emissions for different semiconductor compounds that are usedfor the fabrication of light sources are presented in 38 The base materialsused to fabricate these devices are, e.g., silicon, germanium, indiumgallium arsenide, and lead sulfide. The wavelength ranges of exemplarysemiconductors and semiconductor compounds that are used for photodetectorsare presented in As a photodetector, mainly a photodiode, phototransistor,or charge-coupleddevice (CCD) is used. In addition, CMOS-type  detectors  and mini-spectrometers have increasingly been used in sensingapplications in recent years.As can be seen in In fluorescence spectroscopy, we observe the lightemitted by thetested object; therefore, the light source should be positioned perpendicularto the photodetector or be separated by a set of mirrors and filters.Possible configurations of the light source with respect to the photodetectorare shown in 4amaterial with any external dimension in the nanoscale (length rangeapproximately from 1 to 100 nm) or having internal structure or surfacestructure in the nanoscale\u201d.40 Nanomaterials possess specific chemical, physical, and biologicalproperties.41 They are characterized bylarge surface area to volume ratio, ease of functionalization, porosity,and therefore high loading capacity, and in some cases unique opticalproperties, thanks to which they find applications in the constructionof optical sensors and biosensors, enabling their miniaturizationand improving their performance, limit of detection, and responsetime.43 Functionalization of nanomaterials may additionallyenhance the binding affinity toward the target, improve the stabilityin aqueous solutions, and give desired properties.44Nanomaterials are promising structures for applicationsin sensors.A nanomaterial, as defined by ISO standards, is \u201c45A variety of nanomaterials have found applicationin the constructionof fluorescent sensors 3. The ch4.1hollow nanofibercomposed of carbon\u201d.79 The tubularshape of CNTs comes from curled-up graphene sheets. Depending on thenumber of cylindrical graphene layers, CNTs can be classified intosingle-walled CNTs or multiwalled CNTs . The former attractedspecial attention in sensing and biosensing research. SWCNTs can havea zigzag, armchair, or chiral structure, which are then classifiedas semiconducting, metallic, or semimetallic, respectively. The mostadvantageous properties of these one-dimensional structures include,apart from large surface area and mechanical properties, electronic,electrochemical, and photophysical properties, electrochemical stability,and thermal and electrical conductivity. In comparison to other methodsused in biosensors, such as antigen\u2013antibody interactions,CNTs present a stable, low-cost, reproducible material that does notrequire living organisms for production. In addition, their nanoscalesize may lead to precise targeting of the molecules.82According to ISOStandards, a carbon nanotube is a \u201c84The physical construction of SWCNTs defines their opticalproperties. Because of their nanoscale size (ranging from 0.7 to 3nm in diameter), they are subject to the quantum confinement effect,which leads to near-infrared fluorescence in the range of 900\u20131600nm. This fluorescence is sensitive to its local environment and dependson several factors, such as the diameter and chiral vectors of theCNT, the local dielectric environment, charge transfer, and the presenceof fluorophores or Coulombic interactions. SWCNTs exhibit strong resonanceRaman scattering and possess a broad absorption spectrum, which makesit possible to use them as quenchers for different fluorophores.84 Functionalization creates an organic phase, called a corona, onthe surface of CNTs. A perfect coating should be nontoxic, biocompatible,and stable and possess specific functional groups that allow furthermodification; for example, modification with oxygenated moieties mayleave carboxylate groups on the surface of CNTs. These groups improvewater solubility and can be used for further bioconjugation. Dispersionin aqueous solutions is also possible by using surfactants, such assodium dodecyl sulfate, or soluble molecules, including proteins,nucleic acids, and polysaccharides. In the case of covalent modification,oxidation and cycloaddition are often used.84Initially, SWCNTs are hydrophobic and tend to aggregate. Properlyselected surface modification, covalent or noncovalent, can make themnot only hydrophilic but also biocompatible, adapting them for applicationsin sensors and biosensors. It also tunes the fluorescence propertiesof SWCNTs. Properties, including high photostability, lack of photobleachingand lack of blinking, various chiralities, and biomolecule comparabledimensions, make SWCNTs a good material for sensing applications.83 or glucose levels85), proteins,81 metals,86 or neurotransmitters.67SWCNTs have been applied in opticalsensors for the detection of,e.g., biomarkers  are another promising carbon-basedNM. CDs are nearly spherical, fluorescent carbon materials with onedimension less than 10 nm. Their synthesis is cheap and biofriendly,and they are biocompatible, soluble, and resistant to photobleaching.Because of abundant functional groups, such as hydroxyl, carboxyl,and amine, they can be easily functionalized. They possess high fluorescencestability and strong absorption in the ultraviolet region, expandableto the visible region, they do not blink, and the excitation and emissionwavelengths are adjustable. CDs-based sensors have been developedfor the detection of, for example, metal ions, pesticides, and biomoleculessuch as nucleic acids or for use in food safety.94 In addition, because of their fluorescent properties, granted bythe radiative recombination of electron\u2013hole pairs, they canserve as fluorescent labels or quenchers. The nanoscale allows forthe quantum effects, exciton confinement, and quantum confinementleading to a band gap and edge effects, which lead to optical andelectrical properties unobtainable in classic quantum dots. GQDs canbe easily functionalized, as a large number of sites for potentialmodification is provided by their structure. Functionalization canbe carried out using organic molecules and biomolecules, such as proteins,amines, nucleic acids, or antibodies, or using nanomaterials, creatinghybrid NMs.95Graphenequantum dots (GQDs) are zero-dimensional, fluorescent nanomaterialsconsisting of one or few layers of graphene. As a carbon-based material,GQDs present excellent biocompatibility and low to no toxicity, aswell as good solubility in various solvents, including aqueous solutions.They possess properties characteristic of both graphene and quantumdots. They exhibit good mechanical, electrical, thermal, and opticalproperties, as well as large surface area, chemical inertness, highfluorescence activity, and photoluminescence stability.93 Optical sensors basedon GQDs have been used to detect, for instance, metal ions (such ascopper(II),94 iron(III),96 and mercury(II)97) or organiccompounds .There are two main categories of synthesisof GQDs: the top-downapproach and the bottom-up approach. The first one is a simpler methodthat involves a fragmentation of carbon materials, such as grapheneoxide, CNTs, graphite, or carbon fibers, through, e.g., chemical orelectrochemical oxidation. However, control of the size and morphologyof created GQDs is not possible. The second approach involves severalchemical reactions from smaller precursors, such as citric acid orpolycyclic aromatic hydrocarbon, to create GQDs, e.g., hydrothermaltreatment and chemical vapor deposition. Examples of methods for thesynthesis of GQDs are presented in 4.3crystalline nanoparticles that exhibit size-dependentproperties due to quantum confinement effects on the electronic states\u201d.100 They are semiconducting,inorganic, fluorescent nanocrystals, usually synthesized from elementsof groups II-VI or III-V. Their size is limited by the exciton Bohrradius of the material and usually ranges from 1 to 10 nm. Specialfocus is placed on QDs with the size of 2\u20136 nm, as they resemblebiomolecules.104 Optical properties, which can be tuned by controlling the size ofQDs, are characterized by narrow emission spectra, broad excitationspectra, long fluorescence decay lifetime, high quantum yield, andlack of photobleaching.105 QDs can have a core\u2013shell-like structure, where the coreis surrounded by another layer, usually made of a different compound.The shell protects the core from photo-oxidation and improves thequantum yield.106Quantum dots are definedas \u201c105 Green synthesis has also been described, where toxic compoundsare replaced with environmentally friendly substances and lower temperaturescan be used.107One of the mostpopular methods of synthesis of QDs is the hot-injection method. Inthis technique, precursors of the core compounds are injected intoa hot surfactant, starting the process of nucleation. Then the temperatureis lowered to let the crystals grow, and the process is stopped whenthe nanocrystals reach the desired size. The surfactant moleculesbecome the ligands on the surface of the NPs.108 The ligands on the surface of QDs affect theirsize, shape, optical and physicochemical properties, colloidal stability,and dispersion in polar and nonpolar environments, as well as furtherbioconjugations.109 The simplest modification methodis ligand exchange, where hydrophobic surface molecules are replacedwith molecules with two functional groups, hydrophobic for the surfacebinding  and hydrophilic  for water solubility. Replacement ligands mayinclude, e.g., 3-mercaptopropionic acid or d-penicillamine.112 Silanization is the synthesis of a siloxane layer around a singlequantum dot, and amphiphilic molecules can create a cross-linked polymerlayer around a single QD.110The organometallicsynthesis of QDs causes the requirement of surfacemodification in order to obtain hydrophilic QDs that could be furtherfunctionalized and applied in biomedicine and sensing. Most commonlyused techniques include ligand exchange, silanization, and amphiphiliccoating 5.108 The13 for instance,in the detection of metal ions,113 pesticides114 or toxins,115 sugars,116 microorganisms,117 and biomolecules, such as proteins,118 nucleic acids,119 neurotransmitters,120 or vitamins.121Because of their unique optical properties, QDshave found applicationin many optical sensors,4.4123Gold nanoparticles(AuNPs), also known as colloidal gold, are stable nanoparticles witha size between 1 and 100 nm. They possess unique optical, physicochemical,electronic, and catalytic properties that make them applicable invarious fields. They are characterized by large surface area, distinctshape, stability, strong adsorption, absorption in the visible spectrumregion (with a possible shift to the UV\u2013visible region dependingon the size and morphology), the possibility of functionalization,and optical properties dependent on the size. They present surfaceplasmon resonance, the ability for fluorescence quenching due to fluorescenceresonance energy transfer, and surface-enhanced Raman scattering (SERS).To improve the properties, stability, dispersity, functionality, andbiocompatibility, surface modification is required. Functionalizationis possible through covalent bonding, electrostatic interactions,and molecules binding.124 anionic contaminants,122 pesticides,and drugs.123Because of thesefeatures, AuNPs can be used in sensing and biosensing technologies,for instance, for the detection of metal ions,4.5nano-objects with two external dimensions in the nanoscaleand the third dimension significantly larger\u201d.78 Various morphologies of NFs can be obtained,depending on the process conditions, e.g., smooth, beaded, hollow,or core\u2013shell structures.125 NFsare characterized by excellent mechanical, physical, and chemicalproperties, including a high surface area to volume ratio and porosity.In addition, they offer flexibility in design, easy and low-cost fabrication,and a wide range of functionalization molecules and approaches.127Nanofibers are one-dimensional\u201c128Electrospinning is one of the simplest and therefore commonlyused techniques for NFs fabrication. In short, after being electrified,a liquid droplet generates a jet, which is then stretched and elongated,creating fibrous structures. The basic setup includes a syringe filledwith a solution of a chosen polymer along with a syringe pump, a spinneret, a conductive collector (plate or rotating), anda high-voltage power supply. Charging of the liquid forces the dropletto form a Taylor cone, and the jet is ejected. The jet, initiallyin a straight line, undergoes whipping motions and stretching, solidifies,and is then collected on the collector in the form of nanofibrousmats or membranes.127 The incorporation of carbon NMs, metallic NPs, semiconducting NPs,conjugated polymers, or organic dyes  makesNFs a promising platform for sensing and biosensing  with polymers,followed by electrospinning of the solution, or through surface modificationof NFs by physical adsorption or suitable chemical reaction. The secondapproach is especially useful in sensing and biosensing, as the largesurface area allows the immobiliation or encapsulation of multiplemolecules, leading to improved sensing performance. Moreover, theuse of NFs does not contaminate the solution, as they can be easilyremoved from the sample after the detection.osensing 6.127,129127 environmental toxicants,134 cancer cells,135 and pH.133Nanofibers were applied in optical sensors forthe detection of,e.g., heavy metals,5137 Abnormal levels of these molecules, such as dopamine, epinephrine,serotonin, cortisol, etc. can be found in response to several conditions,such as emotional state  or inthe presence of various diseases, from neurodegenerative and cardiacpathologies to mental disorders and tumors.143 Therefore, it is very important to determine the concentration ofneurohormones in human biofluids  for disease prevention or diagnosis,in order to improve the quality of life and to minimize health risks.Concentrations of neurotransmittersproduced by the nervous systemand of hormones produced by glands are indicators of the body\u2019sstate, and they are related to stress conditions.145 HPLC-MS,147 electrochemical methods,151 and UV\u2013vis spectroscopy,152 have been developed. Despite their selectivityand sensitivity, most of these methods have some non-negligible drawbacks,such as consumption of time, high costs, qualified personnel need,and sometimes the need for additional steps. In this regard, optical(bio)sensors have appeared as promising useful analytical alternatives,possessing a limit of detection in the range of nanomolar or lessand high reproducibility and sensitivity, in addition to rapid response,easy analysis, and low costs. Considering these, fluorescence-based(bio)sensors that use fluorescent nanomaterials are, in general, veryuseful analytical tools thanks to their high sensitivity, biocompatibility,and photostability.141Owing to the low physiological concentrations of these biomarkers,ultrasensitive methods to carry out quantitative analysis in biologicalsamples are necessary. In recent years, numerous analytical strategies,including HPLC,5.1153 In medicine, it is mainly usedin the treatment of heart diseases, bronchial asthma, and anaphylacticshock.56 In free form, EP occurs as anorganic cation in central nervous system tissues and body fluids.154 Many diseases are closely related to changesin adrenaline levels\u2014for example, low levels of adrenalinehave been found in patients with Parkinson\u2019s disease.62 Hence, the quick and sensitive detection ofEP in body fluids is of key importance in medical diagnostics, pharmacology,and the control of nerve physiology.155 Currently, many tests for the determination of EP concentrationsare known, such as electrochemical methods,158 capillary electrophoresis,160 liquid chromatography,162 or chemiluminescence.164 Unfortunately, mostof these methods are limited by high costs, tedious operation, andthe need to prepare the sample for measurements in advance. In thecase of EP, there are almost no existing optical detection systems.It is much more difficult to detect EP because of its rapid metabolism,and in fact, it does not possess any fluorescence properties.155Adrenaline,also known as epinephrine (EP) 7, is one56 For this purpose,water-soluble CuInS2 quantum dots closed with l-cysteine were synthesized. In the next stage, positivelycharged EP was accumulated on the surface of quantum dots throughelectrostatic interactions and hydrogen bonds, resulting in the formationof electrostatic adrenaline\u2013CuInS2 QDs complexes.The constructed system also uses tyrosinase, which can stimulate EPto generate H2O2 and additionally oxidizes adrenalineinto EP quinone. Both H2O2 and epinephrine quinonecan suppress CuInS2 fluorescence through an electron transferprocess. Under optimal conditions, the fluorescence -quenching coefficientwas proportional to the logarithm of the EP concentration in the concentrationrange of 1 \u00d7 10\u20138\u20131 \u00d7 10\u20134 mol L\u20131, while the detection limit was 3.6 nM.Z. Liu and S. Liu have developed a simple fluorescencebiosensorfor the detection of EP.153 A miniature biosensor based on low-temperaturecofiring ceramics technology (LTCC) was designed by the immobilizationof enzymes belonging to the class of oxidoreductases on the surface of a semiconducting polymer -4-(5-hexylthiophen-2-yl)pyridine)).The detection procedure was based on the oxidation of the substratein the presence of the enzyme. An alternative enzyme-free system resultedin the formation of a colored complex between Fe2+ ionsand EP molecules. Under optimized conditions, the sensor was characterizedby very high sensitivity and selectivity over a wide range of concentrationswith a detection limit of 0.14\u20132.10 nM. The constructed systemwas successfully used to determine EP in labeled pharmaceutical samples.Another method of detecting EP was proposed by Baluta and co-workers.2 solution.62 Visual detection was also possible by changingthe color of the solution from pale blue to red-brown. In this case,the CuCl2 solution is used as a probe to simplify the method,and its performance is comparable to that of fluorometric and colorimetricsensors. The created sensor is characterized by simplicity, high selectivity,and repeatable operation. In fluorometric detection, it showed activityin the linear range of 3 \u00d7 10\u20135\u20135 \u00d710\u20137 M, while in colorimetric detection in the rangeof 5 \u00d7 10\u20134\u20132 \u00d7 10\u20135 M. To confirm the commercial applicability, artificial urine andpharmaceutical preparations were successfully analyzed by the constructedtwo-channel sensor.Sivasankaran and Girish Kumar proposed a new EP detection strategybased on colorimetric and fluorescence measurements resulting fromthe formation of copper nanoparticles from a CuCl67 They selected 10 different DNA sequences tomodify the corona phase of CNTs and used near-infrared microscopyto measure the fluorescence of the obtained probes. In the case ofepinephrine, the limit of detection (LOD) was in the range of 0.5\u201333.3nM, depending on the oligonucleotide sequence used for the modification.The same experiment was repeated for dopamine and norepinephrine,with the LOD between 0.7 and 9438 nM and 0.5 and 23.7 nM, respectively.In addition, it was found that selected sensors were able to distinguishdifferent neurotransmitters at low concentrations of 50 nM.Mann et al. used SWCNTs functionalized withDNA to create a fluorescentsensor for the detection of catecholamine neurotransmitters.69 and exhibitedblue fluorescence. These NPs were applied for the detection of neurotransmitters,epinephrine, norepinephrine, and dopamine, via fluorescence recovery.In the proposed strategy, the system\u2019s fluorescence intensitywas quenched by MnO4\u2013 and recovered withthe help of the analytes. The detection limits were 88, 91, and 140nM for EP, NE, and DA, respectively. Additionally, the system showedgood selectivity toward potential interfering agents and good recoveryvalues in real sample analysis.Highly crystalline nitrogen-doped fluorescent carbon nanoparticles(N-CNPs), synthesized from ethylene glycol and alanine anhydride,were developed by Das and DuttaThe sensor systems for the detectionof EP described above aresummarized in 5.264 Thedetermination of NE concentration is extremely important in modernmedical diagnostics, not only for the determination and study of thephysiological processes of this catecholamine but also for the diagnosisand monitoring of the course of treatment of cardiovascular diseasesand mental disorders.165 Moreover, overexpressionof both NE and EP in blood and urine may indicate the presence ofpheochromocytoma located in the adrenal medulla; however, the overexpressionof only NE suggests the presence of a tumor elsewhere. Hence, testsenabling the simultaneous measurement of NE and EP concentration inblood and urine are extremely useful, as thanks to them it is possibleto quickly diagnose the disease and implement appropriate treatment.166 Many methods for the detection of NE have beendescribed, such as electrochemical detection,168 capillary electrophoresis,169 and HPLC-basedmethods.170 The downside to using thesemethods is the need for expensive equipment and manpower. Therefore,it is necessary to find fast and sensitive methods for the determinationof this catecholamine in biological samples.Noradrenaline,also called norepinephrine (NE) 8, like E64 The basis of the detection strategy is the formation ofbrown silver nanoparticles (AgNPs) in the presence of NE, resultingin a strong fluorescent signal. The designed sensor is characterizedby a linear relationship between the absorbance values and the concentrationof NE in the range 1.00 \u00d7 10\u20136 \u22126.66\u00d7 10\u20138 M with a detection limit of 1.79 \u00d710\u20138 M. Additionally, it was found that the fluorescenceintensity was directly proportional to the concentration of NE inthe range 8.92 \u00d7 10\u20133\u20135.66 \u00d7 10\u20135 M with a LOD of 5.59 \u00d7 10\u20136 M. The system constructed in this way has been successfully usedto determine NE in synthetic blood serum, which indicates its potentialuse for diagnostic purposes.Menon et al. developed a two-channel colorimetricand fluorometricsensor for the sensitive and rapid detection of NE.48 During single-wavelengthexcitation, the hybrid nanoprobe generated double-emission peaks belongingto CDs and CdTe-QDs. The basis of the action was quenching of fluorescenceby EP or NE due to electron transfer from QDs to the oxidation productsof catecholamines. At the same time, the fluorescence intensity ofthe CDs remained unchanged. The relative ratios of the fluorescenceintensity of CDs and CdTe-QDs were directly proportional to the concentrationof catecholamines. The new fluorescence detection platform was fastand convenient and was used to detect EP and NE in human serum sampleswith satisfactory linear range results for NE from 0.005 to 10 \u03bcMand a LOD of 2.1 nM.Zhang and his team proposed a differentstrategy. They constructeda ratiometric fluorescent nanoprobe consisting of water-soluble fluorescentcarbon dots and 3-mercaptopropionic acid-coated cadmium telluridequantum dots (CdTe-QDs).2 quantum dots and molecularlyimprinted polymer (MIP) forsensitive and fast detection of NE.51 Theconstructed matrix was characterized by Fourier transform infraredspectroscopy, transmission electron microscopy, and fluorescence spectroscopy.The newly synthesized system was characterized by high selectivityand affinity for NE. The basis of the sensor operation was the measurementof the fluorescence intensity of CdTe@SiO2@MIP in the presenceof the tested catecholamine. The fluorescence intensity of the systemdecreased linearly with the increase of NE concentration in the rangeof 0.04\u201310 \u03bcM. The limit of quantification was set at8 nM. The constructed MIP-based nanoplatform was successfully usedin the analysis of NE concentration in the plasma of rats. Becauseof the simplicity and speed of the gun, it can potentially be usedto determine NE in medical diagnostics.Wei et al. designed a molecular imprintedsensor based on CdTe@SiO52 Forthis purpose, they carried out a MIP-anchoring process on the surfaceof two different colored quantum dots. The platform made of CdTe@SiO2 and CdTe/CdS/ZnS/SiO2 QDs was modified with templatesfrom NE and EP to obtain two matrices, NE-QD@MIP (for NE) and E-QD@MIP(for EP). Such constructed nanosensors were characterized by selectivityand high binding affinity to the appropriate matrix molecule. Thematrix mixture could be excited at the same excitation wavelength,and simultaneous detection of NE and EP could be accomplished by monitoringtwo different color fluorescence signals without spectral overlap.Under optimal conditions, the fluorescence intensity of the systemdecreased linearly with increasing concentration of the standard moleculein the linear range of 0.08\u201320 \u03bcM with a detection limitof 9 nM for NE and 12 nM for EP.Wei et al. also proposedanother MIP-based method for the sensitiveand rapid detection of NE and EP.171 developed astrategy based on excitation\u2013emission fluorescence spectroscopyusing glutathione-capped CdSeS/ZnS quantum dots (QDs-GSH) for thedetection of various neurotransmitters, including EP, NE, GABA, andmore, because of the cross-affinity of the modified nanocrystals towarddifferent chemical structures of neurotransmitters. The proposed assayallowed the quantification of catecholamine neurotransmitters  at the micromolar concentration range.However, in the case of other tested neurotransmitters , the analysis of only specific compoundswas possible, limiting the future use of this method. The authorsstate that further research regarding the LOD, selectivity, and realsample analysis must be done.G\u0142owacz et al.The sensor systems for the detectionof norepinephrine describedabove are summarized in 5.3180Dopamine (DA)9 is an e182 As the concentration of DA in real samples is very low, its determinationrequires very sensitive methods.In agreement with the Human Metabolome Database,the physiologicalconcentration of DA varies in different biofluids: in blood it isless than 130 pM, whereas in human cerebrospinal fluid and urine thelevels of dopamine are \u22485 nM and less than 1 \u03bcmol/mmolof creatinine, respectively.65 who realized a fluorescent biosensor based onin situ bifunctionalized carbon dots with boronic acid and amino groups(B\u2013N-CDs). 3-Aminophenylboronic acid was used as the uniqueprecursor for modified CDs preparation using a simple hydrothermalapproach. The DA-sensing process was based on the interactions betweenthe amino groups on the CDs and DA, which enable the absorption ofDA onto the surface of the CDs through the formation of hydrogen bondsand on those of the boronic acid group (B(OH)2) with thediol moiety of DA (The best detection limit of0.1 pM with a wide linear range (from1 pM to 1 \u03bcM) was achieved by Liu and co-workers,ty of DA 10. Upon 73 using a sensor consisting of polypyrrole/graphenequantum dots (PPy/GQDs) core/shell hybrids , showing very good recovery values (97.6%\u2013103.3%).A sensing process based on similar interactionsbetween oxygen-containinggroups and amino groups with DA was exploited by Zhou et al. hybrids 11, which66 The structure of these materials is still unknown,but their features of photostability, cell permeability, high brightness,and nontoxicity make them particularly suitable for use in sensing.The detection method was based on the quenching of the CNDs fluorescenceat 425 nm when excited at 310 nm. The proposed materials exhibiteda quantum yield up to 58%, allowing the achievement of very good performancesin terms of detection limit .A new class of carbogenic nanomaterials, highlybright multicolorfluorescent sulfur-doped carbon dots (CNDs), obtained by a single-stepreaction, was used for sensitive DA detection by Gupta and Nandi.59 in 2022. In this strategy, dopamine had a doublerole: the analyte and a spacer between Tb(III) and AuNFs, and electrostaticinteractions were mainly responsible for bonding between the Tb3+\u2013DA complex and the NPs. The enhancement of the fluorescencesignal of the system was possible because of the metal-enhanced fluorescence,the Tb3+\u2013DA combination, and the energy transferfrom the analyte to Tb3+. The results showed a wide linearrange (0.80\u2013300 nM), a low LOD of 0.21 nM, and satisfactoryrecovery in serum and DA injection samples.Metal-enhanced fluorescence based on gold nanoflowers(AuNFs) forsensitive and selective DA detection was adopted by Li et al.9 and by Baluta et al.76 It is, in fact, well-known that DA tends to self-polymerize to polydopaminein alkaline conditions, and the polymer possesses fluorescence capability;therefore, it can be used for optical measurements.74 Weng and co-workers in 2015 and Baluta and co-workers in2017 reported rapid and easy fluorescence-sensing strategies basedon the polydopamine thin film formed and absorbed on the surface ofGQDs. In both cases, the detection process was due to the quenchingof fluorescence which occurred through FRET. Baluta and co-workers76 were also responsible for the design and developmentof an enzyme-based fluorescent biosensor consisting of a low-temperaturecofired ceramic platinum electrode (LTCC-Pt) covered with a thin filmof poly(dithienotetraphenylsilane) and GQDs on which laccase had beenimmobilized. The idea to use laccase was derived from the fact thatit catalyzes DA oxidation to dopamine-o-quinone,which is unstable and rapidly polymerizes in an alkaline environmentto polydopamine, which is the desired species for the detection process.Detection limits between 8 and 80 nM were achieved using these strategies.These values do not allow the use of the just-described (bio)sensorsin biological samples, but they are, however, suitable for the analysisof DA-containing drugs .A different sensingprocess approach based on the DA polymerizationwas successfully exploited in different ways by Weng et al.54 in 2016. It is a systembased on [Ru(bpy)2dppz]2+  and thioglycolicacid (TGA)-capped CdTe quantum dots for DA, adenosine, and 17\u03b2-estradioldetection. The mechanism was based on the ionic conjugation betweenthe Ru complex and QDs due to their electrostatic attraction in anaqueous solution that causes a decrease in the fluorescent intensityof QDs. After the addition of an adequate aptamer DNA, the fluorescenceof QDs can be recovered thanks to the strong tendency of DNA to bindto the Ru complex. When the aptamer was first incubated with the targetanalyte, it could not bind to the Ru complex, and the fluorescencewas quenched. It is a very highly selective and sensitive method thattakes advantage of the aptamer specificity and the excellent fluorescenceproperties of CdTe-QDs. In addition, the authors declare that thismethod is one of the simplest and one of the first that can detectthese three analytes by one universal system.Anotherbiosensor, specifically a fluorescent aptasensor, was developedby Huang et al.57 who reported the developmentof a nanosensor for DA and glutathione detection in human serum sampleswith satisfactory results. The particularity of this device is thatit can be considered a double sensor: (i) dopamine\u2013quinonederived from the oxidation of DA, adsorbed on the surface of silicadue to hydrogen bonds and electrostatic interactions, can be determinedby the quenching of the photoluminescence of the modified CdTe-QDs,and (ii) glutathione, which is a strong reducing species, can be determinedthrough the restoring of the fluorescence of QDs due to the reductionof dopamine\u2013quinone.CdTe-QDs coatedwith silica were used as fluorescent probes byXiangzhao et al.,55 and Zhao et al.,53 who synthesized, as DA sensors, adenosine-capped CdSe/ZnSquantum dots (A-QDs) and (3-aminopropyl)triethoxysilane (APTES)-cappedZnO-QDs, respectively. In both cases, QDs were water-soluble, andthe sensing processes were based on the fluorescence quenching ofthe QDs caused by an electron transfer. Mu and co-workers55 achieved a detection limit of 29.3 nM, highselectivity, and satisfactory recovery values (94.80%\u2013103.40%)in human urine samples. Likewise, APTES-ZnO-QDs allowed a LOD valueof 12 nM to be obtained with no significant interference effects froma wide plethora of common molecules present in human blood serum.The optimum features of modifiedQDs were also exploited by Muet al.70 described a novel aptamer-basedbiosensor for dopamine determination using fluorescence energy transferbetween a nanomaterial\u2014single-wall carbon nanohorns (SWCNHs)\u2014anda fluorescein derivative. In the proposed strategy, an aptamer waslabeled with 5-carboxyfluorescein (FAM) and, because of \u03c0\u2013\u03c0interactions, could be absorbed on the surface of SWCNHs, which ledto a decrease in fluorescence intensity. In contrast, in the presenceof DA, a G-quadruplex formed when the analyte bonded to the labeledaptamer could not interact with the NM surface, resulting in the recoveryof fluorescence. The platform exhibited a linear range of 0.02\u20132.2mM and a detection limit of 5 \u03bcM, and its applicability wasconfirmed by the analysis of dopamine-spiked serum samples.Zhang et al.5.4174 Numerous studies have been conducted to find an effective and efficientmethod of production of this neurotransmitter\u2014currently, GABAis produced in pharmaceuticals and food in sprouted brown rice, anaerobicallyincubated tea leaves, or fermented milk products.176 Unfortunately, owing to the zwitterionic nature of GABA (the aminoand carboxyl groups are adequately protonated and deprotonated), thedetection of these compounds is an extremely difficult task; hence,the design of sensor devices is quite a challenge.177Gamma-aminobutyric acid (GABA) 12 is one183 Usually, serotonin contributes to the feelings of happiness, andfor this reason it is also used as a drug for depression therapy.75 However, the actual biological function of serotonin is complexand multifarious, and its low levels can be synonymous of depression,anxiety neurosis, obsessive\u2013compulsive disorder, and migraines.Conversely, extremely high levels of serotonin could cause fatal serotoninsyndrome because of its toxicity, as well as irritable bowel syndrome.63 A typical concentration of 5-HT in whole blood and in platelet-poorplasma was found at 774 \u00b1 249 and 5.17 \u00b1 4.17 nM, respectively,184 between 0.52 and 1.2 nM in cerebrospinal fluid,185 and in the range 10\u201378 \u03bcmol/molcreatinine in urine.186Serotonin 13, also 187 Anomalous increments in cortisolconcentration can inhibit inflammation, depress the immune system,and increase fatty and amino acid levels in blood. Extremely highlevels can cause Cushing\u2019s disease, while decreased cortisollevels lead to Addison\u2019s disease.188 Levels of this hormone in saliva closely reflect the levels of unboundcortisol in blood; hence, saliva is the most preferred sample foranalysis.189 Cortisol levels in the bodyfluctuate during the day between 5 nM and hundreds of nanomoles, reachinga minimum of <2 nM in the evening or at midnight.46Cortisol 14, also As in the case of DA, researchers are trying todevelop increasinglysensitive methods for the determination of GABA, 5-HT, and cortisol.Compared to what has been observed for DA, nanomaterial-based fluorescent(bio)sensors for the detection of GABA, 5-HT, and cortisol are fewer,and the most recent are reported in the following paragraphs.71 In orderto obtain the detection platform, the functionalization of the CDswas performed with 3-aminophenylboronic acid (APBA) and nicotinamideadenine dinucleotide phosphate (NADP+) by means of an ECD/NHScoupling reaction. The CDs modified in this way, together with theenzyme GABase, were used for the determination of GABA by fluorescencequenching due to electron transfer between the enzyme and the substrate,thanks to the formation of a reduced form of NADPH. The system constructedin this way enabled the determination of the GABA concentration inthe linear range of 0\u201390 \u03bcM with a detection limit of6.46 \u03bcM. The performance of the sensor has been confirmed bytesting on biological samples, such as human spinal fluid and serum.Sangubotla and Kim developed a fluorescent enzyme sensor usingnontoxic carbon dots synthesized from corn juice for the sensitiveand rapid detection of GABA.178 The surface of the optical fiberwas modified with QDs via an EDC/NHS coupling reaction. Then, thefunctionalization of the QDs was performed using 3-aminophenylboronicacid and NADP+. The basis of the detection was the measurementof the change in the intensity of the QDs fluorescence, which occursdue to the transfer of electrons from the QDs to NADP+.The use of the GABase enzyme in the system allowed the reduction ofNADP+ to NADPH during the conversion of GABA to succinicacid. The reduction of NADP+ to NADPH made it difficultto transfer electrons, which made it possible to return the fluorescenceintensity of the QDs to the initial state.Zhao et al. constructed a quantum dot fiber optic sensor for thedirect detection of GABA by quenching and fluorescence recovery ofQDs.2+-dopedZnS-QDs modified with silica nanoparticlesbased on molecularly imprinted polymers (SiO2@MIPs) weredesigned and realized by Wang et al.50 Insuch a device, advantages of QDs such as large Stokes shift, low backgroundnoise, and a narrow and symmetric emission spectrum have been combinedwith the peculiar feature of the MIPs to mimic a natural receptor.As a matter of fact, the sensing process was based on the formationof a complex between the amino group present in QDs@SiO2@MIPs and the hydroxyl group of 5-HT, which led to the quenchingof the QD fluorescence  was instead employed by Zhao and co-workers63 Gold nanoclustersas well as QDs possess a large Stokes shift, good photostability,and ultrasmall size. In particular, the system developed by Sha etal. is based on the use of transferrin-encapsulated gold nanoclusters(Tf\u2013Au-NCs) because of the well-known high affinity betweensialic acid (SA) and 5-HT,191 due to the presenceof SA residues in transferrin. Moreover, transferrin, with 40 cysteineand 26 tyrosine fragments in its chain, is a very suitable reducingand stabilizing agent for gold nanoclusters. The obtained biosensoris a turn-on type device, as it showed an aggregation-enhanced emissionof Tf\u2013Au-NCs in the presence of 5-HT. With a detection limitof 0.049 \u03bcM and a linear range of 0.2\u201350 \u03bcM, itwas applied for 5-HT quantification in human serum.A devicefor the highly selective detection of 5-HT, based on anew class of nanomaterials, gold nanoclusters (AuNCs), was developedin 2019 by Sha and co-workers.50 the advantageous feature of the molecular imprintingtechnology to obtain a tailor-made specific binding cavity for a certaintarget molecule was also exploited by Murase and co-workers192 in the construction of a sensor for cortisoldetection. They reported the synthesis of a fluorescence polarizationassay platform based on core\u2013shell-type molecularly imprintedpolymer particles (MIP-NPs) using cortisol-21-monomethacrylate asa template agent. The sensing process was based on the competitivebinding of dansyl-labeled cortisol and cortisol against the cortisol-imprintednanocavities. The just-mentioned device exhibited a detection limitof ca. 80 nM, and it showed no effects of progesterone interference.Similarlyto the 5-HT sensor developed by Wang et al.,46 reported instead two new fluorescence biosensors,one based on cortisol-selective aptamers conjugated on the CdSe/ZnScore\u2013shell QDs surfaces and the other on anticortisol antibodiesconjugated to the same ones. In both cases, the modified QDs werecarried by magnetic nanoparticles in order to facilitate probe conjugationand cortisol detection in saliva samples. In the presence of cortisol,the authors observed fluorescence quenching of QDs sensorics development from a construction pointof view are presented.193 The most approachable in this context are microfluidicdevices, which often are used in miniaturized optical bioanalyticalsystems. Microfluidic approaches, which can be compared to miniaturizedforms of biochemical laboratories, allow the execution of many analysesat the same time, mixing or separation of reagents, biochemical reactionmonitoring, and signal output.195 However, despite theseadvantages, as well as many available sources describing the operationof miniaturized optical sensors/biosensors, also based on microfluidicplatform-assisted miniaturized biosensing systems, there is stilla problem with many analysis steps and with the integration of samplepretreatment. Microfluidic systems could overcome these problems tosome extent; however, the main challenge still remains\u2014to developa fully integrated detection system in a solid and approachable format.Apart from the most important workingparameters of biosensorsfor diagnostic application, which are sensitivity and selectivity,devices for clinical application should allow miniaturization withsimultaneous integration, automatization, and multiplex detectionwithout the need for sophisticated apparatus and trained personnel.197 Additionally, biosensors canbe combined with smartphones as their cameras, light sources, imageprocessing, and communication possibilities can lower costs and simplifydistribution on a large scale and reduce the time of work.198 Such types of chips can be used to measuresignals directly from patient samples, analyze data with personalizedapplications, and wirelessly send the results for interpretation.199 With the use of microfluidic-based biodevices,costs could be reduced thanks to using low-cost material and small-volumereagent requirements.Another important issue is cost-effectiveness. It is importantin sensor development to use ecofriendly, nontoxic, and relativelycheap components. Solutions may be to use stable mass production thatwill reduce costs or to focus on inexpensive disposable chips withreplaceable components that can avoid cross-contamination problemsand complicated cleaning procedures when handling biological samples,i.e., integration schemes that enable disposable cartridges and stand-alonereaders.To summarize, apart from manipulationswithin sensitive and selectivedetection , integration,coherence, and miniaturization are the most important challenges facingbiosensors in the context of their commercialization. The use of fluorescentmethods for the determination of medically important neurohormonesdescribed in this Review only highlights the need to introduce suchdevices to the market as soon as possible, as neurohormones are indicatorsof the body\u2019s homeostasis."}
+{"text": "Since the advent of semiconductor detectors, they have been developed for several generations, and their performance has been continuously improved. In this paper, we propose a new silicon drift detector structure that is different from the traditional spiral SDD structure that has a gap between the cathode ring and the width of cathode ring, increasing gradually with the increase of the radius of the cathode ring. Our new structure of spiral SDD structure has equal cathode ring gap and a given surface electric field, which has many advantages compared with the traditional structure. The novel SDD structure controllably reduces the area of silicon oxide between the spiral rings, which in turn reduces the surface leakage current due to the reduction of total oxide charge in the silicon oxide and electronic states on the silicon/silicon oxide interface. Moreover, it has better controllability to adjust this spiral ring cathode gap to achieve better surface electric field distribution, thus realizing the optimal carrier drift electric field and achieving the optimal detector performance. In order to verify this theory, we have modeled this new structure and simulated its electrical properties using the Sentaurus TCAD tool. We have also analyzed and compared different spiral ring cathode gap structures (from 10 \u00b5m to 25 \u00b5m for the gap). According to the simulation results of potential, electric field, and electron concentration, we have obtained that a spiral ring cathode gap of 10 \u00b5m has the best electrical characteristics, more uniform distribution of potential and surface electric field, and a more smooth and straight electron drift channel. Since the concept of silicon drift detector (SDD) was first proposed by E. Gatti and P. Rehak in the 1980s ,3,4, nucTo optimize the drift behavior of carriers in SDDs and reduce their surface leakage current, we propose a new detector structure based on controlling spiral ring cathode gap, namely, to keep it small and unchanged. Relative to the traditional design that the gap gradually increases with ring radius, this new structure will greatly reduce the silicon oxide area. This in turn can greatly reduce surface leakage current caused by the electronic states in silicon oxide and on the silicon/silicon oxide interface. At the same time, in the design, we can reasonably adjust the given surface electric field according to the actual application to achieve better carrier drift electric field and minimize surface current of the detector, so as to form a fully depleted detector region and realize a high-quality SDD. In this paper, simulations and performance comparisons are performed using the technical computer aided design (TCAD) tool for spiral ring cathodes with equal gaps of 10 \u00b5m and 25 \u00b5m, respectively, for a given surface electric field. Systematic comparative analysis can also be performed for surface electric fields in subsequent work.Considering the dead space and the symmetry of the physical structure of the detector when it is made into an array, we chose a double-sided hexagonal detector structure in our case of simulation. The shape of the detector is hexagonal, with a diagonal length of 3000 \u00b5m and a thickness of 300 \u00b5m. The detector bulk is lightly doped N-type silicon substrate with a doping concentration of An appropriate potential gradient is established for the carrier drifting from the position of incident particle to the collection anode by adjusting the cathode gap effectively and controllably to divide the voltage and to fully deplete the detector. Given the surface electric fields on both surfaces, the depletion region within SDD forms a transverse drift electric field that generates a drift channel. When the incident particle induced free carriers enter the drift channel, they drift to the collection anode through the transverse drift electric field, converting the energy of the incident particle into an electrical output signal. This output signal may be used to detect or identify the incident particle or light.Between each neighboring two spiral rings at a radius of geometry , for a hHere, the helix satisfies the following conditions:Given the surface electric field and equal (constant) gap, the formula of pitch The angle eference :(7)\u03c6(r)=Here, Let:Let:Then If Here, Since, Since:Surface potential Let:Then If Again, if In this paper, the purpose is reducing the surface current and obtaining the minimum leakage current by effectively and controllably adjusting the cathode gap One can solved For the case of The detector structure used in this paper can be derived from calculations using Equation (29) and related parameters in the design. It was mentioned in  that the silicon , and d iUsing this set of bias voltages, we have simulated this structure using the Sentaurus TCAD tool with results shown in Shown in The thick black line in According to the basic principles of physics, we know that the electric field is the gradient of the electric potential, so we can roughly judge the distribution of the electric field according to the potential distribution in The entire detector is in a non-zero electric field environment and is completely depleted. From As shown in The two-dimensional position resolution sensitivity in the detector is achieved by using the timing of electrons arriving at the collection anode to determine one of the dimensions of position, since the distance of arriving electrons to the collection anode is simply the drift time (timing) times the drift velocity (= electron mobility times drift field In this study, we proposed a new semiconductor detector structure of helical silicon drift detector with equal cathode ring gap and given surface electric field. This new structure reduced detector surface area, which in turn reduced the detector surface leakage current and made the detector internal electric field more uniform. We simulated and analyzed the internal characteristics of the detector with different spiral ring cathode gaps. We obtained the potential, electric field, and electron concentration distribution in order to determine the optimal detector parameters. Simulations were performed to compare two detectors with different spiral ring cathode gaps, and we determined that the performance of the spiral ring cathode gap of 10 \u00b5m was superior to the spiral ring cathode gap of 25 \u00b5m.This study provides strong theoretical support for practical fabrication of the detector. The new detector structure can be applied to the fields of space physics and photon science, such as pulsar X-ray detection and X-ray fluorescence spectrometers."}
+{"text": "Thermoplastic cellulose esters are promising materials for bioplastic packaging. For that usage, it is important to understand their mechanical and surface wettability properties. In this study, a series of cellulose esters are prepared, such as laurate, myristate, palmitate, and stearate. The aim of the study is to investigate the tensile and surface wettability properties of the synthesized cellulose fatty acid esters to understand their suitability as a bioplastic packaging material. Cellulose fatty acid esters are first synthesized from microcrystalline cellulose (MCC), then dissolved in pyridine solution, and after the solvent cast into thin films. The cellulose fatty acid ester acylation process is characterized by the FTIR method. Cellulose esters hydrophobicity is evaluated with contact angle measurements. The mechanical properties of the films are tested with the tensile test. For all the synthesized films, FTIR provides clear evidence of acylation by showing the presence of characteristic peaks. Films\u2019 mechanical properties are comparable to those of generally used plastics such as LDPE and HDPE. Furthermore, it appears that with an increase in the side-chain length, the water barrier properties showed improvement. These results show that they could potentially be suitable materials for films and packaging materials. In recent years the demand for bio-derived resources because of overriding environmental contamination and diminishing fossil reserves worldwide has been brought to attention . At pres9 tons of biomass, of which cellulose is 35\u201350%. This concludes that cellulose is the most abundant biopolymer. It is possible to chemically modify it, and the synthesis of cellulose esters allows for obtaining the needed strength and biodegradability and enables the use of cellulose for various applications. However, cellulose\u2019s hydrophilicity, dense fibrous structure, and insolubility in traditional solvents are restricting its full potential for usage  of cellulose solution in cupriethylenediamine hydroxide, CuEn, according to a standard procedure ASTM D1795-13. The MM was then calculated by the Mark-Houwink equation with parameters K = 1.01 \u00d7 10\u22124 dL/g and a = 0.9 [OAc] and stirred at 60 \u00b0C for several hours until the cellulose was completely dissolved to yield a 3.5 wt% solution. To decrease the viscosity, which simplifies the processing, a co-solvent DMSO was added in a ratio of 1:1 to IL. The designed amount of the respective vinyl ester (3 eq./AGU) was added to the cellulose solution in a chemical reactor equipped with a mechanical stirrer and nitrogen flow, and then the reaction was performed at the conditions shown in 1H NMR (hydrogen-1 nuclear magnetic resonance) and 13C NMR (carbon nuclear magnetic resonance) methods. The 1H NMR and 13C NMR spectra of the cellulose fatty acid esters were acquired on an Agilent Technologies DD2 500 MHz spectrometer equipped with 5 mm broadband inverse (1H) or broadband observe (13C spectra) probes. In addition, a 15 min temperature equilibration delay was allowed between sample insertion and NMR acquisition at 40 \u00b0C  or 80 \u00b0C (cellulose laurate in DMSO-d6 and cellulose myristate and laurate in Py-d5) sample temperatures. Typically, for 1H spectra, 32 scans with a 25 s relaxation delay were acquired, and for 13C, 20,000\u201345,000 scans with a 2.5 s recycle delay were acquired to achieve the desired signal-to-noise ratio.DS values were determined with 1H NMR and 13C NMR. In the 1H NMR spectrum, the proton signals from approximately 6.00 to 3.50 ppm are assigned to protons of AGU in cellulose. The signals at 2.32\u20131.93, 1.88\u20131.45, and 1.27 ppm are associated with the methylene protons. The signals at 0.98\u20130.79 ppm are attributed to the methyl protons.The chemical structures of the cellulose esters were confirmed by 13C NMR spectrum of cellulose esters, the signals from 34.78 to 23.34 are assigned to carbons of the aliphatic fatty acid chain, and 14.60 ppm is assigned to the carbon of the ending methyl group of the aliphatic side chain. The AGU carbons C-1, C-1\u2032, C-4, C-2,3,5, C-6, and C-6\u2032 give signals at 104.98, 102.33, 81.50, 76.91\u201374.35, 64.32, and 62.68 ppm, respectively. The signals from 173.89 to 170.98 ppm correspond to the carbonyl carbon at C-7, which provides direct confirmation of the successful attachment of the long-chain fatty acid chain to the cellulose backbone.In the 1H NMR spectrum by taking an integral of the area of terminal methyl groups (ICH3) and AGU signals (IAGU) based on the reported method [The DS of cellulose laurate, -myristate, -palmitate, and -stearate was calculated with equation 1 from the d method . The cal\u22121 . Solutions were then solvent casted on laminated glass plates (250 mm \u00d7 150 mm) using the film casting knife  with a blade width of 100 mm. With the given knife, the casted thickness of 200 \u03bcm was set to achieve the final film thickness of 70\u2013100 \u03bcm. Except for cellulose diacetate, in which a casting thickness of 100 \u03bcm was set due to a higher cellulose concentration. Subsequently, the films were dried for 24 h in the open air under a fume at a room temperature of 20 \u00b0C. The films were removed using a phase immersion method in water after the solutions had been set on the glass plates. Additionally, the pH of the film\u2019s surface was determined with the flat electrode using the Mettler Toledo SevenCompact pH meter S210  to check the acidic levels of each synthesized film. All measurements were conducted at a room temperature of 23 \u00b0C and an air humidity of 40%. Cellulose esters were dissolved in pyridine, and the obtained solutions shear rate ranged from 0.001 to 100 s\u22121. Four scans were taken from each sample with a resolution of 8 cm\u22121 in absorbance mode. For comparison, all spectra were adjusted to the same baseline.The FTIR spectroscopy analysis was carried out on native cellulose and synthesized cellulose esters to evaluate the acylation process. Measurements were performed on an Interspectrum FTIR spectrometer  with the KBr disc method between 500 and 4000 cmThe surface hydrophobicity of the prepared films was estimated by measuring parameters such as equilibrium contact angle. The sessile drop method was used to measure contact angles with the device DataPhysics OCA 20 and the SCA 20 software . All measurements were performed on the top side of the films with distilled water as a liquid agent to create a drop on the surface. The total observation time of the contact angle changing was 40 s starting from the moment when the water drop came into contact with the measured surface. An average of each point of measurement  was taken. A total of six measurements were taken for each variable, and the material average was taken from these six measurements. Before the measurement, the specimens were fixed with clamps on the measuring plate to achieve a smooth surface. All measurements of the contact angle were performed at room temperature and a relative humidity of 40%. p-value was significant, the Bonferroni correction with the new alpha was utilized by dividing the alpha by the given comparisons (t-tests). The new alpha was set at 0.005 (10 comparisons).The tensile test was performed according to ISO 527-3 . The sta\u22121, 2849 cm\u22121, and 1467 cm\u22121 identify the presence of fatty acid long chains and correspond to anti-symmetric C\u2212H stretching, symmetric C\u2212H stretching, and C\u2212H bending vibrations of the cellulose stearate, palmitate, myristate, and laurate [\u22121 (C=O), 1225 cm\u22121 (\u2212C\u2212O\u2212 stretching), and 716 cm\u22121  [\u22121 (OH stretching) is observed for all the synthesized fatty acid esters compared to the native microcrystalline cellulose. laurate . In the  groups) . These b groups) ,17,24,25p < 0.05) compared to fatty acid cellulose esters , which correlates with the observation of chain length increase. Thus, this research shows that all the synthesized cellulose fatty acid esters have the desired hydrophobic properties (contact angle >90\u00b0) for the use of food packaging materials.In general, cellulose esters should be more hydrophobic than cellulose due to acetylation . HoweverTensile properties of the casted films were observed to evaluate the effect of side chain length on cellulose fatty acid esters strength, elongation, and elastic properties. From our study results , it is sWhen compared to the commercially available cellulose acetate, the long sidechains of cellulose esters give a significant decrease in elastic modulus (~70\u201380%), tensile strength (~60\u201370%), and an increase in strain at break (~700\u20131200%). This data indicates that the fatty acids in the cellulose esters could already function as an internal plasticizer. According to Cr\u00e9py and Wen ,16, whenOur research data show some similarities to those of commercial polymers (low-density polyethylene and high-density polyethylene). According to Cr\u00e9py , LDPE haNew cellulose esters  were successfully synthesized in ionic liquid and solvent casted into films. Their surface and mechanical properties were evaluated and compared with those of commercial cellulose diacetate. A lower degree of substitution (<1) results in an increase in stiffness and strength ), which is more like the highly substituted cellulose acetate. However, with the effect of long aliphatic chains, the material obtains a higher strain than commercially used cellulose acetate, which is satisfactory for packaging material. All the synthesised cellulose fatty acid esters showed the desired hydrophobic properties for the food packaging materials. With the increase in side-chain length, the water barrier properties improved. Cellulose palmitate (C16) and cellulose stearate (C18) casted films showed the highest hydrophobic properties. When compared to commercial cellulose acetate (C2), the synthesised cellulose esters had increased elongation and hydrophobicity. Cellulose laurate (C12) and cellulose palmitate (C16) are most similar to LDPE and HDPE, respectively. According to our research data, it is promising that the fatty acid cellulose esters could be utilized for future bioplastic applications."}
+{"text": "Growing concerns about environmental issues and global warming have garnered increased attention in recent decades. Consequently, the use of materials sourced from renewable and biodegradable origins, produced sustainably, has piqued the interest of scientific researchers. Biodegradable and naturally derived polymers, such as cellulose and polylactic acid (PLA), have consistently been the focus of scientific investigation. The objective is to develop novel materials that could potentially replace conventional petroleum-based polymers, offering specific properties tailored for diverse applications while upholding principles of sustainability and technology as well as economic viability. Against this backdrop, the aim of this review is to provide a comprehensive overview of recent advancements in research concerning the use of polylactic acid (PLA) and the incorporation of cellulose as a reinforcing agent within this polymeric matrix, alongside the application of 3D printing technology. Additionally, a pivotal additive in the combination of PLA and cellulose, polyethylene glycol (PEG), is explored. A systematic review of the existing literature related to the combination of these materials  and 3D printing was conducted using the Web of Science and Scopus databases. The outcomes of this search are presented through a comparative analysis of diverse studies, encompassing aspects such as the scale and cellulose amount added into the PLA matrix, modifications applied to cellulose surfaces, the incorporation of additives or compatibilizing agents, variations in molecular weight and in the quantity of PEG introduced into the PLA/cellulose (nano)composites, and the resulting impact of these variables on the properties of these materials. Polylactide, also known as poly(lactic acid) (PLA), stands as a biopolymer derived from renewable and biodegradable sources. It boasts remarkable mechanical characteristics, including a notable ultimate tensile strength ranging from 50 to 70 MPa and a tensile modulus of around 3 GPa ,2,3. TheTo overcome these disadvantages, recent research has investigated the use of cellulose to improve the mechanical, thermal, and rheological properties of PLA ,7,8,9,10The use of cellulose as a natural reinforcement for PLA has been widely studied, as it provides the PLA matrix with better mechanical and rheological properties as well as high crystallinity, which depends on the combination of different factors, such as the mixing processes used and the properties of the cellulose , and the use of compatibilizing agents, surfactants and plasticizers ,5,7. ComWhen producing PLA composites with cellulose, it is common to incorporate additives that enhance material compatibility and modify melt viscosity. This is particularly important as introducing reinforcement, depending on its scale and quantity, has the potential to elevate viscosity, thereby potentially impeding the efficiency of manufacturing processes. One of the most used additives for this type of composite is polyethylene glycol (PEG), which, depending on the molecular weight, can act both as a compatibilizing agent and as a plasticizer . In addiRegarding the methods used in the preparation of PLA/cellulose composites and nanocomposites, the most common are melt mixing  ,14,15,16Additive manufacturing (AM) techniques, which include fused filament fabrication (FFF), are gaining prominence in recent years because they have advantages such as precise control of complex structures, the ability to use various types of plastic materials, and little waste in the process .Hence, this review provides a comprehensive analysis of the existing literature pertaining to the utilization of cellulose in various scales, concentrations, and forms to reinforce the biodegradable PLA matrix. It also explored the impact of incorporating PEG as an additive and examined the application of these composite materials in filament production for 3D printing. Furthermore, this review systematically addresses the array of studies within the literature encompassing the following themes: cellulose-reinforced PLA composites, the role of PEG as an additive to enhance and modify composite properties, the expansion of processing possibilities and potential applications, and the utilization of PLA/cellulose/PEG (nano)composites for filament fabrication in the realm of 3D printing.Biopolymers are defined as sustainable polymers produced from a variety of renewable raw materials such as polysaccharides, lignin, vegetable oils, pine resin derivatives, and proteins as a substitute for petroleum, the most conventional fossil resource ,27. ThisThe biopolymer market has experienced growth in recent years, encompassing both biodegradable and non-biodegradable variants derived from renewable and non-renewable sources. This expansion is attributed to the increasing demand from consumer goods companies, who are striving to offer environmentally friendly products in alignment with sustainability standards ,32.According to the European Bioplastic Report , the totDespite the promising growth, the production of bioplastics still represents less than 1% of the more than 390 million tons of plastic produced annually. This limitation persists due to factors such as the elevated production expenses, insufficient political incentives, and inherent properties that frequently hinder their compatibility with certain applications across various sectors ,32. Among the currently most used biopolymers, PLA stands out for being obtained from a renewable source and presenting biodegradability in industrial composting environments . PLA is Due to the chirality of the lactic acid monomer, PLA exists in different enantiomeric forms , and its properties depend both on the content of optical impurities among the enantiomers and the molecular weight . PolymerThe main applications of PLA are in the production of plastic films, recyclable and biodegradable packaging , garbage bags, wall coverings, fabrics, fibers, and biodegradable medical devices , textile fibers, and 3D printing filaments . Although it has lower toughness; less than 10% elongation compared to conventional polyolefins such as PP, for example; and a low crystallization rate, these properties can be improved with the addition of different filler particles, including cellulose at micro- and nanoscales, which, in addition to improving the thermomechanical properties, do not affect the biodegradability of the polymer ,39,40,41Regarding its application as a filament in additive manufacturing, PLA offers several advantages outlined in Nonetheless, certain limitations, also listed in In polymer composites with a PLA matrix, the cellulose used as filler or reinforcement is added in different amounts, shapes, and scales, such as cellulose fibers ,43,44,45Cellulose can be derived from diverse natural sources like sugarcane bagasse ,45, flaxCellulose fibers are defined as a set of microfibrils where the cellulose molecules are stabilized laterally through hydrogen bonds between the hydroxyl groups . In the The isolation of cellulose nanofibers from lignocellulosic fibers can be performed via different processes, such as electrospinning ,63,64,65Distinct morphologies are achieved contingent on the technique employed to fragment macroscopic fibers into nanofibers. Through acid hydrolysis, a colloidal suspension of aggregates characterized by high crystallinity and an elevated aspect ratio is formed, referred to as microcrystalline cellulose (MCC). Subsequent to acid hydrolysis, the application of sonification disintegrates the fibril aggregates, yielding cellulose whiskers, also recognized as nanocrystalline cellulose (NCC). Utilizing multiple mechanical shearing actions yields microfibrillated cellulose (MFC) or nanofibrillated cellulose (CNF), both comprising interconnected fibrils and fibril aggregates. Additionally, MFC can be obtained through pretreatment involving enzymatic hydrolysis coupled with mechanical shearing .Regardless of the cellulose scale used, there is a chemical incompatibility between hydrophilic cellulose and most hydrophobic thermoplastic polymer matrices. Thus, to improve the compatibility between cellulose and the PLA matrix, surface treatments are often used, which can be chemical, physical, or enzymatic and whose primary function is to increase the hydrophobicity of the cellulose surface, removing some amorphous constituents from lignocellulosic sources or adding some functional group that increases the hydrophobicity of the fiber and improves the chemical and mechanical compatibility with the polymeric matrix. It is also possible to graft polymers on a cellulose surface, decreasing the surface energy and increasing compatibility between cellulose and a polymer matrix .The use of nanocellulose as a natural reinforcement for PLA has been widely studied, as it provides the PLA matrix with better mechanical and rheological properties, which depend on the combination of different factors, such as the mixing processes used and the properties of the cellulose , as well as the use of compatibilizing agents and surfactants ,7. CombiThe use of plasticizers is one of the effective methods to change the properties of polymers and thus increase the variety of applications. PLA is a rigid polymer, which limits its use in many applications, and in this way the use of plasticizers improves the mechanical properties and also helps in the fluidity of the polymeric matrix during processing . PlasticAmong the various plasticizers used for PLA, notable choices include citrate esters, poly(ethylene glycol) (PEG), glycerol, oligomeric lactic acid, and glucose monoesters. PEG, particularly when possessing low molecular weight, distinguishes itself due to its exceptional miscibility, biodegradability, and suitability for applications involving food contact ,12.2CH2)nOH. It is available in a broad range of molecular weights, spanning from 100 to 6000 g/mol [PEG is a nonionic, water-soluble polymer, characterized by the general formula H concentration could maintain stability for four months, after which considerable parameter shifts occurred. Notably, an increased concentration of PEG400, specifically 20% (w/w), extended shellac stability for 6 months.Luangtana-Anan et al.  studied Li et al., 2020  studied In Industry 4.0, 3D printing manufacturing technology, an additive manufacturing technique, also known as layered deposition or rapid prototyping, is a process for manufacturing three-dimensional (3D) objects through the deposition of material in layers, capable of producing parts with complex and customized ergonomic shapes ,76. WithThe thermoplastics most used in this type of additive manufacturing are ABS and PLA, and PLA is more sustainable since it is derived from natural sources and is biodegradable in an industrial composting environment. Furthermore, parts produced by 3D printing using the FFF technique with PLA have fewer warping problems and better mechanical properties compared to ABS . ConversDespite the numerous papers published in this area, much development still needs to be carried out since problems of cellulose agglomeration, the lack of compatibility with the polymeric matrix, and the heterogeneity of the cellulose dispersion within the matrix, as previously mentioned, minimize the capacity of the cellulose to improve the mechanical and rheological properties of PLA while also affecting the printability of the polymer, inducing processing problems, color change, and carbonization .The primary aim of this study is to explore and comprehend the pivotal role of PEG in enhancing the properties of PLA/cellulose (nano)composites when employed in the fabrication of filaments intended for 3D printing. A comprehensive analysis is sought on how the incorporation of PEG affects the mechanical, thermal, and rheological characteristics of these composites, as well as their overall performance as raw materials in additive manufacturing. To evaluate the state of the art in this subject, a bibliometric analysis was carried out of published articles that contained some specific keywords within this theme.A systematic literature search was conducted in the Scopus and Web of Science (WOS) databases, considering English-language documents, and excluding conference papers, reviews, and book chapters. Four distinct searches were carried out using the keywords outlined in The primary function of this systematic review article is to comprehensively investigate and analyze the effects of incorporating PEG into cellulose-reinforced PLA (nano)composites, particularly in the context of filament production for 3D printing. The review critically evaluates the existing literature to understand how different characteristics of PEG, such as molecular weight, percentage added, and the type of cellulose used, influence the mechanical, thermal, and rheological properties of these composites. Additionally, it seeks to identify trends and usage patterns of PEG in these composites by synthesizing findings from a range of studies. The review also highlights research gaps, indicating areas that require further investigation.Furthermore, the article serves as a decision-support tool for optimizing PLA/cellulose composite formulations for 3D printing by providing valuable insights based on a systematic analysis of the literature.This review is intended to provide an informative synthesis of the existing literature, aiming to offer insights and understanding rather than making prescriptive recommendations. Its primary purpose is to comprehensively analyze and distill the available knowledge, facilitating a deeper understanding of PLA/cellulose composites with PEG in 3D printing, without prescribing specific actions or outcomes.The initial search (Search 0) considered general terms, resulting in 1346 documents spanning the past 45 years. Subsequently, the inclusion of the term \u201cPEG\u201d (Search 1) notably reduced the pool to 51 documents published within the last seventeen years. With the incorporation of the term \u201c3D printing\u201d (Search 2), the search yielded 45 documents, all of which are notably recent, originating from 2017 onwards. Finally, the most recent search (Search 3) combined \u201cPEG\u201d and \u201c3D printing\u201d, culminating in the discovery of only two documents published in the last seven years. This clearly underscores a research gap in the literature, presenting a significant opportunity for future research.Regarding Searches 1 and 2, Furthermore, Keyword networking was realized using VOSviewer software (version 1.6.16) considering the frequency and chronological time  with a mAmong the 51 research papers published in the literature using the keywords specified in Search 1 ((\u201cPLA\u201d OR \u201cpolylactic acid\u201d OR \u201cpoly(lactic acid)\u201d OR \u201cpoly(lactide)\u201d AND \u201ccellulose\u201d AND \u201cPEG\u201d), as detailed in Publications with cellulose fibers (or micrometric fibers) reported the use of filler ranging from 15 wt.% to 25 wt.%, while the amount of PEG reported in these papers was between 0.5% to 40%. Moscoso-S\u00e1nchez et al.  used 15 The use of MCC was reported by Jirum and Baimark , Moreno Wang et al.  also notPEG was added in these papers in the range from 5 to 20%, and the molecular weights were 400 g/mol ,85, 600 Most of the research papers therefore used nanocellulose as a reinforcement, both NCC and CNF. Only one publication reported the use of cellulose nanosphere (CNS) . With thThe use of PEG as a coupling agent to change the surface properties of cellulose and increase the compatibility with PLA was reported by Macke et al. , Yu et aSrisawat et al.  comparedEicher et al.  reportedIn general, higher plasticizer molecular weights are associated with increased resistance to migration, prolonged utility lifespan, and reduced volatilization. Nevertheless, based on the results published in the literature for PLA/cellulose composites and nanocomposites, PEG can act as a plasticizer, its most common use. Additionally, PEG serves as a compatibilizer between hydrophilic cellulose and hydrophobic PLA. Thus, the effect of PEG addition can vary according to its molecular weight, the added content, as well as the specific type and scale of cellulose employed as a filler or reinforcement.The primary manufacturing processes reported in the publications arising from Search 1, for composites reinforced with fibers and MCC or MFC, were the combination of mixing processes such as melting blending using an internal mixer or twin-screw extruder, with molding processes such as solvent casting, compression, extrusion, and injection . For nanIn the systematic examination of the literature using the terms specified in Search 2 ((PLA\u201d OR \u201cpolylactic acid\u201d OR \u201cpoly (lactic acid)\u201d OR \u201cpoly(lactide)\u201d AND \u201ccellulose\u201d AND \u201c3D printing\u201d), a total of 45 published papers were identified , 18% involve MCC or MFC, and another 18% incorporate cellulose fibers sourced from various natural origins (as depicted in 2SO4-SE-steam explosion). Their evaluation encompassed both the potential for ethanol production and the enhancement of economic viability in the reed biorefinery process. The outcomes of this analysis led to the identification of three specific pretreatment processes: RES (enzyme hydrolysis-processing residue treated with NaOH), REP (enzyme hydrolysis-processing residue treated with p-TsOH), and RED (enzyme hydrolysis-processing residue treated with H2SO4-SE). Subsequently, these treated residues were harnessed as the reinforcing phase for the material utilized in 3D printing.Within the subset of eight publications that explored cellulose at the micrometric scale (cellulose fibers), the content of fibers integrated into the PLA matrix spanned from 3 to 30 wt.%. Notably, merely two of these publications documented the implementation of surface modifications to the fibers. Jiang et al.  documentIt is worth mentioning that two other publications, related to the keywords of Search 2 and using cellulose fibers as a filler, were identified in the literature, although they did not appear in the searches carried out in the databases used, probably because they did not contain the exact keywords used in this search. In these two publications, the authors also used surface treatment of the cellulose fiber, such as treatment with tetraethyl orthosilicate (TEOS) to increase the compatibility between PLA and the fiber  and chemThe application of PEG was documented by Ma et al. , who incIn terms of employing MCC or MFC, the database search yielded eight publications. Within this set, seven studies employed MCC, with only one publication opting for MFC. Winter et al.  presenteThe amount of MCC and MFC reported in these works varied from 1 wt.% to 50 wt.%, and the use of a superficial modification of cellulose was reported by Murphy et al. . Murphy From Search 2, 20 documents related to the use of nanocellulose as a filler to PLA matrix. Besides NCC and CNF, the use of carbonized cellulose nanofibers  and TEMPThe effect of NCC or CNF addition on the PLA matrix, however, depends on different factors such as amount of filler added and the use of a coupling agent or some cellulose surface modification. According to Dong et al. , the incAmong the papers resulting from Search 2, not all reported studies considered the 3D printing process; some publications reported only filament production, using in most cases a twin-screw extruder . In someAs in Search 1, some papers resulting from Search 2 did not necessarily report the production of PLA/cellulose composites or nanocomposites but rather the formation of blends and/or the use of cellulose derivatives. Pis et al. , for exaFinally, from Search 3 (\u201cPLA\u201d OR \u201cpolylactic acid\u201d OR \u201cpoly(lactic acid)\u201d OR \u201cpoly(lactide)\u201d AND \u201ccellulose\u201d AND \u201cPEG\u201d AND \u201c3D printing\u201d), only two research papers were identified. These two works are from the same authors, in which they discussed the use of PLA with NCC concerning the printability of the composite plasticized with PEG  and alsoIn the realm of PLA/cellulose (nano)composites and 3D printing, there are numerous promising avenues for future exploration and research.Firstly, the potential of cellulose in these composites can be further unlocked by varying its scale, content, and sourcing from different natural origins. By tailoring the type and source of cellulose, researchers can fine-tune the composite properties, adapting them to specific applications. Furthermore, there is a critical need to delve deeper into understanding how different molecular weights of polyethylene glycol (PEG) influence PLA/cellulose (nano)composites. This investigation is essential for optimizing composite properties and ensuring they meet desired performance criteria.In the context of 3D printing, one intriguing prospect is addressing the challenges associated with multiple extrusion cycles, a common practice for achieving better composite uniformity. Given PLA\u2019s sensitivity to temperature and potential degradation, exploring the effects of multiple extrusion cycles is vital to preserving material integrity. The rapidly evolving landscape of 3D printing, as an emerging manufacturing technology, necessitates increased attention and diversified research. This entails exploring innovative approaches, materials, and processes, particularly within the realm of composites.Environmental considerations are becoming increasingly important. Future research should encompass comprehensive assessments of the environmental impact of PLA/cellulose composite filaments and the final printed products. Sustainability, carbon footprint, and eco-friendliness throughout their lifecycle should be thoroughly evaluated. Understanding the end-of-life implications of PLA/cellulose (nano)composites is equally crucial. This includes studying the disposal and recyclability aspects to ensure these materials align with sustainable practices and do not contribute to environmental harm.Lastly, evaluating the residue generated during filament production and 3D printing processes is vital. Effective waste management strategies should be developed to minimize environmental impact and maximize resource utilization.In summary, the prospects for future research in the field of PLA/cellulose (nano)composites and 3D printing are expansive, encompassing various opportunities from material composition enhancement to sustainable practices and environmental impact assessment. Addressing these prospects will drive progress in the field, optimizing composite performance and promoting sustainability in 3D printing. Future research should also focus on the environmental impact of the PLA/cellulose composite filaments and final printed products, as well as their end of life. Also, the residue generated throughout the filament and printing process should be evaluated.The versatility of PLA/cellulose (nano)composites, particularly when enhanced with additives like polyethylene glycol (PEG), extends to a wide array of industries and uses. In the realm of packaging, these composites offer an eco-friendly alternative. They can be employed for biodegradable packaging solutions, including food packaging ,150,151,In the biomedical field, the biocompatibility of these (nano)composites makes them suitable for creating biodegradable medical devices. They have applications in biodegradable implants, drug delivery systems, and tissue scaffolds for regenerative medicine ,153,154.In the realm of 3D printing, neat PLA is already widely employed for producing biodegradable and environmentally friendly 3D-printed objects. This includes a wide range of items from prototypes to functional parts and artistic creations. However, when combined with cellulose as a reinforcement, the literature predominantly focuses on material characterization and filament production, with limited reporting on specific practical applications. Nevertheless, the PLA/cellulose (nano)composite shows significant promise for 3D printing applications, especially when incorporating additives like PEG. These additives enhance the composite\u2019s rheological, mechanical, and thermal properties, making it an attractive candidate for a wide range of 3D printing applications ,142,143.In summary, PLA/cellulose (nano)composites have some other less common yet promising applications including textiles, automotive, agriculture, consumer goods, and construction. These applications offer sustainable solutions and align with environmental responsibility. Ongoing research is expected to reveal more innovative uses in the future.This review covered the main concerns on PLA/cellulose/PEG composites. Firstly, an overview of each material was given, emphasizing its recent trends. Then, a systematic review was provided according to the most current databases in the field (Scopus and Web of Science), in which three searches were highlighted and discussed over the work.Cellulose, as a valuable compound from natural sources, has been widely applied in the PLA composites field with articles published in the past 45 years (848 documents), while the use of PEG (Search 1) and the composite\u2019s application in 3D printing (Search 2) are recent subjects. Regarding the 3D printing topic, Search 2 presented 39 documents, with an increasing tendency in the last 6 years, emphasizing that this subject should be explored more in future works, focusing on improving the PLA/cellulose filaments\u2019 performance in printing. Also, Search 3 showed that the use of PEG to improve the 3D printability of PLA/cellulose composite represents a significant gap in the literature as well as the use of other plasticizer/coupling agents.The discussion of the articles resulting from Searches 1 and 2 provided significant information about the scales/content of cellulose that have been used, the principal properties of the composites, and possibilities for improvement by using a plasticizer with different molecular weight. Also, printing parameters and performance were widely reported. Therefore, this work not only provided valuable information to guide future studies but also emphasized 3D printing as an innovative feature for PLA reinforced with natural filler composites, showing that there is much more than the commonly used PLA filament."}
+{"text": "In the last few years, new sustainable fractionation processes have been developed that enable the obtaining of cellulose fibers in a more eco-friendly way. As a raw material, cellulose\u2019s use is widely known and established in many areas. Additionally, its products/derivatives are recognized to have a far better environmental impact than fossil-based materials. Examples are textiles and packaging, where forest-based fibers may contribute to renewable and biodegradable substitutes for common synthetic materials and plastics. In this review, some of the main structural characteristics and properties of cellulose, recent green extraction methods/strategies, chemical modification, and applications of cellulose derivatives are discussed.Cellulose is the most abundant renewable polymer on Earth and can be obtained from several different sources, such as trees, grass, or biomass residues. However, one of the issues is that not all the fractionation processes are eco-friendly and are essentially based on cooking the lignocellulose feedstock in a harsh chemical mixture, such as NaOH + Na The rapid population growth and rise of globalization have been followed by the depletion of fossil fuel reserves, increasing health/environmental concerns. These have led researchers worldwide to look for new renewable resources for a more sustainable future. Cellulose, as the main component of plants , is the most abundant biopolymer on earth. Due to its high availability, low cost , biodegradability, appealing physical properties, and chemical reactivity , cellulose has been receiving great attention from the research community over the last decades. Recent renewed interest has arisen due to its potential use as a renewable energy platform and for the development of cellulose-based materials.2 [A crucial contribution to the global climate challenge comes from forests and forest-based products, which store ca. 447 million tons of CO2 . In fact2 . For exa2 .However, cellulose processing is challenging due to some important disadvantages, such as its insolubility in water and in most common solvents and its low resistance against microbial attacks . FurtherIn the current review, we explore the (1) fundamental structural characteristics and properties of cellulose from various sources; (2) sustainable extraction processes; (3) chemical modifications used in the preparation of cellulose derivatives; and 4) various applications for cellulose derivatives. Special attention is given to recent sustainable strategies engaging extraction, dissolution, and modification of cellulose, with a particular focus on the utilization of deep eutectic solvents. In the literature, it is possible to find different reviews dealing with cellulose derivatives and their applications. However, most of them are focused on specific applications, such as biomedical applications  various , or wastCellulose is generated at the plasma membrane in the form of paracrystalline microfibrils . The hieCellulose can be obtained from various biomass sources, such as hardwoods , softwoods , forestry residues, agricultural wastes, or grasses. Most properties of the obtained cellulose are strongly dependent on the source of biomass; one of these properties is the molecular weight, which has deep effects on cellulose application and processability. For example, hardwood raw materials typically present degrees of polymerization (DPs) ranging from ca. 1400\u20131790 , but it The extraction process used to obtain cellulose also has an impact on the DP of the recovered cellulose. For instance, the sulfite process leads to less depolymerization than the kraft process ,16. AnotIn addition to cellulose, lignocellulosic biomass is composed of two other structural polymers (hemicellulose and lignin), along with other minor compounds such as proteins and fatty acids ,21. CellConsidering what is referred to above, the choice of the cellulose source will depend on the desired properties and application, its availability, and economic purposes. Nowadays, most of the cellulose fibers used worldwide are extracted from wood. Nonetheless, wood is not widely available in some regions, and there is also a competing interest among several industries related to construction, furniture, pulp and paper, and the burning of wood for energy harvesting. Thus, it can be challenging to supply the required quantities of wood at reasonable prices to all sectors . This enThere are various lignocellulosic biomass fractionation processes that allow the separation and isolation of the components; the choice of the most efficient method depends on the target polymer, source, and desired properties of the final product. Usually, cellulose is obtained by dissolving lignin and hemicellulose, along with low-molecular-weight compounds.Conventional methods for biomass fractionation, such as those used in the pulp and paper industry , are very efficient for the extraction of cellulose, but the nature of the solvents used and the harsh treatment conditions employed have led the scientific community to search for new environmentally friendly alternatives. The use of green solvent systems, such as ionic liquids (ILs) and deep eutectic solvents (DES), has been reported for biomass fractionation and demonstrated to be very promising systems , not onlILs, first reported by Paul Walden in 1914, are known as \u201cmolten salts\u201d because they present a low melting point, usually below 100 \u00b0C , and arew/w) of the total lignin present in different sources, such as corn straw [In this context, DES have emerged as promising solvent systems due to their greener profile and high efficiency for biomass fractionation ,61. The rn straw . Systemsrn straw , ChCl anrn straw , and ChCrn straw  are partrn straw . Despitern straw .Cellulose is a high-molecular-weight linear homopolymer composed of D-anhydroglucopyranose units (AGU) connected by \u03b2(1\u20134)-glycosidic bonds . Each AGAs shown in The reactivity of the hydroxyl groups combined with their tendency to establish hydrogen bonds is responsible for some of the characteristics of cellulose, such as its highly cohesive nature and remarkable mechanical features . Throughw/w) of the total cellulose fiber, and it is very dependent on the cellulose source and extraction conditions [The crystalline fraction typically ranges between 40% and 70%  their inherent chemical reactivity; (2) steric effects that may arise from the reacting agent; and (3) steric effects that are driven by the supramolecular structure of cellulose . In mostOne important class of reaction in cellulose is esterification. In this respect, it has been found that the OH at the C6 position is more prone to react than the OHs at the other positions. Moreover, the OH at the C2 position reacts twice as fast as the OH at the C3 position in esterification reactions . In compDespite the favorable presence of reactive OH groups, reactions involving cellulose are typically not easy, mainly because cellulose is highly heterogeneous in nature. As discussed above, different parts of its constituent fibrils display very different accessibilities to the same reagent  . The accThe efficiency of the activation process (swelling/dissolution) deeply influences the capacity to facilitate and control reactions with the three hydroxyl groups in each AGU . It is iCellulose is a fascinating polymeric material and possesses several favorable features, but it also presents some drawbacks, such as its poor solubility in common solvents and its lack of thermoplasticity and antimicrobial properties. To overcome such limitations, controlled chemical modification of the cellulose structure is often a suitable strategy ,85.Dimensionally speaking, cellulose derivatives fall into two main categories: macromolecular cellulose derivatives and nanoscale particles . EtherifTo improve cellulose reactivity and allow further modifications, pretreatments are often performed to introduce more reactive groups into the cellulose structure. One example is the oxidation of cellulose to convert the hydroxyl groups into more reactive aldehyde groups that can then undergo other derivatizations. Regarding the oxidizing reagents used in cellulose chemistry, many can be enumerated, such as nitrogen oxides, alkali metal nitrites and nitrates, ozone, permanganates, and peroxides. These agents usually lead to reactions with low selectivity. However, cellulose oxidation with periodates presents a very high selectivity , while mSeveral reaction parameters may influence the properties of the obtained DAC, such as the concentration of periodate , temperature, and reaction time. The pH effect is also an important parameter to control during the oxidation of cellulose. It was reported that, in acidic conditions (pH < 3), the hydrolysis of cellulose is enhanced, resulting in superior degradation of the fibers . UsuallyAlthough periodate oxidants are toxic, environmentally harmful, and relatively expensive, their recycling and reuse may make the process more sustainable and feasible, both from environmental and economic perspectives . MoreoveCellulose, usually obtained as cellulose fibers from wood sources, is typically negatively charged due to the ionization of the hydroxyl groups. The functionalization of cellulosic materials with cationic moieties has been a chosen strategy to confer affinity toward other negatively charged molecules/particles and expand the applicability of cellulose derivatives. Cationic celluloses have been applied as bio-based flocculant and/or adsorbent alternatives for water treatments ,95. AddiTwo main strategies are described in the literature for the cationization of cellulose. The first one involves the physical adsorption of cationic polymers into the cellulose surface , and theAlthough cations from various atomic elements can be used for cationization , most of the literature focuses on the use of nitrogen-derived compounds. Depending on the derivative, the charge can be pH-dependent, with the cationic group being formed due to the protonation of amines  or heterocyclic compounds (pyridine and imidazole) under acidic conditions. On the other hand, quaternary ammonium derivates present a permanent pH-independent positive charge ,103.One common method for cellulose cationization is based on direct modification by dissolution of short-chain cellulose molecules in aqueous solutions , which are pre-cooled to sub-zero temperatures, followed by cationization in a homogeneous medium with N-(3-chloro-2-hydroxypropyl)trimethylammonium chloride (CHPTAC). In this system, the reactive epoxy reagent is prepared in situ by reacting CHPTAC with alkali A. The epAn alternative approach considers the cationization of pre-modified cellulose by using, for example, DAC , CA, or w/w)). The solubilization of the fibrils was not detected.The direct cationization of wood cellulose fibers with CHPTAC and the indirect method with GT were both tested by Pedrosa et al.  as a preSirvi\u00f6 et al.  reportedEmam et al.  reportedAlthough cellulose anionization is not as well explored as cationization, there are some procedures described in the literature. For example, Rajalaxmi et al.  and GrenIn Grenda et al. , after tw/w) water was found to be the most promising mixture. The use of ball milling in the process reduces the cellulose particle size, increases the surface area of cellulose, promotes the interaction between DES and the cellulose molecules, and disrupts the crystalline structure, thus increasing the carboxylic content in the modified cellulose.Cao et al.  have synCellulose molecules possess a great number of hydroxyl groups, leading to fibers with a strong polarity and high water sorption capacity. However, most of the synthetic polymeric matrices are nonpolar, such as plastics (polyethylene or polypropylene are among the most common) . Therefo31P NMR) can be achieved under mild conditions for acetylated and carbanilated wood, respectively [Homogenous acetylation and carbanilation reactions of wood-based lignocellulosic materials in ILs have also been investigated, resulting in highly substituted lignocellulosic esters . A high ectively . The optThe preparation of hydrophobically modified cellulose from renewable feedstocks (based on green chemistry principles) can be met using plant oils. Plant oils are triglycerides with hydrophobic long hydrocarbon chains, which have been exploited as sustainable alternatives to materials derived from non-renewable resources . Yoo et 4)2Ce(NO3)6) [Another widely used way to modify the surface of polysaccharides and make them more hydrophobic relies on the introduction of acrylates and methacrylates into the chain. Littunen et al. studied the filling of various acrylates and methacrylates as monomers through a free radical copolymerization initiated by ammonium cerium (IV) nitrate ((NHe(NO3)6) . InitiatAn important advantage of this method is that the entire synthesis can be performed in an aqueous medium. The macrostructures formed by the grafted polymers ranged from a thin coating to a continuous matrix completely enveloping the fibrils. This type of modification can offer a simple way of improving the compatibility between lignocellulosic materials and synthetic polymers.13C NMR spectrum in The introduction of siloxane groups into the cellulose structure is another suitable approach to enhancing the hydrophobicity of the molecule. Schuyten et al. introduced, almost 70 years ago, the first cellulose derivative, trimethylsilylcellulose (TMSC) ,122,123.Later, some improvements in the TMSC synthesis led to products soluble in organic solvents, such as chloroform, 1,1,1-trichloroethane, and o-xylene . The disFrom the examples discussed above, it is clear that lignocellulosic materials can be modified in many different ways. The choice of the most suitable method will depend on the desired properties of the cellulose derivatives and the application foreseen.Chemical modification of low-cost, naturally occurring raw materials, such as cellulose, is an important and promising route for the development of green value-added products. Cellulose derivatives are currently used in different areas, such as food formulations, coatings, or films with barrier properties ,126,127 An edible cellulose-based film for probiotic entrapment was prepared by Singh et al.  using soIn the biomedical area, the use of cellulose derivatives as controlled drug release systems is very appealing ,132. NanThere are numerous cellular species that can be cultured on nanocellulose biomaterials, such as hydrogels, electrospun nanofibers, sponges, composites, and membranes . Among tFuller et al.  studied Cellulose derivatives are also reported as efficient flocculants, namely for the flocculation of pigments  and calcIn the textile industry, cellulose modification can also be applied to enhance dye uptake into textile fibers. A potentially environmentally friendly dyeing method using a cationization method in combination with mercerization was proposed by Fu et al. . The catCellulose is the most abundant natural polymer on Earth. Due to its wide availability, it is a very promising raw material for the replacement of non-renewable feedstocks. To overcome some limitations of its application and expand its valorization and utilization, cellulose can be chemically modified to improve its chemical and/or physical properties. Numerous studies reporting the modification and application of cellulosic materials are available in the literature. In the present review, some of the most common chemical modifications of cellulose  are briefly described, and selected applications of these derivatives are presented. Cellulose and its derivatives can be obtained from different sources, including biomass such as agroforestry residues, and are suitable for a wide range of applications. A brief review of possible fractionation procedures is also presented, including greener alternatives. By understanding cellulose structure and reactivity, it is possible to tune the properties of the resultant material, such as by modulating the hydrophilic/lipophilic balance, charge, or degree of polymerization, to obtain materials with improved performance for the intended application. The overall range of material applications for cellulose derivatives is virtually limitless.In summary, cellulose appears to be a sustainable, environmentally friendly feedstock with valuable properties such as its biocompatibility, non-toxicity, and wide availability, and the study of new routes to improve its properties and applications is of great interest. The cellulose derivatives are suitable for the replacement of fossil-based products and are an important alternative to reduce the environmental problems derived from the use of petroleum-based materials and fuels. Indeed, cellulose and its derivatives can provide the biological, chemical, physical, and engineering communities with new opportunities for exciting advancements and discoveries."}
+{"text": "Therefore, COF-PLA degradation does not cause pollution, making it a promising sand-control material.A new high-strength, thermally stable, and degradable covalent organic framework (COF) -modified polylactic acid fiber (PLA) material (COF-PLA) was constructed for reinforcing the PLA material, to be used to produce environmentally friendly sand barriers. The micrographs, structure, thermal stability, and photodegradation products of COF-PLA were investigated. The results indicated that the COF material was compatible with PLA, and that the COF-PLA material took on the merits of the COF, so that it had a more regular arrangement, smoother surface, and smaller size, and was more thermostable than PLA alone. The successful incorporation of the COF improved the thermal stability of PLA. The initial pyrolysis temperature of the COF-PLA material is 313.7 \u00b0C, higher than that of the PLA material at 297.5 \u00b0C. The photodegradation products of COF-PLA and PLA indicated that the COF and PLA materials were mixed in a complex manner. After photodegradation, the COF-PLA material can produce melamine molecules that can neutralize the lactic acid and CO Land desertification is an environmental and ecological issue, and one of the greatest threats to humanity in the 21st century . Most laVarious plant, mechanical, and chemical measures have been used to combat desertification. Mechanical sand-control measures, such as sand barriers, are usually used in harsh deserts where plants struggle to survive. These methods can stop the forward movement of sand dunes, stabilize the shifting sands, and create stable terrain for plants to grow . Mechani2 and H2O, and therefore does not cause secondary pollution to the environment [Many studies have been reported on mechanical sand barriers , includiironment ,10. Compironment .However, the development and application of PLA are limited due to its high price, unstable mechanical properties, and low thermal stability, which are in urgent need of improvement ,13. CompTherefore, the modification of PLA with plant fibers can not only improve the mechanical properties of PLA products, but also reduce their cost while maintaining their reported benefits. These composites can greatly increase the scope of PLA applications, and many studies have focused on preparing PLA-based composites using natural plant fibers as reinforcements. Ghazali et al.  preparedHowever, plant fiber, or cellulose, is usually produced by chemical extractions, such as the alkali cooking method with the chemical extraction process, which has many extraction steps and may cause chemical contamination during production ,23. CovaBhadra et al.  found thIn recent years, COFs have been widely used in storage adsorption ,28, hydrIn this study, chemically stable and cost-effective COF materials were prepared using a one-step method, and the micrographs, structure, thermal stability, and photodegradation of PLA modified by a highly structured COF material were studied.Lactic acid . Zinc oxide . Ethyl acetate . Stannous octoate . Trichloromethane . Methyl alcohol . Melamine/1,3,5-Triazine-2,4,6-triamine . 2,4,6-Triformylphloroglucinol aldehyde . Acetic acid . \u03b1-cellulose . Dimethylacetamide . Dimethylsulfoxide .Fourier transform infrared spectrometer . Thermal gravimetric analyzer . Circulating water multi-purpose vacuum pump . Ampere bottle . Vacuum drying oven . Thermo Exactive-GC mass spectrometer . Brunauer\u2013Emmett\u2013Teller automatic surface and porosity analyzer . X-Ray Diffraction, XRD .First, 200 mL of lactic acid was distilled to dehydration in a vacuum pump under reduced pressure at 100 \u00b0C. Zinc oxide was dried at 500 \u00b0C and then added to produce a 1% by volume solution in the lactic acid. The temperature was then adjusted to 150 \u00b0C and the mixture was distilled for 3 h to yield lactide. Next, 5 g lactide was dissolved in 10 mL ethyl acetate at 45 \u00b0C, then cooled at room temperature to obtain pure lactide with a yield of approximately 75%.Next, pure lactide was vacuumed and frozen in an amperometric flask. Stannous octanoate with a mass fraction of approximately 0.1% lactide was added. The mixture was thawed slowly and then heated for thorough mixing. The mixture was taken through three cycles of thawing, vacuum, and a high-purity nitrogen gas exchange operation before the amperometric flask was sealed under vacuum conditions and placed in an oven at 220 \u00b0C for 48 h to produce the PLA. The resulting PLA is shown in At 150 \u00b0C, 38 mg of melamine (Tt), 63 mg of 2,4,6-triformylphloroglucinol aldehyde (Tp), 2 mL DAMc, 1 mL DMSO, and 0.3 mg of Hac (acetic acid) catalyst were placed in a test tube to react for 48 h. During this period, the aldehyde groups in phloroglucinol and trimylamine reacted to form a structurally stable reticular COF structure with multiple aldehyde groups. A schematic representation of the COF synthesis is shown in One gram of PLA was dissolved in 50 mL of chloroform and refluxed to thoroughly dissolve the solid. Next, 2 mg of COF nanomaterial was added to the refluxing mixture. After the complete dissolution of the solid, the mixture was allowed to cool. The PLA-COF solution was added dropwise while stirring into 150 mL of methanol. After the separation of the solid product from the solution was observed, the flocculent product was washed with methanol and the resulting COF-PLA was obtained, as shown in According to the solubility parameter close principle, the COF can be dissolved in chloroform since both COF and chloroform are polar. Additionally, the dissolution did not change the COF structure. For example, the structure of a polymer dissolved in toluene remains unchanged. In addition, COF affords ultrahigh chemostability in strong acid, alkali, and boiling water, as well as \u03b3 radiation . TherefoHowever, as COFs have a stable structure and large molecular weight, their solubility in chloroform is poor. Therefore, we prepared different COF-PLA materials with weight ratios (COF/PLA) of 1:300, 1:500, and 1:1000.Micrographs of the samples were obtained using scanning electron microscopy. The samples were sprayed with gold before imaging, and an acceleration voltage of 10 kV was applied.Brunauer\u2013Emmett\u2013Teller (BET) analysis was used to determine the surface area (SBET) of the COF.The crystalline structures of the synthesized nanomaterials were identified using powder X-ray diffraction (PXRD).\u22121.Fourier-transform infrared spectroscopy (FT-IR) was used to study the molecular structure and chemical composition of the samples in the range of 500\u20134500 cmThe thermal decomposition process, thermal stability, and compositional changes in the samples were analyzed by TGA using a thermal analyzer. The temperature ranged from room temperature to 500 \u00b0C with a heating rate of 10 \u00b0C/min, a nitrogen flow rate of 30 mL/min, and a sample volume of approximately 5 mg.First, 50 g of sand was washed, dried, and placed in a flat quartz boat. Second, 0.1 g PLA or 0.1 g COF-PLA composite material was dissolved in chloroform and slowly dripped onto the sand in the quartz boat. Subsequently, a stable layer of the PLA or COF-PLA was formed on the sand surface after the solvent evaporated. The PLA membrane was colorless and the COF-PLA membrane was light yellow. Next, the PLA and PLA-COF quartz boats were doused with a small amount of water and placed under a wired light source with a current of 10 A. As irradiation proceeded, the color of the PLA quartz boat became more yellow, and the COF-PLA quartz boat became lighter, indicating that both PLA and COF-PLA were degraded. Finally, the degradation products of the PLA and COF-PLA materials were analyzed using liquid mass spectrometry.Micrographs of the synthesized materials are shown in 2/g. The surface area (SBET) of the COF was determined by the Brunauer\u2013Emmett\u2013Teller automatic surface and porosity analyzer. The Brunauer\u2013Emmett\u2013Teller (BET) analysis of the COF revealed that it had a surface area (SBET) of 169.20 m\u22121, which correspond to -OH stretching and bending vibration peaks, respectively. The troughs at 2947 and 2998 cm\u22121 correspond to the -CH symmetric and asymmetric stretching vibration peaks, respectively. The peak at 1213 cm\u22121 was caused by the C-C stretching vibration. A C-O stretching vibration peak was observed at 1127 cm\u22121. The characteristic peak of the amorphous phase for crystalline is shown at 871.2 cm\u22121, and the peak at 756 cm\u22121 is caused by the crystalline phase. This spectrum is in agreement with the spectroscopic results of a previous study [us study , confirm\u22121. The peak at 3210 cm\u22121 is characteristic of the hydroxyl group on the benzene ring. The peaks at 1615 and 1651 cm\u22121 were attributed to the C=O double bonds in the COF material. The vibration peak of the trimylamine ring group was observed at 832 cm\u22121, and the vibration peak at 808 cm\u22121 was attributed to the benzene ring. These results confirmed the successful synthesis of new COF materials containing both melamine and benzene ring structures.\u22121, amorphous phase correlation peak at 871 cm\u22121, and crystalline phase correlation peak at 756 cm\u22121, but also the vibration peaks of the new COF material, including the C=O double bond vibration peak at 1619 cm\u22121 and the benzene ring vibration peak at 810 cm\u22121. Hence, As shown in 3-CH-C=O group from the PLA structural units and belongs to a metastable structure. The fragment with a mass of 182 was formed by removing a hydrogen proton and a sodium ion from the metastable structure mentioned above. The fragment with a mass of 455 was formed by removing a hydrogen proton from six PLA structural units and one sodium ion. A fragment with a mass of 504 was formed from seven PLA structural units. The fragment with a mass of 527 was composed of seven PLA monomers and one sodium ion, which belongs to a metastable structure. A fragment with a mass of 670 was formed from nine PLA structural units and one sodium ion, with a hydrogen proton removed. A fragment with a mass of 631 was formed by removing one oxygen atom from the nine PLA structural units belonging to the steady-state structure.As shown in As shown in 2. The remaining PLA can be biodegraded and the remaining unstable CO2 can be absorbed by the atmosphere or plants. This process allows the acid\u2013base balance of sandy soils to be maintained even as the material degrades, thus supporting plant growth. The photodegradation products of COF-PLA and PLA in the spectra confirmed that the COF and PLA materials were mixed in a complex manner. The degradation of COF-PLA resulted in the formation of melamine monomers that neutralized the lactic acid and carbon dioxide degraded from PLA. This is a useful balancing process because all stable melamines can be neutralized with PLA and COAs can be seen from In addition, due to the low solubility of COF in chloroform, lower weight ratios between COF and PLA result in a better doping effect and, therefore, better thermal stability of COF-PLA. Furthermore, the presence of water vapor may lead to the production of nitrogen in the COF pyrolysis process. Nitrogen is an essential nutrient for plant growth, and this process may allow the COF-PLA to act as a fertilizer for sand-fixing plants while also contributing to sand fixation.In this study, we prepared a highly chemically stable and cost-effective COF material through a one-step method, and modified PLA with this COF to prepare a new degradable COF-PLA material with good thermal stability for use in high-strength sand barriers. Compared to other materials used as sand barriers, COF-PLA materials are simple to prepare, pollution-free, and economical. Furthermore, the experimental results illustrated that the new COF-PLA material had a stable structure, regular arrangement, smooth surface, and strong heat resistance. During the degradation process, the COF-PLA produces a melamine monomer which can neutralize the lactic acid and carbon dioxide produced during the degradation of PLA. This may help maintain the acid\u2013base balance between sandy soil and plant growth. This new COF-PLA material has great potential applications for sand fixation in desert regions."}
+{"text": "Polylactic acid (PLA) is a biodegradable polyester polymer that is produced from renewable resources, such as corn or other carbohydrate sources. However, its poor toughness limits its commercialization. PLA composites can meet the growing performance needs of various fields, but limited research has focused on their sustainable applications in sports. This paper reviews the latest research on PLA and its composites by describing the characteristics, production, degradation process, and the latest modification methods of PLA. Then, it discusses the inherent advantages of PLA composites and expounds on different biodegradable materials and their relationship with the properties of PLA composites. Finally, the importance and application prospects of PLA composites in the field of sports are emphasized. Although PLA composites mixed with natural biomass materials have not been mass produced, they are expected to be sustainable materials used in various industries because of their simple process, nontoxicity, biodegradability, and low cost. The continuous advancement of science and technology has increased the global demand for natural resources, leading to frequent problems, such as material shortages and environmental pollution. Rapidly depleting oil reserves, greenhouse gas emissions, and the large-scale use of oil-based products have resulted in a lack of biodegradable products, prompting researchers to explore biodegradable, renewable, and recyclable materials. Polylactic acid (PLA) is a biodegradable bio-based aliphatic polyester that can be extracted from 100% renewable resources, such as corn, potatoes, and sugarcane ,3,4,5.2/kg PLA) of the released CO2 in the PLA life cycle is released during its conversion. By optimizing the conversion process of PLA, there is tremendous potential for PLA to become a low-carbon material [PLA is an extracted thermoplastic that is suitable for manufacturing composite materials using various methods, such as injection molding, extrusion molding, and compression molding . Increasmaterial .This report discusses the latest developments in the research and development of PLA and its composites. It first outlines the basic structure, characteristics, production, degradation process, and latest modification methods of PLA, and it discusses the inherent advantages of selecting PLA composite materials and focuses on the relationship between different biodegradable materials and their performance in the final PLA composite materials. Then, it introduces the application prospects of PLA composite materials in the field of sports. Finally, it discusses the challenges faced by PLA composite materials and competing materials.2O and CO2. After photosynthesis, CO2 and water are converted back into substances such as starch, which can be used as raw materials to resynthesize PLA, thereby realizing a carbon cycle process [PLA is an entirely biodegradable polymer hailed as one of the most promising bio-based polymers because of its biocompatibility, biodegradability, high mechanical strength, nontoxicity, nonirritation, and processability. PLA can be synthesized by low-energy processes, and it is independent of petroleum resources. Microorganisms can decompose waste PLA into H process  that doeLactic acid molecules contain a chiral asymmetric \u03b1-carbon atom and exhibit optical activity that can be divided into two configurations: left-handed (L) and right-handed (D). The dehydration of two lactic acid molecules forms three optical isomers of lactide: L-lactide, D-lactide, and meso-lactide. L-lactide is cheaper because it is naturally occurring. The content of D-lactic acid changes the crystallization behavior of PLA, including different crystallization rates, multiple crystal types, different scales, and layer thicknesses. The crystal morphology is closely related to the mechanical properties of the polymer: the larger the PLA crystal, the more defects in the interior and on the surface of the crystal, and the poorer the mechanical properties of the resulting material . Like L-Mw). When the molecular weight doubles from 50 kDa to 100 kDa, the PLA\u2019s tensile strength and elastic modulus also double [PLA is a member of the family of aliphatic polyesters and has the essential characteristics of universal polymer materials. PLA has a tensile strength similar to that of polyethylene terephthalate (PET), approximately 54 MPa, while its tensile modulus is 3.4 GPa, which is slightly higher than that of PET ,15. The o double . The meco double ,18.2O and CO2.The environmental degradation process of PLA occurs in two steps: hydrolysis and microbial degradation. PLA first undergoes the hydrolytic cleavage of ester bonds, degrading into PLA oligomers (OLAs) . The hydIn the late 1980s, the advancement of direct condensation technology significantly increased the global production of PLA and greatly reduced its costs. Direct condensation involves preparing PLA by dehydrating and condensing lactic acid molecules. The disadvantage of this method is that the reaction system is in a dynamic equilibrium between condensation and depolymerization, and the high viscosity of the system makes it difficult to remove the water by-product. The unremoved water causes the depolymerization reaction to proceed, even under vacuum conditions, making it difficult to extract water and increasing the molecular weight of the PLA. Under a high temperature (>200 \u00b0C), the PLA will undergo depolymerization, discoloration, and racemization accompanied by a series of side reactions, such as ester exchange, which may form differently sized cyclic products. This results in reduced product properties and poor mechanical properties, which limit their industrial applications. However, the use of direct condensation to produce PLA is a short and inexpensive method. Chen et al. used a combination of direct condensation and melt polymerization using tetra butyl titanate as a catalyst. They used different vacuum periods, esterification, and condensation reactions. The results showed that this method reduced the system\u2019s viscosity, thus helping to remove water and increase the molecular weight .In the early 1990s, Cargill Inc. applied for a patent for a solvent-free process and new distillation technology based on ROP to convert lactic acid into high-molecular-weight polymers. This made PLA the second-highest volume bioplastic after starch-based materials. By utilizing specific microbial strains, natural agricultural materials can undergo fermentation to produce lactic acid (LA), which is a precursor for PLA ,35. The According to Refs. ,38,39, tPLA is a thermoplastic polymer whose processing temperature is generally between 170 and 230 \u00b0C. In recent years, researchers have produced self-reinforcing PLA through techniques such as melt extrusion, stretching, and injection molding without the need for additives, which retain the biocompatibility and biodegradability of PLA. This method can also solve the trade-off between the toughness and strength and compatibility of blends. In addition, Cao et al.  designedChrissafis et al.  added 2.Researchers can improve the mechanical properties of polymers by changing the structure and composition of copolymers. Adjusting the ratio of lactic acid and other monomers in the copolymer system can produce copolymers with the desired mechanical strength to improve the mechanical properties of PLA. By utilizing the hydroxyl and carboxyl groups on the lactic acid segment, different monomers, such as caprolactone (CL), ethylene oxide (EO), ethylene glycol (EG), and trimethylene carbonate (TMC), can be used to synthesize PLA copolymers with improved mechanical properties, especially toughness. Li et al.  prepared2 and H2O, requiring temperatures near the Tg (60 \u00b0C) of the polymer and a high relative humidity [2 emissions are offset by the initial absorption during PLA production. Under such conditions, the degradation time can be as short as 30 days.PLA is a biopolymer that can also undergo biodegradation under certain conditions without producing environmental pollution ,46. Polyhumidity . The CO2Ea = 53.2 kJ mol\u22121), and the second mechanism was associated with a self-catalytic effect of increasing carboxylic acid groups during the depolymerization process (Ea = 36.9 kJ mol\u22121). This effect was previously noted in PLA hydrolysis and lowered the solution pH. The group\u2019s further work modeled the hydrolysis of PLA at higher temperatures (170\u2013200 \u00b0C). The kinetic model described the batch erosion of PLA and subsequent hydrolysis of oligomers, and the model accurately predicted the conversion and concentration of oligomers. Under these conditions, PLA could be completely transformed within 90 min.Piedmont and Gironi  studied Although PLA has excellent mechanical properties, renewability, biodegradability, and low costs , Figure Natural fibers can be divided into plant and animal fibers according to their sources . GeneralCellulose nanocrystals (CNCs) are rod-shaped nanoparticles extracted from cellulose through acid hydrolysis. A wide range of sources, including bleached wood pulp, cotton, and hemp fibers, can be used to produce CNCs ,62. BecaSince Favier et al.  first atLignin is the most abundant aromatic biomass in nature, accounting for 20\u201330% of the weight of wood ,68. MostSpiridon et al.  obtainedSilk fiber is a natural animal protein fiber with a higher crystallinity, toughness, and tensile strength than plant fibers . In addiZhao et al.  prepared2 and H2O and are nontoxic to the soil and air [As a new bio-based polymer material, PHA has diverse structures, various sources, and biodegradability, biocompatibility, optical activity, piezoelectricity, and gas barrier properties. They can be naturally biodegraded into CO and air ,78. Curr and air ,80.Zembouai et al.  studied gT), indicating the poor compatibility of unmodified PBAT/PLA blends. In experimental studies, the preparation of PBAT/PLA blends usually involves melt blending. At high temperatures and sufficient time, ester exchange reactions occur between the two polyesters, thereby improving their compatibility [PBAT is a biodegradable material produced on large scales and widely used in packaging materials and biomedical fields. PBAT has good processability and can toughen and modify other polyesters , but comtibility . By incrtibility .Arruda et al.  preparedMicrocellular injection molding was first proposed in the 1980s by Nam et al. . The forExtrusion molding can be divided into continuous and intermittent types based on the different pressures used during extrusion. Continuous extrusion applies pressure with the rotation of a screw to uniformly plasticize the material inside the barrel. The material undergoes mixing and heating through the action of the screw during the extrusion process, resulting in good material uniformity . IntermiCompression molding is a standard processing method for PLA. During compression molding, PLA particles are placed in a heated mold, and pressure is applied to liquefy and flow the material at high temperatures . As the The global production capacity of all biodegradable plastics, including PLA, is expected to increase rapidly to approximately 1.33 million in 2024 , with prPLA fiber is a biodegradable synthetic fiber that is refined and fermented from starch sugar in corn, beets, or wheat. It is a new type of polyester fiber in the textile industry. PLA fiber is 100% compostable and reduces the Earth\u2019s carbon dioxide levels throughout its entire life cycle. The cross-section of PLA fiber is generally circular with a smooth surface. Its load\u2013elongation curve is similar to that of wool, while its toughness is lower than that of cotton. PLA fiber has good core absorbency and fast moisture management. Therefore, by blending PLA fiber with cotton, the moisture transmission properties of cotton fabrics can be improved. Guruprasad et al.  developeRaykar et al.  used PLATraditional protective sports gear has a structure consisting of a hard outer shell made of a thermoplastic material and an inner soft foam padding. Currently, there are new \u201csoft shell\u201d technologies for sports protectors based on the use of soft polymer foams typically made of polyurethane or polyacrylate with good cushioning properties. During the manufacturing process of sports protectors, soft polymer foams can be combined with PLA. Soft polymer foams are used as the internal cushioning material. In contrast, PLA can be used as the outer shell material to improve sports knee protectors\u2019 lightweight, breathability, and comfort properties, thus achieving better protection results. Yang et al.  used tenThe source of power for a surfboard comes from the movement of waves, and the significant impact force generated by an impact wave can often break a surfboard, mainly when materials such as fiberglass are used in its production. In recent years, researchers have turned their attention to biodegradable materials. Soltani et al.  used theBecause of the biocompatibility and biodegradability of PLA when in contact with mammalian bodies, it has been widely used in the biomedical and pharmaceutical fields  to manufCompared with traditional printing materials, PLA produces almost no harmful gases and has a lower shrinkage rate, making it ideal for 3D printing sports equipment. Protective gear, such as mouthguards, helmets, and shin guards , can be Compared with traditional petroleum-based plastic sports equipment, the green disposal of idle sports equipment meets the requirements of sustainable development. Sports equipment made of PLA composites can be used safely and decomposes after being discarded, which can prevent environmental pollution. In addition, PLA has a lower density, allowing for the production of relatively lightweight sports equipment. Through 3D printing, PLA enables personalized customization, offering more possibilities for the innovative design of sports equipment. However, as a linear thermoplastic polyester, PLA\u2019s strength may not meet the requirements of certain sporting equipment in specific environments. For example, because of PLA\u2019s high brittleness and low elongation at break , sports PLA is a natural, renewable, and low-cost biodegradable material, but its inherently poor toughness limits its broader applications. By adding reinforcement materials to develop PLA composites, it can adapt to the increasing performance requirements of various fields. Compared with most inorganic and synthetic fibers, natural fibers have abundant sources, low prices, complete degradability, low energy consumption, and environmental friendliness. In the future, appropriate additives, modifications to polymerization conditions, and reinforcement techniques will be employed to enhance the strength of PLA and meet specific needs. At the same time, by developing low-cost reinforcement materials and optimizing formulations and processing methods, the manufacturing costs of PLA composites can be reduced. Their performance can be improved to meet various environmentally friendly applications, including sports equipment manufacturing. Currently, the application of PLA composites in the sports field is expanding. Compared with petroleum-based materials, the mechanical properties of PLA composites still need to be improved. However, as biodegradable alternatives to petroleum-based plastics, they still have tremendous potential."}
+{"text": "In the history of cellulose chemistry, hydrogen bonding has been the predominant explanation when discussing intermolecular interactions between cellulose polymers. This is the general consensus in scholarly textbooks and in many research articles, and it applies to several other biomacromolecules\u2019 interactions as well. This rather unbalanced description of cellulose has likely impacted the development of materials based on the processing of cellulose\u2014for example, via dissolution in various solvent systems and regeneration into solid materials, such as films and fibers, and even traditional wood fiber handling and papermaking. In this review, we take as a starting point the questioning of the general description of the nature of cellulose and cellulose interactions initiated by Professor Bj\u00f6rn Lindman, based on generic physicochemical reasoning about surfactants and polymers. This dispute, which became known as \u201cthe Lindman hypothesis\u201d, highlights the importance of hydrophobic interactions in cellulose systems and that cellulose is an amphiphilic polymer. This paper elaborates on Bj\u00f6rn Lindman\u2019s contribution to the subject, which has caused the scientific community to revisit cellulose and reconsider certain phenomena from other perspectives. In 2010, a paper by Lindman, Karlstr\u00f6m and Stigsson discussed the mechanisms of cellulose dissolution, pointing out some discrepancies in the common literature, where hydrogen bonding was argued as the most crucial interaction to overcome. The paper (re)introduced hydrophobic interactions and cellulose amphiphilicity as essential to consider for the successful development of future cellulose solvents . Since tOocystis, and, due to this, the synthesis machinery was designated as the terminal complex (TC) . Th. Th+) orThe use of amphiphilic cations or species of intermediate polarity, such as urea or thiourea, typically enhances the stability of the cellulose dopes (delay of gelation), which is very important for many applications, such as fiber spinning ,79. ThisApart from the enhanced stability of cellulose solutions, the use of amphiphilic-like cations has been observed to improve dissolution and allows reaching a molecularly dissolved state of cellulose b. On the+, it was anticipated that if their beneficial effect in cellulose dissolution is related to weakening hydrophobic interactions, then once the cations are hindered or removed from the solution, cellulose\u2019s solubility should be compromised. This hypothesis was successfully tested using different cyclodextrins (CDs) as host agents to form complexes with TBA+ cations [+ complexes was proven by NMR and the worsening of the solvent quality with CD addition was clearly observed from the polarized light microscopy and turbidimetry measurements. Overall, data support the hypothesis that the amphiphilic properties of TBA+ are determinant for the efficient dissolution of cellulose.Returning to the alkaline-based systems containing inorganic or amphiphilic-like cations, PTssNMR confirmed the higher degree of dissolution in the latter case; an intense \u201cinsensitive nuclei enhancement by polarization transfer\u201d (INEPT) signal coming from dissolved cellulose . This is clearly seen in The driving forces responsible for cellulose regeneration and the formation of crystalline domains have been evaluated by different authors ; for insAt this stage, it is clear that in order to develop novel solvents for cellulose or improve existing ones, different aspects need to be considered. One of these important, but often neglected, effects that has been already mentioned is related to the ionization of cellulose. Note that, since hydroxyls were thought to be solely engaged in the H-bonds, it was disregarded that they could also be involved in other phenomena, such as ionization. The role of the cellulose charge and the concomitant ion entropy effects should not be underestimated . Contrar+ cations bind to cellulose with ca. 1.2 TBA+ ions/AGU [+ ions solvating the cellulose molecules [+ cations are suggested to be the main driving forces [+, it is reasonable to also anticipate a significant contribution from the hydrophobic effect regarding the favorable TBA+\u2013cellulose interactions. These works strongly suggest that cellulose is, to a large extent, charged in a concentrated alkaline medium, an overlooked but relevant factor to rationalize its solubilization behavior.Further NMR studies have shown that TBAions/AGU . This waolecules ,96. Fromg forces . NeverthOrganic co-solvents, such as dimethyl sulfoxide (DMSO), have been observed to enhance cellulose\u2019s dissolution, allowing the use of much less alkaline media ,97,98,99+, which demonstrates the preferential interaction of TBA+ with cellulose [+ suggests the weaker adsorption of the former. As mentioned, DMSO facilitates cellulose dissolution, not only by tuning the solvent viscosity (enhancing mass transport) but also by solvating cellulose , which facilitates further interaction between the TBA+ ions and cellulose. The highly polar character of the S-O bond in DMSO drives the overall negative charge density in the oxygen atom. On the other hand, the sulfur atom, despite displaying a positive charge density, carries a pair of non-bonding electrons [+, which further substantiates the preferred interaction of cellulose for the latter.The work of Idstr\u00f6m et al. is particularly interesting since cellulose\u2013DMSO contacts were found to be three times longer than the DMSO\u2013DMSO interactions . Neverthellulose . The faclectrons . Thus, bAs an emerging class of biomass solvents, deep eutectic solvents (DES) are interesting. Mixtures of natural bio-sourced cations and anions , such as those obtained from natural organic acids, amino acids, non-nutritive sweeteners or natural compounds including choline or betaine, are particularly appealing due to their environmental benignity and relatively low cost when compared to, e.g., the synthetic ILs . However3PO4 (aq) [Cellulose is a versatile source of natural emulsifiers and can be utilized as such over the whole hierarchical size range, from cellulose particles and gels to macromolecules; see PO4 (aq) ,121,122.PO4 (aq) ,124,125.PO4 (aq) .One means of producing emulsions using dissolved cellulose is by following a dissolution\u2013emulsification \u201cin situ\u201d regeneration approach, where the oil is directly dispersed in the cellulose solution and regeneration takes place at the oil\u2013water interface (in situ). A continuous-like coating is formed around the oil droplet, giving it a smooth appearance ,121,124.Several researchers have also demonstrated the ability of cellulose particles to self-assemble at oil\u2013water interfaces and to stabilize w/o emulsions without the aid of classical surfactants ,127,128.Cellulose dissolved in a good solvent is solidified when its solvency is decreased by any means, and this can be attained in several ways. Most commonly, different non-solvents (antisolvents) are being used to obtain this efficiently. Regeneration can also be imposed by, e.g., the addition of different salts or a combination of an antisolvent and salt. Remarkably, regeneration can also be triggered by temperature changes, without the addition of any other substances. In an aqueous solvent system comprising alkali and urea, gelation occurs via a sol\u2013gel transformation when increasing the temperature of the cellulose solution , similarThe above-mentioned methodologies can be utilized when dissolved cellulose undergoes regeneration into the solid state to control the degree of crystallinity. Since crystallinity affects many different material properties of the solidified cellulose, such as the surface morphology, transparency and haze, mechanical strength, density, water contact angle, moisture uptake and gas permeability ,132, thiGlobal warming and the accumulation of microplastics in the oceans have driven research efforts toward developing bio-based and biodegradable materials to replace petroleum-based products. At the beginning of 2023, the European Polysaccharide Network Of Excellence (EPNOE) released the Research Roadmap 2040, a strategic document that summarizes the most relevant scientific questions to be answered for biomass and polysaccharides during the coming decades . CelluloRecently, a promising breakthrough in IL development for the dissolution of cellulose was achieved by research groups in Finland, led by Profs. Herbert Sixta and Ilkka Kilpel\u00e4inen . The metThe first isolation of nanocellulose occurred in the mid-20th century , while tConsidering the advances over the past twenty years, several challenges have been identified that hinder the transition of nanocellulose production and utilization to a large industrial scale. To overcome the difficulties, improvements in the production efficiency with environmentally friendly approaches and less energy-consuming processes to lower the cost are strongly recommended ,156,157.N,N-dimethylacetamide [The term all-cellulose composite was first introduced by Nishino et al. . By distcetamide . Via thecetamide . Regenercetamide .2SO4 [\u00ae80. Thereafter, the sol\u2013gel transition was triggered by increasing the temperature of the w/o emulsion and lowering the solubility of the biopolymer, to solidify the cellulose\u2013chitosan droplets into micrometer-sized spherical nanocomposites. The two types of chemically or physically crosslinked microparticles obtained were thereafter washed, solvent-exchanged, freeze-dried from tert-butanol and thoroughly characterized.Yang et al. prepared transparent and flexible cellulose\u2013montmorillonite nanocomposite films by dissolving cellulose in a montmorillonite-dispersed LiOH\u2013urea aqueous solution, followed by regeneration in acetone . The hig2SO4 . Composi2SO4 ,164. Fro2SO4 . The out2SO4 . ParticlQi et al. reported on a sensor system prepared from cellulose dissolved in alkali\u2013urea mixed with multi-walled carbon nanotubes (MWNT) and regenerated. MWNT\u2013cellulose films displayed both flexible and conducting MWNT characteristics in an interval of 2\u201310 wt% MWNTs. Films with multifunctional sensing ability for the monitoring of stress\u2013strain, temperature and humidity were obtained, with potential for electronic, magnetic, semiconducting and biotechnological applications and as water sensors ,168.Cellulose-based fibers from a combination of hydrophilic cellulose and hydrophobic polyaniline (PANI) were prepared to yield antistatic fibers ,170. PAN\u22123 was reached in anisotropic nanofiber-structured cellulose films (ACFs). The well-aligned and densely packed cellulose nanofibers significantly decreased the interstitial spaces and minimized light scattering, giving ACFs with high optical clarity (91%), low haze (<3%) and birefringence behaviors. The simple methodology was argued to be scalable in fabricating high-strength, super tough and transparent cellulose films for emerging biodegradable next-generation packaging, flexible electronics and optoelectronic applications.Ye et al. recently presented a green route to the fabrication of strong and tough regenerated cellulose films . The fil2+ ions in a dispersion of alkali\u2013urea-dissolved cellulose. The nucleation of the NPs was suggested to be initiated in the vicinity of the deprotonated hydroxyl groups in cellulose under highly alkaline conditions. Well-dispersed and spherical Cu/Cu2O NPs of a narrow size distribution decorating the cellulose were obtained. The hybrid material showed efficient antibacterial properties and its potential for catalytic and electronic applications was highlighted.An interesting approach to synthesize cellulose hybrid materials in situ has been described by Eivazihollagh et al. . A facil2 reduction, as with many other aspects of sustainability, are receiving increased attention scientifically and technically. Since the energy and climate crisis are two major challenges that we are facing, where the transition to green energy is urgently needed, this is an emerging field for cellulose-based materials. Cellulose can be utilized in many ways in energy storage and harvesting, and the whole value chain from the produced energy to the customer can thereby become more sustainable.An electrical energy supply and utilization are essential for the functioning of society. In May 2021, the International Energy Agency (IEA) released its roadmap to global net-zero emissions, analyzing the implications of existing net zero pledges and showing a pathway to net zero emissions in electric energy production globally by 2050 . In connCellulose in different forms has been widely used as a separator, electrolyte, binder and dispersion agent in material for energy storage devices . NanocelSupercapacitors are considered one of the potential candidates in the domain of energy storage devices for future generations. There is a wide variety of applications, such as storing waste energy to power electric vehicles and other electronic systems and balancing the electric grid from fluctuations that arise from renewable energy sources such as solar and wind power. Supercapacitors have generated great interest due to their high power density, long cycle lifetime and fast charge\u2013discharge ,178. A sHajian et al. suggested that there is an association between nanocellulose and carbon nanomaterial, and the attraction increases with the surface charge of the cellulose . CNC wasBatteries are power sources with high energy density, consisting of one or more electrochemical cells, with external connections that can be used to power electrical devices, such as electric cars and mobile phones. A battery consists of a negative electrode (anode), a positive electrode (cathode) and an electrolyte, and a separator is sometimes used to prevent short-circuits. In batteries, a binder is used to prevent electrode swelling and mechanical degradation and to protect the active material against the electrolyte, with maintained ion transport throughout the binder . One of Historically, due to its excellent dielectric properties, cellulose (paper) has been and still is widely used as an insulator material in high-power transformers ,200. Bes\u22122 could be reached. This is a striking result that shows that, once processed correctly, cellulose can fulfil the demands regarding the performance to replace fossil-based polymers in TENGs. Nevertheless, there is still a limited fundamental understanding of how the structural characteristics relate to the electric properties of cellulose and affect the output in triboelectric applications. However, in a newly published study, the dependence of cellulose\u2019s physical structure on different length scales was elaborated more systematically with regard to the triboelectric performance [Cellulose was recently demonstrated as a very potent triboelectric material . In a TEformance . SpecifiBy utilizing triboelectric counter-layers with different mechanical softness, effects from both surface roughness and surface polarity could be followed in triboelectric power generation. The assigned peaks at 2\u03b8 12.3\u00b0, 20.5\u00b0 and 22\u00b0 in the XRD diffractograms shown in Cellulose, growing on land and in the oceans, is the largest biomaterial resource on Earth and therefore an exceptionally important renewable raw material for humanity. With Prof. Lindman\u2019s profound revisiting of the fundamentals of cellulose, scientific discussions of the interactions and mechanisms in cellulose systems have been facilitated. Cellulose\u2019s amphiphilicity has been highlighted, and hydrogen bonding, which often has been used as the sole explanation for cellulose\u2019s intramolecular interactions in aqueous systems, has been reconsidered theoretically as well as empirically in many papers. This has led to a deeper understanding of the hierarchical cellulose and its behavior, from molecules to macrofibers. This work paves the way to new thinking in all areas of cellulose technology, which is vital for the continuous and incremental optimization of different processes involving cellulose, as well as for the future development and design of novel cellulose materials to replace petroleum-based ones, with the aim of sustainable development."}
+{"text": "We mainly evaluated whether preserving the inferior mesenteric artery (IMA) sheath to dissecting IMA root lymph nodes  would benefit patients in terms of comparable lymph-node yield removed during operation and postoperative complications in laparoscopic radical resection of rectal cancer.t test, chi-square test or Fisher\u2019s exact test between the 2 groups.This is a prospective study included 141 rectal cancer patients who received laparoscopic radical resection during September 2018 to December 2020. All patients were randomly assigned to the preserved group (n\u2009=\u200971) and the peeled group (n\u2009=\u200970). The baseline characteristics, pathological features, intraoperative and postoperative data outcomes and complications were analyzed by independent samples P\u2009=\u20090.002), a shorter lymph node dissection time (P\u2009<\u20090.001), less intraoperative bleeding (P\u2009=\u20090.004), an earlier time to first flatus (P\u2009=\u20090.013), an earlier time to fluid intake (P\u2009=\u20090.033) and a shorter length of hospitalization (P\u2009=\u20090.012) than the peeled group. The differences between the 2 groups were not statistically significant (P\u2009>\u20090.05) in regard to the total number of lymph nodes cleared, positive lymph nodes, bleeding, anastomotic leakage, pneumonia, wound infection, abscess, ileus, urinary retention, urinary tract infection and chyle leakage.The baseline characteristic and pathological features had no statistical difference between the 2 groups. The preserved group had a shorter operative time   There were 4.064\u00a0million new cases of cancer and 2.413\u00a0million new deaths occurred in 2016 and showing a continuing upward trend in China [Even so, there are numerous ways to treat rectal cancer, including surgery, radiation, chemotherapy, molecular targeted therapies, immunotherapies, and Chinese herbal medications. Some molecular targeted drugs, such as cetuximab, panitumumab and bevacizumab, have been shown to play important roles in the treatment of rectal cancer \u20137. Some Different scholars have different opinions regarding laparoscopic radical surgery for rectal cancer, and many studies have been performed on the dissection of No.253 lymph nodes, but few clinical studies have been conducted on the preservation of the IMA sheath during the dissection of No.253 lymph nodes. In the past, we thought that peeling the IMA sheath would increase the detection rate of No.253 lymph nodes in patients with rectal cancer . But nowWe designed this prospective randomized controlled study to investigate whether preserving the IMA sheath is more advantageous in lymph node dissection treatment and postoperative complications compared to peeling the IMA sheath to clean the lymph nodes at the root of the IMA.This is a prospective randomized controlled trial included 141 rectal cancer patients who received laparoscopic radical resection during September 2018 to December 2020, in the Department of Gastrointestinal Surgery, The First Affiliated Hospital of Wannan Medical College. All patients were prospectively and randomly assigned to the preserved group (n\u2009=\u200971) and the peeled group (n\u2009=\u200970) by envelope method. Both groups of patients underwent Dixon surgery for laparoscopic radical resection of rectal cancer, while the patient\u2019s left colic artery (LCA) was preserved during the surgery. Patients with preserved IMA sheaths were placed in the preserved group, and patients with peeled IMA sheaths were placed in the peeled group. All patients and their families communicated with providers and signed informed consent forms before surgery. This study had been performed in accordance with the principles stated in the Declaration of Helsinki. The study was approved by the Ethics Committee of The First Affiliated Hospital of Wannan Medical College and registered by the China Clinical Trials Registry (ChiCTR2200060830).The inclusion criteria were as follows: preoperative pathology showing adenocarcinoma, preoperative diagnosis of stage T1-T3(according to the American Joint Committee on Cancer (AJCC) 7th edition TNM tumor staging criteria) rectal cancer by CT or MRI with a range of 5-15\u00a0cm from the anal verge, single rectal tumor lesion with no distant metastases, preoperative American Society of Anesthesiologists (ASA) rating of I-III, no emergency surgery, and no preoperative radiotherapy or other antineoplastic treatment.Exclusion criteria were as follows: conversion to open surgery during laparoscopic surgery; intraoperative finding of mesenteric metastases or local invasion; intraoperative IMA root ligation for bleeding; and patients with comorbid systemic diseases that are inoperable, such as severe coagulation dysfunction, severe liver disease, severe renal disease, severe cardiac disease, severe pulmonary disease or other systemic malignancies. All patients received oral antibiotics 3 days prior to surgery to prevent infection and a cleansing enema 1\u00a0day prior to surgery. Those with one or more of the above conditions were excluded. A flow diagram for the study participant screening and grouping is shown in Fig.\u00a0The researchers, not the operators, put one of the two surgical schemes into an opaque envelope which has the same appearance and size and 100 envelopes are allocated for each scheme. After these envelopes are completely messed up, write a code on the outside of the envelope, seal it and give it to the operator. When a research object enters the study, if it meets the inclusion criteria and exclusion criteria, number the patient, open the corresponding numbered envelope, and perform the surgery according to the grouping scheme in the envelope. The treatment plan received by each subject was determined by the generated random sequence.All the operations were performed by the same team of surgeons following the established grouping requirements. The patient was placed in a supine head-high, leg-low position under general anesthesia via tracheal intubation. Disinfecting the skin, and laying sterile sheets routinely, the abdominal cavity was routinely explored, peeling the right paramedian rectal sulcus with an ultrasound knife. The left mesorectal was separated along the abdominal aorta, the inferior mesenteric plexus was exposed, the IMA was exposed, and lymph node dissection from the beginning of the IMA to the beginning of the LCA without peeling the IMA sheath was performed in the preserved group. In the preserved group, the IMA sheath was not peeled, and the lymph nodes and adipose tissue in the No.253 lymph nodes area were dissected outside the IMA sheath. In the peeled group, the IMA sheath was peeled, and the lymph nodes were dissected at No.253 lymph nodes, the LCA, sigmoid artery (SA) and superior rectal artery (SRA) were exposed. To peel the IMA sheath, we gently peeled the IMA sheath from the root of the IMA with the ultrasound knife and then carefully and gently pushed the nonworking side of the ultrasound knife through the gap between the sheath and the vessel to the bifurcation of the IMA and the LCA, peeling away the intact IMA sheath. Before peeling the IMA sheath, we would carefully free the lymph and adipose tissue at the root of the IMA, expose and protect the root nerve plexus that runs along both sides of the IMA root. Generally, we operated from the point 0.5\u00a0cm away from the connection between the IMA and the abdominal aorta to avoid thermal damage to the nerves and blood vessels. The surrounding adipose tissue and lymph nodes were gently dissected, completing the dissection of the No.253 lymph nodes  or Fisher\u2019s exact test. The difference was statistically significant at P\u2009<\u20090.05.All categorical data were measured in numbers or percentages. Microsoft Excel was used for clinical data collection. Statistical Package for Social Science  software was used for statistical analysis of the data. Values were expressed as \u203ex\u2009\u00b1\u2009s, and independent samples A total of 141 patients underwent laparoscopic radical rectal cancer treatment in our hospital between September 1, 2018, and December 31, 2020. They were randomly divided into the preservation group and the peeled group, and the general clinical data of the 2 groups were compared in Table\u00a0P\u2009<\u20090.001), a shorter operative time , and less intraoperative bleeding  , earlier time to fluid intake (P\u2009=\u20090.033) and shorter hospitalization (P\u2009=\u20090.012). The differences between the 2 groups were not statistically significant  plays an important role in the treatment strategy and prognosis of colorectal cancer, including in peel or laparoscopic surgery or even surgery assisted by the da Vinci robot. Before the appearance of distant metastases, lymph node metastasis is a critical prognostic factor for patients with colorectal cancer and is related to the subsequent treatment plan and course of treatment . ChineseThe lymph nodes at the root of the IMA are classified as third station lymph nodes of the sigmoid colon or rectum. Most scholars think that the area for No.253 lymph nodes dissection is delineated by the IMV on the left, the abdominal aorta on the right, the duodenum on the cephalic side and the area of the angle between the IMA and the LCA on the caudal side , 21. No.The surgical approach of this trial was performed in accordance with the standard laparoscopic radical rectal cancer surgery (Dixon procedure) with preservation of the LCA, while the lymph nodes in No.253 lymph nodes were cleared according to the established requirements for the preserved or peeled groups, and the LCA was preserved because previous research has shown that preserving the LCA in radical rectal cancer surgery can increase the blood supply to the proximal IMA of the colon, provide better conditions for the growth and healing of the anastomosis, reduce the incidence of anastomotic leak and anastomotic stenosis postoperatively and is convenient during laparoscopy \u201326 .The influence of heteromorphosis of IMA vascular branches on the surgical approach to peeling the sheath should be noted when dissecting No.253 lymph nodes. For IMA vascular typing, Yada  and ShenThe IMA sheath is the tissue that lies between the outer surface of the IMA and the collagen layer that connects the outermost nerve fibers. It contains collagen fibers, nerves, microvasculature, lymphatic vessels and adipose tissue. Studies have shown that microscopically, the IMA sheath contains only lymphatic vessels without the presence of lymph nodes, and no tumor cells are observed within the IMA sheath in lymph node metastasis-positive cases , 31. HowOn the basis of dissection of the IMA sheath. There are currently two types of lymph node dissection for No.253 lymph nodes, and one is to preserve the IMA sheath surgical approach. Li  and GongIn this study, the preserved group had a shorter total operative time than the peeled group. The preservation of the IMA sheath for dissection reduces the operative time, makes the procedure simpler, and reduces the risk of intraoperative anesthetic accidents in patients with a long smoking history, especially those with comorbidities of diabetes, heart disease, kidney or liver problems, which helps to reduce operative risk, improve perioperative safety and improve patient prognosis . The preThe preserved group had significantly shorter times to first flatus and fluid intake than the peeled group. Due to the presence of numerous capillaries and lymphatic vessels within the vascular sheath , 38, we P\u2009=\u20090.735). There was no significant difference in terms of urinary tract infection between the 2 groups. With the use of an ultrasound knife in laparoscopic surgery, the number of patients with chyle leakage in laparoscopic rectal cancer surgery decreased significantly, but there were still cases of celiac disease in the reserved and peeled groups . However, in this study, there was no statistically significant difference between the two datasets.There were no cases of postoperative mortality or deep vein thrombosis after the operation. Although there were cases of postoperative bleeding in the preserved and peeled group, they were all controlled by appropriate treatment. There were four anastomotic leakage patients in the peeled group  and only two in the preserved group . This might be due to the peeling of the sheath affecting the blood flow to the anastomosis, while the influence of the physical condition of the individual patients could not be excluded. Two of the patients with anastomotic leakage were older (both older than 70 years) or had underlying diseases such as hypertension and diabetes. Both groups had three patients with postoperative pneumonia, and all six patients had a long history of smoking. Data from the 2 groups showed no significant difference in the incidences of wound infection and abscess. The number of patients with postoperative ileus is lower than that in the past because surgeons pay more and more attention to the application of enhanced recovery after surgery (ERAS) concept in clinical work. Only two and three patients in both groups developed postoperative ileus; however, the symptoms were quickly alleviated with appropriate and timely treatment. Urinary retention infrequently occurred in both groups, and the data were not significantly different between the 2 groups (5/71, 7.0% vs. 6/70, 8.6% Although this study shows some significant results, there are still some limitations. First, this study is an independent single-center prospective randomized controlled trial, and further validation of the results is needed in a multicenter prospective study with a large sample size. Second, due to limitations in inclusion or exclusion criteria, further data were not available for some patients who had undergone preoperative radiotherapy or emergency cares, and a separate clinical trial could be designed in the future to verify the efficacy of surgery in this population. Finally, in order to reduce the impact of different surgical methods on No.253 of lymph node dissection, the surgical method adopted in this trial was to preserve the LCA for low anterior resection of rectal cancer. Whether the data brought by other surgical methods are similar to this trial needs further prospective trial to verify.Preserving of the IMA sheath in laparoscopic radical surgery for rectal cancer will reduce the total operation time and the length of hospitalization. This surgical method could lead to lower complication rate and faster recovery."}
+{"text": "Rutin is a collection of natural compounds that possess a distinctive polyphenolic structure of flavonoids. The present preliminary study aimed to investigate the impact of dietary rutin supplementation on the growth performance, antioxidant capacity, and intestinal health of yellow catfish. Three diets supplemented with different levels of rutin  were fed to juvenile tilapia for 56 days. Results revealed that supplementing with 100 mg/kg rutin enhanced growth, antioxidant capacity, and intestinal health in yellow catfish. However, the administration of rutin at a dosage of 500 mg/kg did not yield any additional advantages but potentially exhibited adverse effects on yellow catfish. This study has demonstrated the potential of rutin as a novel feed additive in aquafeed.Pelteobagrus fulvidraco). Rutin was added to the basal diets at doses of 0 (control), 100 mg/kg, and 500 mg/kg. Each diet was fed randomly into three tanks, each tank containing 30 fish with an initial body mass of (10.27 \u00b1 0.62) g. The feeding trial was conducted in an indoor recirculating aquiculture system at 28 \u00b0C for 56 days. According to the findings, the inclusion of 100 mg/kg rutin significantly improved the growth performance of yellow catfish and reduced the feed conversion ratio; however, the growth promotion effect was diminished when the diet was supplemented with 500 mg/kg of rutin. The inclusion of 500 mg/kg rutin in the diet significantly reduced the level of crude lipid and protein of the whole fish. Serum activities of alkaline phosphatase, albumin, and total protein were all significantly increased when fish were fed the diet supplemented with 500 mg/kg rutin, while serum glucose was significantly lower compared to the control group. Meanwhile, dietary rutin at a concentration of 500 mg/kg significantly induced the hepatic mRNA expressions of antioxidant-related genes  and inflammatory-associated genes . Incorporating rutin at doses of 100 mg/kg and 500 mg/kg into the diets resulted in a notable increase in superoxide dismutase (SOD) activity, while simultaneously reducing malondiadehyde (MDA) content in the liver and intestine. Intestinal villus height, villus width, muscular thickness, and lumen diameter were significantly increased with the administration of 500 mg/kg of dietary rutin. Gut microbial diversity analysis indicated that supplementing diets with 100 mg/kg and 500 mg/kg rutin significantly enhanced the abundance of Cetobacterium while decreasing Plesiomonas richness. In conclusion, dietary rutin levels at 100 mg/kg could enhance the growth, antioxidant capability, and intestinal health of yellow catfish under present experimental conditions.This research aimed to examine the effects of dietary rutin supplementation on growth, body composition, serum biochemical indexes, liver enzyme activities and antioxidant-related genes expression, intestinal morphology, and microbiota composition of juvenile yellow catfish ( Ctenopharyngodon idella) [Rhamdia quelen) resulted in increased antioxidant enzyme activities in the brain, liver, kidney, and gills while also promoting growth [Oreochromis niloticus) [Rutin is a collection of natural compounds that possess a distinctive polyphenolic structure of flavonoids . It exhi idella) . The admg growth . Furtherloticus) .Pelteobagrus fulvidraco) is a widely cultured fish species in China. Its production reached about 0.58 million tons, which ranked tenth in total freshwater fish output [Yellow catfish . Based on the previous literature ,12, dietn = 30) were chosen and randomly divided into 9 tanks . Three tanks were randomly divided into groups and fed one of the test diets. The fish were fed three times a day , and the feeding rate was 3\u20135% of body weight. The fish were weighed once every two weeks, and the feeding rate was adjusted according to the change of body weight. The feeding trial went on for 56 days. Feed intake and mortality of test fish were recorded daily. The filter sand tank of the RAS was backwashed with about 10% fresh water before feeding every morning and afternoon. The water temperature was kept at 28 \u00b0C. During the culture experiment, water quality parameters were measured once a week. The main water quality parameters were dissolved oxygen > 5 mg/L, pH: 6.5\u20137.5, total ammonia < 0.2 mg/L, nitrite < 0.05 mg/L.The experimental yellow catfish were provided by a local fish breeding facility. The experimental fish were acclimated for 4 weeks in an indoor recirculating aquiculture system (RAS) at Yangtze River Fisheries Research Institute, Chinese Academy of Fishery Sciences. During the domestication period, the fish were hand-fed three times a day with apparent satiation. Prior to the official feeding trial, the fish were fasted for 24 h. Afterwards, 270 yellow catfish with initial weight , hepatosomatic index (HSI), and condition factor (CF). Part of the liver and midgut of 3 fish per tank were placed in frozen tubes, frozen in liquid nitrogen, and stored in the refrigerator at \u221280 \u00b0C for the determination of liver metabolomics, antioxidant indexes, and intestinal microorganisms. An additional portion of the liver and the midgut of 6 fish per tank was used for the preparation of HE tissues sections for histological observation.At the end of the feeding trial, the fish were fasted for 24 h, then counted and weighed in buckets, and the weight gain rate and specific growth rate were calculated. Six fish per tank were anesthetized with 75 mg/L MS-222, and their body length and weight were measured. Blood was drawn through the tail artery and allowed to stand for 4 h, then centrifuged at 960\u00d7 Approximate analyses of whole fish and experimental diets were performed according to the method of the Association of Official Agricultural Chemists . In briThe contents of serum total protein (TP) , albumin (ALB) , triglyceride (TG) , total cholesterol (TCHO) , glucose (GLU) , and the activities of alkaline phosphatase (ALP) , aspartate aminotransferase (AST)  and alanine aminotransferase (ALT)  were detected by an automatic biochemical analyzer . Diagnostic reagents were obtained from Sysmex Wuxi Co., Ltd.  according to standard protocols.w/v) of ice-cold saline solution; it was centrifuged at 960\u00d7 g for 10 min at 4 \u00b0C to separate the supernatant. Superoxide dismutase  and malondialdehyde  were measured by biochemical kits .The tissue was thoroughly homogenized with 10 volumes . Five fields were randomly selected from each intestinal section to measure the number, height, width, diameter of mucosal fold, and muscular layer thickness using Image pro plus 6.0 software .The midgut samples from the R0, R100, and R500 groups were collected for DNA extraction. After the measurement of DNA integrity and purity, about 2 ng/\u03bcL DNA for each sample was used for 16S rRNA sequencing. The primer sets including 338F (5\u2032-ACTCCTACGGGAGGCAGCA-3\u2032)/806R (5\u2032-GGACTACHVGGGTWTCTAAT-3\u2032) were utilized to amplify variable V3\u2013V4 regions of the 16s rRNA. The sequencing was accomplished by the staff of Shanghai Meiji Biotechnology Co., Ltd.  with the Illumina Miseq platform. Bioinformatics analysis was described in detail following our recently published literature . RepreseTotal RNA was extracted from liver samples of groups R0, R100, and R500 using Trizol reagent , according to the manufacturer\u2019s instructions. RNA integrity was verified by 2% agarose gel electrophoresis, and the concentration and purity were determined using a Nanodrop spectrophotometer . Two \u03bcg of RNA from each sample was reverse transcribed to first-strand cDNA using the primer-scriptTM RT kit (Takara Biotechnology).Cu/Zn-SOD), superoxide dismutase 2 (Mn-SOD), catalase (CAT), glutaredoxin 3 (GPx), alpha-induced protein 8-like 1 (TNF\u03b1), IL-10 (interleukin 10), lysozyme g-like (LYZ), and ribosomal protein L13a (Rpl13a) were selected as target genes for detection. These primer sequences are listed in \u03b2-actin was used for gene normalization. Relative mRNA expression levels were calculated according to the 2\u2212\u0394\u0394Ct method [qRT-PCR was performed using an ABI 7500 Real-Time PCR system with a reaction volume of 20 \u00b5L, 10 \u00b5L SYBR Premix Ex Taq II (Takara Biotechnology), 8.2 \u00b5L double-distilled water, 1 \u00b5L cDNA template, and 0.4\u00b5L primer (10 \u00b5M) included. All reactions were performed three times. Superoxide dismutase 1 (t method .n = 3), and p < 0.05 indicated significant difference.SPSS20.0  was used for one-way analysis of variance (one-way ANOVA) of the experimental data, and Duncan\u2019s multiple comparison analysis was used to analyze the significance of the difference between groups. All data results were expressed as mean \u00b1 standard deviation  than the fish fed diet R0. The fish fed with diet R100 exhibited a significantly higher weight gain rate and specific growth rate compared to the fish fed with diet R0 (p < 0.05), and there was no significant difference between group R500 and group R100 (p > 0.05). There were no significant differences on the hepatosomatic index, condition factor, and the viscerosomatic index of yellow catfish among the groups (p > 0.05).p < 0.05). The crude lipid content of the whole fish in the R500 group was significantly lower than that of the other two groups (p < 0.05). Dietary rutin supplementation did not affect the moisture and ash contents of the whole body (p > 0.05).The proximate compositions of whole fish are presented in p < 0.05). Fish in the R100 and R500 groups had notably reduced levels of glucose (GLU) and activities of alanine transaminase (ALT) compared to the fish in the control group (p < 0.05). Nonetheless, the experimental groups did not display any noteworthy variations in serum total protein (TP), triglyceride (TG), total cholesterol (TCHO), and aspartate aminotransferase (AST) activity (p > 0.05).p < 0.05), and the MDA content was significantly lower in the R100 and R500 groups compared to the R0 group (p < 0.05).The superoxide dismutase (SOD) activity and malondiadehyde (MDA) content of yellow catfish are displayed in Cu/Zn-SOD, Mn-SOD, CAT, and GPx) and inflammation-related genes  in the R500 group were significantly up-regulated among the three groups. The R100 group had the lowest transcriptional expression of Mn-SOD, CAT, and GPx among the three groups. However, there was no significant difference in the expression of inflammation-related genes between the R100 group and the control group.The results of genes expression analysis are shown in p < 0.05).As shown in p > 0.05). The Chao and Ace index in the R500 group was significantly higher than that in R0 group (p < 0.05). At the phylum level, Fusobacteria, Proteobacteria, and Firmicutes were the dominant phylum in the gut flora of yellow catfish . In addition, the change in blood glucose is correlated with hepatic enzymes activities, which may suggest that rutin has a beneficial influence on liver function. Notably, reduced AST activity observed in fish fed with rutin further supports its potential for improving overall liver function when compared to the control diet.Serum biochemical parameters generally reflect the fish physiological and metabolic conditions under nutritional manipulation or environmental stress . TP coulH&E staining is commonly used for histopathology evaluation in fish due to that it can clearly illustrate cellular structures with remarkable signatures . ExcessiCetobacterium, Plesiomonas, and Peptostreptococcaceae were identified as dominant genera within the gut microbiota of yellow catfish. Cetobacterium can produce vitamin B12 in the process of carbohydrates fermentation and play crucial functions in the nutritional metabolism [Peptostreptococcaceae predominated in gut diseases [Cetobacterium and the decreased Plesiomonas richness were determined on fish fed the diets with 100 mg/kg or 500 mg/kg rutin. These data suggested that dietary rutin has positive effects on the improvement of intestinal structure and microbial composition, which is beneficial to the gut health of yellow catfish.There exists a substantial population of microorganisms within the fish intestine which has established a dynamic and intricate microenvironment over an extended period of evolution . The guttabolism . Pseudomdiseases . The preIn summary, the present results demonstrated that dietary supplementation of rutin at 100 mg/kg could improve the growth, antioxidant capability, and intestinal health of yellow catfish. However, the administration of rutin at a dosage of 500 mg/kg did not yield any additional advantages but potentially exhibited adverse effects on yellow catfish. The determination of the optimal quantity of rutin to be included in the diet of yellow catfish will be conducted in forthcoming investigations."}
+{"text": "Psoriasis is an immune\u2010mediated chronic inflammatory disease, and currently it is widely believed that the IL\u201023/IL\u201017 axis and Th17 cells play a critical and central role. However, increasing evidence suggests that neutrophils may interact with a variety of immune cells to play an indispensable role in psoriasis.We searched the recent literature on psoriasis and neutrophils through databases such as PubMed and CNKI, and summarized the findings to draw conclusions.Neutrophils can promote the development of psoriasis by secreting IL\u201023, IL\u201017, and cytokines with TH17 cell chemotaxis. Activated keratinocytes (KCs) can attract and activate neutrophils, induce the formation of neutrophil extracellular traps (NETs). KCs can also expose self\u2010antigens which lead to strong autoimmune reactions. The granule proteins secreted by activated neutrophils can activate IL\u201036, which converts vulgaris psoriasis to generalized pustular psoriasis (GPP).The function of neutrophils components and the interaction between neutrophils and immune cells play an essential role in the pathogenesis of psoriasis. The aim is to provide a theoretical basis for the exploration of targeted clinical treatments and fundamental research on the pathogenesis of psoriasis. They play an indispensable role in anti\u2010infective and inflammatory processes through mechanisms such as antigen presentation, reactive oxygen species (ROS) generation, release of granular proteins, formation of neutrophil extracellular traps (NETs), and production and release of cytokines.1.1Neutrophils, as a type of innate immune cell, are the first to reach the site of inflammation in the early stages of inflammation. Neutrophil lifespan is short, and apoptosis begins after entering the blood for 24\u00a0h, but during the period of psoriasis inflammation, the lifespan of neutrophils can be significantly extended.1.1.1IL\u201023, composed of subunits p40 and p19. Macrophages, dendritic cells, and KCs in psoriatic skin tissue can secrete IL\u201023After binding to IL\u201023R, IL\u201023 can exert various biological effects. IL\u201023R is expressed on the surfaces of lymphocytes and myeloid cells .1.1.2It is currently believed that IL\u201017 plays an important role in psoriasis, and IL\u201017 antagonists such as secukinumab have been widely used in clinical practice.Among other diseases comorbid with psoriasis, IL\u201017\u2010producing neutrophils have also been observed. IL\u201017+ neutrophils were found in atherosclerotic plaques.1.2Neutrophils can be recruited by various chemokines and migrate to the lesion site. KCs can produce neutrophil chemokines, but previous studies have shown that neutrophils themselves can also produce chemokines under the stimulation of inflammation,1.3MPO gene defects have decreased ability to produce NETs.The composition of neutrophil granule proteins is relatively complex and can be divided into three major categories, including primary azurophilic granules (myeloperoxidase (MPO) etc.), secondary specific granules, and gelatinase granules  etc.).1.41.4.1NETs are fibrous networks released by neutrophils that contain various components such as neutrophil granule proteins, LL\u201037, RNA, and cytokines.The formation of extracellular traps by neutrophils is called NETosis.The uncontrolled release of NETs may contribute to various diseases such as psoriasis, systemic lupus erythematosus,1.4.2NETs contain MMP\u20109,Both mast cells and neutrophils release IL\u201017 during the formation of extracellular traps, but neutrophil NETs contain higher levels of IL\u201017 than MCETs do.1.5In traumatized or microbially infected skin, mast cells recognize viruses, bacteria, and other pathogens through TLRs.KC in the skin has antigen\u2010presenting function.When activated by pathogenic factors in the internal and external environment, KCs release neutrophil\u2010derived chemokines, which results in the recruitment of neutrophils to the skin. When neutrophils arrive at the vessels in the skin, they interact with the VECs. The MMP\u20109 produced by neutrophils destroys cell connections between VECs, causing vascular dilatation and increased permeability. Along with changes on vascular endothelial cells, neutrophils are activated,2Neutrophils are innate immune cells that play a critical role in the early stages of psoriasis inflammation. They can interact with other immune cells such as KCs and mast cells through the production of IL\u201017, formation of NETs, and release of granule proteins. Neutrophil lifespan is significantly extended, and KCs and TH17 cells can attract neutrophils, amplifying the inflammatory effect.Previous studies have identified the role of NETs in psoriasis, atherosclerosis, uremia, inflammatory bowel disease, and chronic obstructive pulmonary disease. NETs also play an important role in the pathogenesis of comorbidities. Today several drugs that can inhibit NETs have been developed for possible blocking NETosis, including cannabidiol [99],The authors have no relevant financial or nonfinancial interests to disclose."}
+{"text": "Loss of the tumor suppressive activity of the protein phosphatase 2A (PP2A) is associated with cancer, but the underlying molecular mechanisms are unclear. PP2A holoenzyme comprises a heterodimeric core, a scaffolding A subunit and a catalytic C subunit, and one of over 20 distinct substrate-directing regulatory B subunits. Methylation of the C subunit regulates PP2A heterotrimerization, affecting B subunit binding and substrate specificity. Here, we report that the leucine carboxy methyltransferase (LCMT1), which methylates the L309 residue of the C subunit, acts as a suppressor of androgen receptor (AR) addicted prostate cancer (PCa). Decreased methyl-PP2A-C levels in prostate tumors is associated with biochemical recurrence and metastasis. Silencing LCMT1 increases AR activity and promotes castration-resistant prostate cancer growth. LCMT1-dependent methyl-sensitive AB56\u03b1Cme heterotrimers target AR and its critical coactivator MED1 for dephosphorylation, resulting in the eviction of the AR-MED1 complex from chromatin and loss of target gene expression. Mechanistically, LCMT1 is regulated by S6K1-mediated phosphorylation-induced degradation requiring the \u03b2-TRCP, leading to acquired resistance to anti-androgens. Finally, feedforward stabilization of LCMT1 by small molecule activator of phosphatase (SMAP) results in attenuation of AR-signaling and tumor growth inhibition in anti-androgen refractory PCa. These findings highlight methyl-PP2A-C as a prognostic marker and that the loss of LCMT1 is a major determinant in AR-addicted PCa, suggesting therapeutic potential for AR degraders or PP2A modulators in prostate cancer treatment. Loss of PP2A activity is often associated with cancer but the underlying mechanism remains unclear. Here, the authors show that decreased methylation of PP2A catalytic C subunit caused by loss of LCMT-1 in prostate cancer abrogates the tumor suppressor activity of PP2A on AR/MED1-dependent gene expression, proposing decreased methyl-PP2A-C as a prognostic marker for prostate cancer progression. Though the inappropriate activation of numerous oncogenic kinases plays a causal role in transformation, metastasis, and therapy resistance and several of these kinases have been successfully targeted for cancer treatment, much less is known about the function and mechanisms of regulation of phosphatases in cancer. The highly conserved protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase with a complex heterotrimeric composition involved in the regulation of numerous critical aspects of cellular function2. PP2A is one of the most abundant enzymes, accounting for up to 1% of the total cellular protein in some tissues and represents the majority of serine/threonine phosphatase activities in many tissues3. PP2A is comprised of a core dimer consisting of a scaffolding \u201cA\u201d subunit (PPP2R1/PP2A-A), and a catalytic \u201cC\u201d subunit (PPP2C/PP2A-C), which form a heterotrimeric complex with one of at least 20 substrate-directing regulatory \u201cB\u201d subunits clustered into four structurally distinct families PPP2R5/B56, PPP2R2/B55, PPP2R3/PR72/PR130, and Striatin4. The AC heterodimers are not considered the functional phosphatase holoenzyme in vivo, with the binding of the B subunit conferring target specificity and allowing PP2A to dephosphorylate target proteins.Reversible protein phosphorylation plays a fundamental role in numerous biological processes and is regulated by a dynamic interplay between protein kinases and phosphatases. Disruption of this balance due to aberrant activation of kinases and inactivation of phosphatases is a hallmark of cancer6. PP2A dysregulation is commonly observed in cancer, typically through one of several mechanisms, including somatic mutations, haploinsufficiency and/or decreased expression of PP2A subunits, and increased expression of endogenous PP2A inhibitors such as CIP2A10. PP2A heterotrimerization is regulated through post-translational modifications of the catalytic C subunit, which influence the binding affinity of the B subunits to the AC dimer and thus the substrate specificity. While PR130 and Striatin binds to the AC dimer in a methylation-independent manner, a central evolutionarily conserved mechanism that leads to heterotrimerization of AC and B55, and B56 subunits is the S-adenosylmethionine dependent PP2A-C \u03b1-carboxymethylation at the terminal leucine residue (L309) by Leucine Carboxyl Methyltransferase 1 (LCMT1)14. The presence of methylation on L309 neutralizes the negative charge on the terminal carboxylic acid, allowing for the binding of both B55 and B56 family members to the PP2A AC dimer. Conversely, Protein Phosphatase Methylesterase 1 (PME-1) mediated removal of this methylester from the PP2A-C dimer results in the retention of the negatively charged terminal carboxylic acid creating steric hinderance that prevent B55 and B56 family member binding, thus adding another layer to the regulation of PP2A biogenesis16. Given the previous studies demonstrating the role of the B55/B56 methyl-sensitive PP2A heterotrimers in regulating gene expression by targeting multiple signaling pathways such as PI3K/AKT/mTOR activation20, one critical function of C subunit methylation by LCMT1 could be to coordinately regulate both B55/B56 containing heterotrimers to negatively affect transcription and survival signals in cancer. Interestingly, LCMT1 is the only enzyme known to catalyze methylation of not only PP2A-C but also two other PPP family members, the PP4 and PP6 catalytic subunits22. Nevertheless, the status of LCMT1 expression, regulation, and its role in cancer development and progression remains elusive.PP2A is widely considered a tumor suppressor and plays a fundamental role in regulating cellular homeostasis by dephosphorylating a broad array of protein targets, from mitotic spindle components, chromatin regulators to intracellular signaling mediators23. PCa relies on AR driven transcription for growth and survival, forming the basis for effective androgen deprivation therapy (ADT) as a first-line of treatment in the primary stages of the disease. However, restoration of AR signaling through AR point mutations, AR overexpression, AR genomic amplification, and constitutively active AR splice variants contributes to an AR-addicted castration-resistant prostate cancer (CRPC)26. Next-generation AR signaling inhibitors (ARSIs) such as abiraterone, enzalutamide, darolutamide, or apalutamide are used in combination with ADT to treat metastatic and nonmetastatic CRPC28. Despite the clinical success surrounding ARSI, resistance to these therapies invariably emerges through various mechanisms that restore AR-signaling, resulting in poor clinical outcomes31. AR-mediated transcriptional addiction is, therefore, a hallmark of a vast majority of refractory CRPC, where AR functions in a multi-protein complex comprising transcriptional coactivators and chromatin-associated proteins33. Additionally, greater than 50% of CRPC demonstrates PI3K pathway activation through either biallelic loss of Pten or activating mutations in PIK3CA and AKT134. Nevertheless, how this recurrently activated oncogenic survival pathway converges upon AR signaling is unclear. There remains an impending need for a better understanding of the mechanism that restores AR signaling and to discover novel ways to target AR addiction in refractory CRPC.Prostate cancer (PCa) is the second most common cancer in men worldwide, and the eighth leading cause of cancer-related deathsIn this study, we report the association of decreased PP2A-C \u03b1-carboxymethylation of L309 with rapid biochemical recurrence and metastasis in PCa. Our results show that reduced AB56\u03b1C heterotrimers lead to increased AR activity and castration-resistant tumor growth in vivo. Furthermore, we demonstrate LCMT1-dependent methyl-sensitive AB56\u03b1C heterotrimer targets AR and its critical coactivator MED1 for dephosphorylation, resulting in the decruitment of the AR-MED1 complex from the chromatin and loss of target gene expression. In addition, we identified S6K1/\u03b2-TRCP mediated degradation of LCMT1 as a mechanism for the restoration of AR signaling in antiandrogen refractory prostate cancer cells. Together, these results demonstrate that the loss of LCMT1/decreased PP2A-C \u03b1-carboxymethylation is a major determinant of AR-addicted PCa and supports the therapeutic use of AR degraders and selective small molecule modulators of PP2A for refractory prostate cancer treatment.22. To better define the role of carboxymethylation in regulating B56 holoenzyme formation, we specifically immunoprecipitated B56\u03b1 . This was different for the B56\u03b1 subunit, which showed similar total expression levels in wild-type and Lcmt1 null cells. However, B56\u03b1 had a reduced ability to interact with non-methylated PP2A-C/PP2A-A dimers indicating a high methyl-PP2A-C affinity/preference of B56\u03b1. Re-expression of V5-tagged LCMT1 in Lcmt1 null cells resulted in a quantitative rescue of the defects in PP2A-C methylation as well as in B56 holoenzyme assembly  with antibodies against p-MED1 and p-AR revealed nuclear puncta for both factors in LNCaP cells expressing LacZ, however, a significant reduction in the puncta and total fluorescence accompanied by increased methyl-PP2A-C staining was observed in cells overexpressing LCMT1  and four (isoleucine) to alanine (2A)  using CRISPR. Ectopic expression of HA-tagged MED1 and Halo-tagged AR showed increased p-MED1 and p-AR in the LCMT1 null cells compared to Cas9 control, and interestingly, the phosphorylation of endogenous MED1 was substantially higher in the null cells, suggesting a direct role of LCMT1 via PP2A in regulating MED1 and AR phosphorylation Fig. . Next, iMT1 Fig. . Next, cMT1 Fig. . Since P AR Fig. . The two2A) Fig. . Co-tran2A) Fig. . These o2A) Fig. , which e2A) Fig. . Collect2A) Fig. , thereby2A) Fig. . We haveant Fig. , thus suant Fig. .Fig. 3LCklk3 and others  or cullin neddylation inhibitor (MLN4924) led to the accumulation of LCMT1, implying a potential cullin-RING E3 ubiquitin ligases (CRLs) dependent mechanism in regulating LCMT1 stability and degradation amino]propan-2-olato dizinc(II) specifically binds to phosphorylated amino acids and generates a mobility shift on acrylamide gels proportional to incorporated phosphates48, we observed a slow-migrating band(s) for LCMT1, which was eliminated upon phosphatase ) treatment  such as phosphorylation50. Noticeably, we identified a conserved phosphorylation consensus R/X/R/X/X/S/T sequence (R4QRESS9), for the protein kinase AKT-mTOR-ribosomal protein S6 kinase 1 (S6K1) in its N-terminal IDR  Pten null prostates develop high-grade prostate intraepithelial neoplasia (HGPIN) phenotype- the main precursor lesion to adenocarcinoma53, and they displayed a marked increase in p-MED1 staining, along with higher MYC, p-AKT and p-S6 staining - a characteristic pattern associated with LCMT1 loss/reduction  used in the study showed no significant pathological changes. Similar to observations made in human cells, PI3K-AKT-mTOR pathway inhibitors stabilized LCMT1 in mouse fibroblast cells , apalutamide (Apa), and darolutamide (Daro) are efficacious in the treatment of both nonmetastatic and metastatic CRPC27. However, the disease invariably recurs through restoration of AR-signaling55. Our observation that decreased LCMT1 expression leads to increased p-AR/p-MED1, reduced sensitivity to enzalutamide, and castration-independent growth  in LNCaP and VCaP cells by growing them continuously in the presence of the drug for close to six months and collecting the cell fractions at regular intervals. As expected, following a quiescence associated reduction in p-AR/AR and p-MED1/MED1 for the first 6-7 weeks, a steady increase in their level was evident, paralleled by a gradual loss in LCMT1 and methyl-PP2A-C, as cells evolved to attain the EnzaR state  engineered from phenothiazine parent compounds have shown to drive dephosphorylation of select pathogenic substrates, including AKT and S6K162. From our finding implicating S6K1 in directly regulating LCMT1 expression resulting in stable AR transcriptional complex formation in refractory CRPC cells, we postulated that inhibition of AKT-S6K1 signaling by these PP2A modulator molecules will have a feedforward effect on LCMT1-dependent AB56\u03b1C heterotrimer assembly and AR/MED1 dephosphorylation , the human genome codes for far fewer (\u223c50) putative serine/threonine phosphatases (PSPs) comprising three major families: phosphoprotein phosphatases (PPPs), metal-dependent protein phosphatases (PPMs), and the aspartate-based phosphatases represented by FCP/SCP 38. Our data show a reduction in the AB56\u03b1C/methylation-sensitive heterotrimers due to phosphorylation-mediated degradation of LCMT1 involving S6K1\u2014a downstream effector of the PI3K pathway. This strongly suggests a paradigm where persistent oncogenic PI3K signaling in cancer cells is achieved by the elimination of its negative regulator AB56\u03b1C and also other methylation-sensitive heterotrimers such as AB55C through S6K1-mediated degradation of LCMT1. Another important implication of these findings is that the PI3K signaling could also result in the inactivation of other phosphatase family members, such as PP4 and PP6, since LCMT1 is also required for the methylation of PP4-C and PP6-C catalytic subunits21.Moreover, AB56\u03b1C/methylation-sensitive heterotrimers negatively regulate the PI3K-AKT-mTOR-S6K signaling axis, and a high proportion of prostate cancers exhibit increased activation of this critical survival pathway73. However, though the dogma of PP2A-regulated transcription is well established, we here report the presence of canonical PP2A heterotrimer components on the chromatin. In this regard, our mechanistic data show LCMT1 catalyzed methylation-sensitive AB56\u03b1C heterotrimers on the chromatin with consequent dephosphorylation of AR-MED1, thus providing the evidence for a direct PP2A-substrate interaction on the chromatin and a consequent change in gene expression. Recently, a noncanonical PP2A holoenzyme, comprising the AC dimer and the multi-subunit RNA endonuclease integrator, Integrator\u2013PP2A complex (INTAC), was shown to regulate and fine-tune transcription and RNA maturation by antagonizing the action of the CDK9 kinase activity on targets like DSIF and RNAPII74. However, it is unclear whether the interaction between the AC dimer and Integrator complex is methylation dependent; if so, then LCMT1 could be expected to facilitate not only the canonical substrate-specific PP2A heterotrimers but also Integrator-mediated transcriptional control directly through L309 methylation of PP2A-C.Methylation-sensitive PP2A heterotrimers have also been implicated in the regulation of oncogenic transcription through negative regulation of chromatin modulators and transcription factors such as BRD4, HDACs, SWI/SNF, and MYC30. Though restoration of AR signaling through AR point mutations, overexpression, genomic amplification, and constitutively active splice variants is shown to promote resistance to AR antagonism, the underlying molecular basis for AR transcriptional activity and its requirement for the survival of ARSI-resistant cells is unclear56. The findings here implicate AKT/S6K1-mediated LCMT1 degradation as key to developing resistance to ARSI in CRPC cells that continue to rely on AR activity for survival. Furthermore, our data showing the sensitivity to AR degraders in the ARSI-resistant cells illustrate the incomplete targeting of AR by enzalutamide, apalutamide, or darolutamide. Therefore, further targeting of the AR through PROTAC-based therapies that are under investigation75, may be clinically beneficial in patients who develop resistance to next-generation ARSI. Furthermore, the loss of methylation-sensitive AB56\u03b1C heterotrimers, and potentially AB55C heterotrimers which is known to negatively regulate AKT76, leads to increased PI3K/AKT signaling in ARSI refractory CRPC cells. This suggests that drugs targeting PI3K/AKT could be beneficial in treating ARSI refractory CRPC.Despite recent advances in treating CRPC with second-generation ARSI such as enzalutamide, darolutamide, and apalutamide, de novo resistance is observed in 20\u201340% of patients, and most patients invariably acquire resistance to these therapies77 based therapeutics could provide additional opportunity for targeted activation of methyl-sensitive biased PP2A heterotrimers in treating transcription addicted cancers.Beyond their biological significance, the results presented in this work strongly support the use of SMAPs in treating ARSI-resistant CRPC. The SMAP induced biased PP2A reactivation coupled with feedforward activation of LCMT1 through AKT/S6K1 inhibition provide an excellent opportunity for targeted eviction of the hyperphosphorylated AR transcriptional complex from chromatin. While target-specific molecular glues like SMAPs are still in their early phase of development, our study suggests that combining PI3K/AKT/S6K1 pathway inhibitors with antiandrogen therapies cannot be ruled out in AR-addicted prostate cancer. Furthermore, the mechanistic insights presented in this work indicate that direct stabilization of LCMT1 by phosphorylation-targeting chimeras (PhosTACs)Pten knockout mice were generated by crossing Pbsn-Cre male mice Tg (Pbsn-cre) 4Prb/J  with Pten floxed mice . All mice were housed in a pathogen-free animal barrier facility and all in vivo experiments were initiated with male mice aged 5-8 weeks.Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at The University of Pennsylvania and/or The University of Michigan. Animal use and care was in strict compliance with institutional guidelines and all experiments conformed to the relevant regulatory standards by the Universities. The maximal tumor size in our study models (mice) never exceeded 1\u2009cm in diameter as allowed by the above ethics committee. NOD SCID or NCI SCID/NCr athymic nude mice were obtained from the Jackson Laboratory (strain code: 005557) and Charles River (strain code: 561). Prostate-specific The maintenance of mice and the experimental procedures (immunizations) have been conducted according to the Austrian Animal Experiments Act and have been approved by the Austrian Federal Ministry of Science and Research BMWFW-66.009/0211-WF/V/3b/2015 and the animal experiments ethics committee of the Medical University of Vienna.78. LNCaP, LNCaP-AR, VCaP, and LAPC4 enzalutamide-resistant cell lines were generated by culturing their parental lines in presence of enzalutamide (10\u2009\u00b5M) for 4\u20136 months in vitro. Surviving enzalutamide-resistant polyclonal pools were maintained in 10\u2009\u00b5M enzalutamide throughout. Apalutamide- and darolutamide-resistant LNCaP, LNCaP-AR, VCaP, and LAPC4 lines were generated by culturing the cells in 10\u2009\u00b5M of the respective drugs for at least two months.The cell lines used in this study were obtained from American type cell culture (ATCC) unless mentioned. LNCaP-AR cells were a generous gift from Dr. Charles Sawyers- Memorial Sloan-Kettering Cancer Center, New York, NY. LNCaP, LNCaP-AR, and LAPC4 prostate cancer cell lines were grown in RPMI 1640 , VCaP- prostate cancer line, HEK293-T, and NIH-3T3, cells were grown in DMEM with Glutamax . HAP1 wild-type and LCMT1 CRISPR Cas9 deletion cells  were grown in Iscove\u2019s Modified Dulbecco\u2019s Medium . The medium was supplemented with 10% of FBS  or FCS  and 1% of Penicillin Streptomycin Solution . LNCaP-AR enzalutamide-resistant cell lines were derived from enzalutamide-resistant tumor xenografts as described previously2. The complete list of cell lines used is indicated in Supplementary Data\u00a0All experiments using antiandrogen-resistant pools were done in the presence of enzalutamide, except when cells were treated with AR-PROTAC ARD-69. shLCMT1 or non-targeting control shNTC cell lines were created transducing shLCMT1-GFP or shNTC-GFP lentiviral vectors in LNCaP, VCaP, LAPC4, and LNCaP-AR cells. Cells were sorted for GFP expression. Knockdown was confirmed by immunoblotting. LCMT1 overexpression or control cell lines were generated by transducing pLX304-LacZ or pLX304-LCMT1 in LNCaP cells. Cells were selected using Blasticidin. Overexpression was confirmed by immunoblot. All the cell lines were routinely tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection kit  and were maintained in a humidified incubator at 37\u2009\u00b0C and 5% COThe complete list of the reagents used throughout the study and their commercial suppliers are given in Supplementary Data\u00a079.A set of tissue microarray (TMA) slides (section thickness 3\u2009\u00b5m) containing a single 0.6\u2009mm tissue punch from each of 17,747 patients was constructed at the Institute of Pathology, University Medical Center Hamburg-Eppendorf, Germany. The composition of the TMA is given in Supplementary Table\u00a02O2 and protein exposure to remove non-specific binding. The slides were exposed overnight to the antibody . After washing with TBST. The slides were exposed to DAKO Envision+ (Goat antimouse polymerized HRP) and subsequently the chromogen, diaminobenzidine (DAB). After counterstaining with hematoxylin, the slides were digitally scanned using an APERIO scanner. In order to determine the specificity of the antibody, the antibody was pre-incubated with 10x molar excess of the methylated and unmodified peptides prior to incubation with slides.Immunohistochemistry was performed on a DAKO autostainer. TMA slides were deparaffinized and rehydrated in a graduated series of ethanol. Slides were subjected to epitope retrieval using 10\u2009mM Tris\u2013HCl buffer pH9.0 containing 1\u2009mM EDTA. The slides were then subjected to HThe digital slides were examined using QuPath and the H-score and Allred scores were submitted back to the originator of the TMA slides to be correlated with the clinical data.2O2 in a sodium acetate buffer pH\u2009=\u20096. The reaction was stopped by the addition of 1\u2009N H2SO4 and the absorbance was measured at 450\u2009nm, for background correction the absorption of 560\u2009nm was subtracted. For ELISA quantification the signals of the antibodies were normalized to the signal of the cognate target peptide of the antibody which was artificially set to 1.ELISA 96-well plates  were coated with 50\u2009\u00b5l peptide solution (2\u2009\u00b5g/ml in TBS) at 4\u2009\u00b0C over night. The plate was blocked with 2% BSA in TBS for 1\u2009h at RT and incubated with primary antibodies in TBS for 1 to 2\u2009h at room temperature. Incubation with secondary peroxidase conjugated antimouse was performed for 1\u2009h at RT followed by detection with TMB  and HMeasurements were performed using Biacore T200 instrument. The sensorgrams obtained were analyzed with Biacore T200 Evaluation Software (version 3.1). Flow cells were coated with 30\u2009\u00b5g/mL antimouse IgG antibody (in immobilization buffer) using the mouse antibody capture kit  via amine coupling according to the manufacturer\u2019s protocol. The immobilization levels obtained were 11605.9 and 11333.9 RU. The antibody (7C10) was applied with a concentration of 50\u2009\u00b5g/ml and reached a response level of approx. 1200 RU. The antigens were used in different concentrations:PP2A Me 5\u2009nM, 10\u2009nM, 20\u2009nM, 40\u2009nM, 80\u2009nM and 160\u2009nM for 7C10PP4 Me 50\u2009nM, 100\u2009nM, 200\u2009nM, 400\u2009nM, 800\u2009nM for 7C10Antibody capturing time was adjusted to reach approx. 1200 RU, antigen association time was 360\u2009s, dissociation time was 450\u2009s. The flow rate was 40\u2009\u00b5l/min. Regeneration of the chip was performed injecting 10\u2009mM glycine-HCl pH 1.7 for 180\u2009s with a flow rate of 20\u2009\u00b5l/min. The value of the equilibrium dissociation constant (KD) was obtained by fitting a plot of response at equilibrium (Req) against the respective concentration of the analyte .Peptides were purchased from Pichem. The sequences of the peptides used in this study are as follows:309: Ac-His-Val-Thr-Arg-Arg-Thr-Pro-Asp-Tyr-Phe-Leu-OHLeu309: Ac-His-Val-Thr-Arg-Arg-Thr-Pro-Asp-Tyr-Phe-Leu-OMemeLeu309: Ac-His-Val-Thr-Arg-Arg-Thr-Pro-Asp-Tyr-Phe-Leu-NH2amLeu307: Ac-Ile-Pro-Ser-Lys-Lys-Pro-Val-Ala-Asp-Tyr-Phe-Leu-OHLeu307: Ac-Ile-Pro-Ser-Lys-Lys-Pro-Val-Ala-Asp-Tyr-Phe-Leu-OMemeLeu307: Ac-Ile-Pro-Ser-Lys-Lys-Pro-Val-Ala-Asp-Tyr-Phe-Leu-NH2amLeu305: Ac-Ile-Pro-Pro-Arg-Thr-Thr-Thr-Pro-Tyr-Phe-Leu-OHLeu305: Ac-Ile-Pro-Pro-Arg-Thr-Thr-Thr-Pro-Tyr-Phe-Leu-OMemeLeu305: Ac-Ile-Pro-Pro-Arg-Thr-Thr-Thr-Pro-Tyr-Phe-Leu-NH2amLeuFor immunoblotting (IB), cells were harvested and lysed by sonication in RIPA buffer - , supplemented with protease inhibitor cocktail  and phosphatase inhibitor . Protein concentration was determined by the standard BCA method, and an equal amount of protein from each sample was boiled in a sample buffer and subjected to SDS\u2013PAGE. The proteins were transferred to PVDF membrane  or nitrocellulose membranes, blocked with PBS-T - 5% (w/v) nonfat milk in PBS containing 0.1% Tween-20, probed with the relevant antibodies for 2\u2009h at room temperature or overnight at 4\u2009\u00b0C, and subsequently washed and probed with species-specific secondary antibodies coupled to horseradish peroxidase  at room temperature for 2\u2009h. For Methyl-PP2A-C protein detection, IP lysis buffer  was used instead of RIPA buffer, and the antibodies were probed in 0.5% nonfat milk in PBS-T. Immunoreactive proteins were detected by an enhanced chemiluminescence system as per the manufacturer\u2019s protocol  or Kwik Quant Imager (Kindle Biosciences).2, 10% Glycerol) containing protease and phosphatase inhibitors and the lysates were incubated with anti-FLAG M2 beads for 2\u2009h at 4\u2009\u00b0C or used for V5 antibody pull-down assay. For B56a and B55a/d immunoprecipitations one to two 15\u2009cm cell culture dishes with approximately 70% confluent cells or tumor tissues were lysed in lysis buffer (10% (v/v) glycerol; 135\u2009mM NaCl; 20\u2009mM Tris, pH 8.0; 1% Nonidet P-40; 1\u2009mM PMSF; 0.03 U/ml aprotinin (Sigma), 1\u00d7 Complete (Roche)) by 2\u2033 sonication, centrifuged at 10956 \u00d7 g at 4\u2009\u00b0C for 10\u2009min. Supernatant of lysates was adjusted to a protein concentration of 2 to 2.5\u2009\u00b5g/\u00b5l. 1/10 of the lysate (input) was boiled in protein sample buffer and 1\u2009ml of lysate was incubated either with B56\u03b1 (1D3-E12) or B55\u03b1/\u03b4 (2G9) or B56\u03b1 (ab89621) antibody crosslinked to protein G-Sepharose beads  to immunoprecipitate endogenous B55\u03b1/\u03b4 (clone 2G9) or B56\u03b1 (clone 1D3-E12) for 1\u2009h. The immune complexes were washed once with lysis buffer, 3\u00d7 with Tris-buffered saline . For PP2A-A-V5 coimmunoprecipitation experiments, cells were harvested, and coimmunoprecipitation was performed per Dynabeads CoImmunoprecipitation protocol . V5 antibody (Bio-Rad) was coupled at a concentration of 7 \u03bcg/mg of Dynabeads. Cell lysate was incubated with the Dynabeads for 30\u2009min, rotating, at 4\u2009\u00b0C. Fresh conjugated beads were prepared for each biological replicates. The immunoprecipitate was boiled for 5\u2009min at 95\u2009\u00b0C in protein sample buffer. After that, SDS\u2013PAGE, and immunoblotting was performed as described above. Densitometric quantification was performed using the Image J software and Image Lab Software.For immunoprecipitation (IP) experiments, nuclear or whole cell protein extracts were obtained from cells using NE-PER nuclear extraction kit  or RIPA buffer, respectively. The nuclear pellet was then lysed in an IP buffer by sonication. Nuclear lysates (0.5\u20132.0\u2009mg) were pre-cleaned by incubation with protein G Dynabeads  for 1\u2009h on a rotator at 4\u2009\u00b0C. Next, an antibody (2\u20135\u2009\u03bcg) was added to the pre-cleared lysates and incubated on a rotator at 4\u2009\u00b0C overnight. The following day, Protein G Dynabeads were added to the nuclear lysate-antibody mix for 1\u2009h. Beads were washed twice in IP or RIPA buffer containing 300\u2009mM NaCl, resuspended in 40\u2009\u03bcL of 2\u00d7 loading buffer, boiled at 100\u2009\u00b0C for 5\u2009min, and subjected to SDS\u2013PAGE and immunoblotting. For FLAG-tag immunoprecipitation, the cells were lysed in NP40 buffer  and a Tukey\u2019s honestly significantly different (HSD) post hoc test for multiple comparisons, or unpaired Students The antibodies used in this study are given in Supplementary Data\u00a0CDK7 inhibitor-THZ1 , R1881C-III , Trametinib , Enzalutamide , Apalutamide , Darolutamide , MLN4924 , Torin1 , LY294002 , MK-2206 , MG132 , DT-061 , Cycloheximide  and Rapamycin  were dissolved and aliquoted in DMSO . EGF  was dissolved and aliquoted in water.For Epidermal Growth Factor (EGF)  stimulation, the cells were plated in complete media at 60\u201370% confluency. After 24\u2009h cells were washed with PBS, serum starved for 24\u2009h and then treated with 100\u2009nM EGF for the desired time points. Similarly, for androgen starvation, the cells were cultured in a medium without Phenol Red  and 10% charcoal-dextran stripped FBS  for the required time points post 24\u2009h growth in a regular medium.For gene knockdown experiments, cells were seeded in six-well plates and transfected with 100\u2013200\u2009nM ON-TARGET plus SMART pool siRNA targeting S6K , \u03b2-TrCP  and non-targeting pool as a negative control  using lipofectamine RNAiMAX  according to the manufacturer\u2019s instructions. The cells were then harvested 48/72\u2009h post-transfection and used for the data presented.For the cell proliferation assay, the cells were seeded at a density of 50,000 per well in 12-well plates (n\u2009=\u20093). Twenty-four hours after seeding the cells were treated with ARD-69 or Enzalutamide or DT-061 or DMSO. Medium with the drug/compound was changed after every 48\u2009h. The cells were harvested, and live cell numbers were counted in the conditions used by Countess II FL .n\u2009=\u20093) were seeded and treated with the required drugs/compounds or vehicle for 12-14 days. Media was replenished every 3\u20134 days. Colonies were fixed and stained using 0.5% (w/v) crystal violet  in 20% (v/v) methanol for 30\u2009min, washed with distilled deionized water, and air-dried. After scanning the plate, the stained wells were destained with 500\u2009\u03bcL 10% acetic acid and the absorbance was determined at 590\u2009nm using a spectrophotometer . For VCaP or VCaP-Enzalutamide-resistant cells, viability was assessed by Cell-Titer GLO .For the colony formation assay, approximately 10,000 cells/well in six-well plates  supplemented with 0.1% Triton-X-100. After incubation on ice for 10\u2009min, the nuclear pellet was collected by centrifugation at 1300 \u00d7 g for 5\u2009min at 4\u2009\u00b0C, washed in Buffer A, and resuspended in Buffer B  with the same centrifugation settings, and incubated on ice for 30\u2009min. The chromatin pellet was collected by centrifugation at 1700 \u00d7 g for 5\u2009min at 4\u2009\u00b0C, washed and resuspended in Buffer B with 150\u2009mM NaCl, and incubated on ice for 20\u2009min. After centrifugation at 1700 \u00d7 g for 5\u2009min to remove proteins soluble in 150\u2009mM salt concentrations, the pellet was then incubated in Buffer B with 300\u2009mM NaCl on ice for 20\u2009min and centrifuged again at 1700 \u00d7 g to obtain the final chromatin pellet. The chromatin pellet was dissolved in a sample buffer, sonicated for 15\u2009s, and boiled at 95\u2009\u00b0C for 10\u2009min. Immunoblot analysis was conducted on samples as described previously. All buffers were supplemented with Pierce protease inhibitor and Halt protease & phosphatase inhibitors.Chromatin-bound proteins were extracted following a protocol previously describedhttp://bioinfo.ut.ee/primer3-0.4.0/primer3) and synthesized by Integrated DNA Technologies. The primer sequences for the SYBR green assays qPCR can be found in the Supplementary Data\u00a0The miRNeasy Mini Kit  was used to isolate total RNA from cells and the SuperScriptIV  kit was used to synthesize cDNA from 1\u2009\u00b5g total RNA. qRT-PCR was performed using Fast SYBR Master Mix  or Taqman Fast Advance MMIX , and analyzed on QuantStudio3 . The \u0394\u0394Ct method was used to quantify target mRNA expression, as normalized to GAPDH transcript levels. Primers were designed using Primer3 Input (version 0.4.0) , pAR (1:100), and methyl C (1:50). Coverslips were washed three times with PBS-T and incubated with the respective Alexafluor secondary antibodies (1:200) for 1\u2009h at room temperature followed by counterstaining with DAPI (Sigma). Coverslips were mounted on glass slides using VECTASHIELD PLUS Antifade Mounting Medium (Vector Laboratories) and cured overnight. Images were captured on a Zeiss LSM 880 confocal microscope. FIJI software was used to integrate the signal for quantitative image analysis. For specific quantification of nuclear staining, regions of interest were overlaid with the DAPI signal, and only co-localizing regions were included in the integration to exclude background cytoplasmic signals.2 according to the manufacturer\u2019s instructions. The Phos-tag gels were washed with transfer buffer containing 4\u2009mM EDTA for 20\u2009min and then were washed with transfer buffer containing 0.1% SDS for 5\u2009min before transfer onto PVDF membranes. The membranes were then immunoblotted with specific primary antibodies overnight followed by imaging and analysis.The protein samples were resolved on 6% PAGE gel containing 10\u2009\u03bcM Phos-tag acrylamide AAL-107  and 50\u2009\u03bcM MnCl2HPO4, and 2\u2009mM KH2PO4 at 4\u2009\u00b0C. Upon equilibration purified GST-tagged protein was loaded on the beads by incubating at room temperature for 45\u2009min on a shaker. Following that 2\u20133\u2009mg of nuclear protein, lysate was incubated with protein-bound glutathione beads at 4\u2009\u00b0C for overnight on a shaker. The next day, the bead-bound complex was washed four times with PBS to remove unbound protein, and the bound proteins were eluted from beads by boiling the sample in SDS loading dye at 100\u2009\u00b0C. Elute was resolved on SDS\u2013PAGE and proteins of interest were detected using immunoblotting.Glutathione Sepharose beads  were equilibrated by incubating for 10\u2009min (3\u00d7) in PBS -137 mM NaCl, 2.7\u2009mM KCl, 8\u2009mM NaFor in vivo ubiquitylation assay, HA- \u03b2-TrCP1, Flag-LCMT1, and His-ubiquitin expression plasmids were co-transfected into HEK293-T cells. After 36\u2009h, 5\u2009\u03bcM MG132  was added for 6\u2009h before harvesting the cells. Transfected cells were lysed with 1% SDS and sonicated. The cell lysates were diluted with NP10 buffer and incubated with Ni-NTA beads at 4\u2009\u00b0C for 2\u2009h. Next, beads were washed three times with the NP40 buffer, boiled in 50\u2009\u03bcl of 2\u00d7 SDS loading buffer for 5\u2009min, followed by western blot analysis with anti-V5-LCMT1 antibody to detect polyubiquitination.\u22121 Ubc3, 10\u2009ng\u2009\u00b5l\u22121 Ubc5, 2.5\u2009\u00b5g\u2009\u00b5l\u22121 ubiquitin (Boston Biochem), S6 kinase, and 1\u2009\u00b5l of unlabeled in vitro transcribed/translated LCMT1 and/or \u03b2TrCP1. The reactions were incubated at 30\u2009\u00b0C for 1\u2009h, subjected to SDS\u2013PAGE, then analyzed by immunoblotting with an anti-LCMT1 antibody.For in vitro ubiquitylation assay, the ubiquitylation of LCMT1 was performed in a volume of 10\u2009\u00b5l, containing 50\u2009mM Tris pH 7.6, 5\u2009mM MgCl2, 2\u2009mM ATP, 1.5\u2009ng\u2009\u00b5l\u22121 E1, 10\u2009ng\u2009\u00b5lhttps://www.agilent.com/store/primerDesignProgram.jsp), and Site-directed mutagenesis was performed using the QuikChange II XL site-directed Mutagenesis Kit  as per the manufacturer\u2019s protocol. All the mutations were verified by Sanger sequencing.The pLX304-V5-LCMT1-WT plasmid was purchased from DNASU (HsCD00441432) and pLX304-V5-LCMT1-S8A, pLX304-V5-LCMT1-S9A, pLX304-V5-LCMT1-S12A, pLX304-V5-LCMT1-S8.9.12\u2009A constructs were created from it through Site-directed mutagenesis. Similarly, pWZL hygro FLAG-HA TRAP220-WT  was used to create pWZL-HA-MED1-2A-a, pWZL-HA-MED1-2A-b, and pWZL-HA-MED1-2A-ab plasmids. Primers, shown in the Supplementary Data\u00a0Other plasmids used in this study are V245 pCEP-4HA B56\u03b1 , PPP2R1A plasmid in pLX304 , Flag-FBXW7, Flag- \u03b2-TrCP, pFN21K-AR-Halo, and MSCV-myrAkt (Addgene plasmid_ 49267-deposited by Lawrence Kane & Arthur Weiss), pLX304-LacZ , pLX304-EGFP , shNTC and shLCMT1 plasmids . The plasmids were transfected into HEK293-T or LNCaP cells using Lipofectamine 2000  as per the manufacturer\u2019s protocol. The cells were then harvested 48/72\u2009h post transfection and subjected to immunoprecipitation or immunoblotting or used for Lentivirus production. Lentiviral particles were generated in the lab at the University of Pennsylvania (UPenn) or in collaboration with the Vector Core at the University of Michigan. Following lentiviral production, viral particles were incubated on cells in penicillin/streptomycin-free media for 24\u2009h, followed by media replacement with normal media. After 72\u2009h, cells were selected in 16\u2009\u00b5g/mL Blasticidin-Invivogen (pLX304 constructs) or sorted by FACS for GFP positivity (shLCMT constructs) to generate stable cell lines. Knockdown or overexpression of proteins was confirmed by immunoblotting.RNA-seq libraries were constructed using the TruSeq sample Prep Kit V2.5 (Illumina) according to the manufacturer\u2019s instructions. In brief, 1\u2009\u03bcg of purified RNA was poly-A selected and fragmented with a fragmentation enzyme. Poly-A selected/fragmented RNA templates were used to synthesize the first and second strands, and end-repair to PCR amplification was performed according to the manufacturer\u2019s protocol. Libraries were purified and validated for appropriate size on a 2100 Bioanalyzer DNA 1000 chip . DNA library concentration was assessed using Qubit and normalized to 4\u2009nM before pooling. Libraries were pooled in an equimolar fashion and diluted to a loading concentration of 1.8 pM. Library pools were clustered and run on the Nextseq500 platform (Illumina Inc.) with single-end reads of 75 bases.80 with default parameters to either the HG19 or MM10 reference genomes. To remove low-quality/duplicate reads and sort the aligned BAM files, Samtools (v1.13) was used. Filtered BAM files were converted to BigWig files to view on the UCSC browser using bamCoverage (v3.5.1) from the Deeptools suite. Cufflinks, Cuffquant, and Cuffnorm v2.2.181 were used to assemble, quantify, and normalize transcripts (FPKM) to assemble the count tables, respectively.Single-end reads were demultiplexed using Illumina\u2019s bcl2fastq CLI tool. Fastqc was used to quality check the fastq files. Reads were then aligned using STAR v2.7.3.ahttps://bioconductor.org/packages/release/bioc/html/DESeq.html). DESeq282 performs internal normalization to account for library size and RNA composition bias provided the raw counts. Genes with a fold change of at least 1 and a q-value\u2009<\u20090.05 were considered to be differentially expressed.Differential expression analysis was done using the Bioconductor package, DESeq2 , LCMT1 overexpressing vs LAZ cells (LNCAP), and PTEN vs PTEN-Null (mouse organoids) using Hallmark signatures that are available in the Molecular Signatures Database (MSigDB) by BROAD institute.Gene enrichment analysis or GSEA is a tool that determines overrepresented genes in predefined gene sets that are biologically related using statistical methodsChromatin immunoprecipitation (ChIP) was performed using the iDeal ChIP-seq kit for Transcription Factors  according to the manufacturer\u2019s instructions. In brief, cells were collected and subjected to DNA-protein cross-linking using formaldehyde. After cell lysis, the cross-linked chromatin was fragmented and immunoprecipitated using Protein A magnetic beads and antibodies targeting transcription factors of interest. IPure magnetic beads were used to purify eluted DNA, which then underwent library preparation and sequencing. Library preparation for Illumina NextSeq was performed using the NEBNext Ultra II DNA library prep kit (NEB) as per the manufacturer\u2019s protocol. The quality of the library prepared was assayed using a BioAnalyzer-2100  and quantified using Qubit prior to loading of samples onto an Illumina NextSeq-500 to perform sequencing of 75 nucleotide paired-end reads at 40 million read depth.84. To overlap peaks and remove problematic regions known to produce enriched signals in several next-generation sequencing experiments, known as blacklists (https://github.com/Boyle-Lab/Blacklist), bedtools intersect (v2.30.0) was used.Paired-end reads were demultiplexed using Illumina\u2019s bcl2fastq CLI tool. Fastqc was used to quality check the fastq files. Reads were then aligned using Bowtie2 (v2.2.5) with default parameters to the HG19 reference genome. MarkDuplicates from the genome analysis toolkit  was used to mark duplicates. To remove low-quality/duplicate reads and sort the aligned BAM files, Samtools (v1.13) was used. Filtered BAM files were converted to BigWig files to view on the UCSC browser using bamCoverage (v3.5.1) from the Deeptools suite. MACS2 (v2.2.6) was used to compute and find enriched peaks in BAM files83. Gene signature scores were computed using singScore85. Samples were split into those pre-/post-ARSI. The post-ARSI patients were further split based on high/low survival from the date of biopsy, and high/low time to progression on ARSI. Analysis of variance (Anova) and Wilcoxon signed-rank tests were used to determine differences between groups.From 4/2005-7/2021, 268 prostate cancer patients older than 18 years of age underwent tissue collection for tumoral RNA-sequencing at the University of Michigan . Clinical data including information on ARSI treatment regimens  and survival was collected from 05/2021-01/2022. RNA-sequencing data for 313 RNA libraries across 290 biopsies was processed using Turnkey Precision Oncology, a flexible in house computational pipeline. Gene signatures for androgen response and mTORC1 signaling were obtained from the Molecular Signatures DatabasePten knockout mice, we crossed Pbsn-Cre male mice Tg (Pbsn-cre) 4Prb/J  with Pten floxed mice . The genotype of the mice was confirmed using the primers provided in the Supplementary Data\u00a0To generate the prostate-specific 86.Eight-week-old wild-type C5B6 and Pten floxed mice were euthanized and the prostate glands were dissected in DMEM 10% FBS. The prostate gland was transferred to a new petri dish with fresh dissecting media and minced with a razor blade. The minced tissue was digested with collagenase on a shaker at 37\u2009\u00b0C for 2\u2009h. Tissue chunks were spun down and digested with Trypsin/0.05% EDTA for 5\u2009min at 37\u2009\u00b0C. The cell/tissue suspension was passed through a 40\u2009\u00b5m nylon mesh filter to get a single-cell suspension. 10,000 cells in 40\u2009\u00b5L PrEGM media were mixed with 60\u2009\u00b5L Matrigel and seeded to the rim of a 12-well plate, followed by adding 800\u2009\u00b5L warm PrEGM in 30\u2009min. In 10 days, both wild-type and Pten floxed P0 organoids were digested with Dispase and Trypsin into single-cell suspension and treated with Adeno-cre for 20\u2009min to induce the knockout in the Pten floxed cells. Ten thousand treated cells in each group were seeded in 12-well plates, images were taken on day 10 and organoid sizes were measured using ImageJ. Genomic DNA and protein lysates were harvested on day 10 for the indicated molecular analysis2O2 for 5\u2009min. After three times 5\u2009min TBST washing, slides were blocked with blocking buffer (1.25% of goat serum) at room temperature for 1\u2009h. Slides were applied with diluted primary antibody and incubated in a humidified chamber at 4\u2009\u00b0C overnight. After three 10\u2009min TBST washes, slides were incubated with secondary antibody  for 30\u2009min at room temperature the color of the antibody staining was revealed by peroxidase-based detection  and the sections were counterstained with hematoxylin . Representative photographs were taken on a Keyence BZ-X Series All-in-one Fluorescence Microscope with 20x and 40x objectives. Images were assessed and quantified in ImageJ.Mouse prostate tissues were fixed in 4% of formaldehyde for 48\u2009h, and paraffin-embedded through Molecular Pathology and Imaging Core at UPenn. Paraffin-embedded sections from the prostate of Pten floxed control and Pten knockout mice were deparaffinized with 3 changes of xylene for 5\u2009min each. Slides were then rehydrated in 100% alcohol for 10\u2009min, 95% alcohol twice for 10\u2009min each, and 70% alcohol and distilled water for 10\u2009min each. Slides were subjected to citrate-based (pH 6.0) antigen retrieval  at 95\u2009\u00b0C for 30\u2009min and followed by blocking endogenous peroxidase activity with 3% H6 VCaP cells stably overexpressing LCMT1 along with the control VCaP cells were injected into the dorsal flanks of the non-castrated NOD-SCID mice . The chi-square- or t-test was used to evaluate the association of individual tumor characteristics with engraftment.Eight million LNCaP expressing shNTC, shLCMT (knockdown study) were subcutaneously injected into the right flank or both the dorsal flanks of 5\u20138-week-old male non-castrated and castrated NCI/NOD SCID/NCr mice  in 50% Matrigel . Castration surgery was performed by Charles River before receipt of the mice. The tumor measurement study ended at 40 days, and mice with tumors were sacrificed due to body condition or used for subsequent analysis. Tumor tissue was harvested and was snap frozen in liquid nitrogen for immunoblotting analysis. Mice that did not form tumors at this time continued to be monitored for tumor growth and body weight for first detection tumor analysis, up to 80 days. Eight million LNCaP LacZ or LCMT1 (overexpression study) were subcutaneously injected into the right flank or both the dorsal flanks of 5\u20138-week-old male non-castrated NCI/NOD SCID/NCr mice . Similarly, 2\u2009\u00d7\u2009106 VCaP-EnzaR prostate cancer cells suspended in 80\u2009\u03bcL of RPMI 1640 with 50% Matrigel were implanted subcutaneously into the dorsal flanks of the mice. When the tumor volumes reached approximately 80\u201390 mm3 mice were randomized into three treatment groups viz 15\u2009mg/kg body weight DT-061, 50\u2009mg/kg body weight DT-061 and vehicle-DMA (10%). The treatment was given orally b.i.d for 5 days a week for 3 weeks. In all studies tumors were monitored and recorded by digital caliper twice weekly, and animals were weighed twice weekly. The tumor volumes were estimated using the formula (\u03c0/6) (L \u00d7 W2), where L = length and W = width of tumor. At the end of the treatment regimen the mice were sacrificed, and the tumors were extracted for further analysis.For in vivo efficacy studies of DT-061 2\u2009\u00d7\u200910Further information on research design is available in the\u00a0Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting SummarySource Data"}