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+{"text": "C. elegans embryos is a powerful method for identifying genes involved in cell division processes. Here we present a functional analysis of the gene B0511.9, previously identified as a candidate cell polarity gene in an RNAi videorecording screen of chromosome I embryonic lethal genes.RNA interference coupled with videorecording of cdc26\u0394 mutant.Whereas weak RNAi inhibition of B0511.9 causes embryonic cell polarity defects, strong inhibition causes embryos to arrest in metaphase of meiosis I. The range of defects induced by RNAi of B0511.9 is strikingly similar to those displayed by mutants of anaphase-promoting complex/cyclosome (APC/C) components. Although similarity searches did not reveal any obvious homologue of B0511.9 in the non-redundant protein database, we found that the N-terminus shares a conserved sequence pattern with the N-terminus of the small budding yeast APC/C subunit Cdc26 and its orthologues from a variety of other organisms. Furthermore, we show that B0511.9 robustly complements the temperature-sensitive growth defect of a yeast C. elegans APC/C subunit CDC-26.These data demonstrate that B0511.9 encodes the  For the time course in Table emb-27/Cdc16, RNAi feeding was for 20 hours. For weak RNAi of B0511.9 in Figure RNAi was carried out by feeding  as in 3 using RNVideorecordings were done as in . Antibodcdc26\u0394::KanMX4 strain and the corresponding wild-type are in the BY4741 genetic background (MATa his3\u03941 leu2\u03940 met15\u03940 ura3\u03940) and were obtained from the European Saccharomyces cerevisiae Archive for Functional Analysis (EUROSCARF). The doc1\u0394::KanMX4 deletion allele was introduced into the W303 background .The C. elegans B0511.9a in yeast, a cDNA in the donor vector pDONR201 was obtained from [URA3 marker, the yeast 2-micron origin, and expresses B0511.9a from the PGK promoter. Translation starts at the second methionine of the original B0511.9a sequence where the homology with other Cdc26 orthologues begins (see Figure CDC26 and DOC1 were cloned into the vectors YCplac33 [To express ned from  and thenned from . The resYCplac33  and pRS4YCplac33 , respectYCplac33 . To deteDatabase searches were performed at the National Center for Biotechnology Information with Tblastn and Blastp . MultiplYD participated in the design of the study, performed the experiments described in Figures"}
+{"text": "The geometry of the CuII atom can be described as tetra\u00adgonal-pyramidal derived from the calculation of the value \u03c4 = 0.102. The three N atoms of the pyridine and ethane-1,2-diamine ligands and one Cl atom belong to the basal plane and the other Cl atom represents the axial position of the pyramid. The Cu atom is displaced by 0.2599\u2005(2)\u2005\u00c5 from the basal plane towards the axial Cl atom. In the crystal, mol\u00adecules are linked into chains by inter\u00admolecular N\u2014H\u22efCl and C\u2014H\u22efCl hydrogen bonds.The title complex, [CuCl DOI: 10.1107/S1600536809010149/si2162Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:"}
+{"text": "Trypanosoma brucei cell cycle, measuring changes in mRNA abundance on a transcriptome-wide scale. We developed a \u201cdouble-cut\u201d elutriation procedure to select unperturbed, highly synchronous cell populations from log-phase cultures, and compared this to synchronization by starvation. Transcriptome profiling over the cell cycle revealed the regulation of at least 430 genes. While only a minority were homologous to known cell cycle regulated transcripts in yeast or human, their functions correlated with the cellular processes occurring at the time of peak expression. We searched for potential target sites of RNA-binding proteins in these transcripts, which might earmark them for selective degradation or stabilization. Over-represented sequence motifs were found in several co-regulated transcript groups and were conserved in other kinetoplastids. Furthermore, we found evidence for cell-cycle regulation of a flagellar protein regulon with a highly conserved sequence motif, bearing similarity to consensus PUF-protein binding motifs. RNA sequence motifs that are functional in cell-cycle regulation were more widespread than previously expected and conserved within kinetoplastids. These findings highlight the central importance of post-transcriptional regulation in the proliferation of parasitic kinetoplastids.Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising questions about the extent of cell-cycle regulation in these organisms and the mechanisms whereby regulation is achieved. Here, we analyse gene expression over the  In the eukaryotic cell division cycle, many proteins involved in the replication of the cell and its components are specifically expressed exactly when required, ensuring tight control over replicative processes and increasing cellular efficiency. Regulation at the level of transcription has been thoroughly documented: for example, in yeast, at least nine transcription factors central to cell-cycle regulation operate in a network to control the expression of each other and of downstream effectors of cell cycle progression Trypanosoma brucei, the causative agent of African Sleeping Sickness (trypanosomiasis), differentiates through at least seven distinct cell types as it progresses through its life cycle, passing from mammalian hosts to the Tsetse fly vector The kinetoplastids are an early-branching group of unicellular eukaryotes including several important parasitic pathogens of humans and animals. Their life-cycles involve alternation between two very different hosts, typically vertebrates and biting insects, each of which represent considerable, but very different, challenges to the parasites' survival. During parasite adaptation, the cell shape can change from long, spindle-shaped cells with flagellar-driven motility to almost spherical, immotile cells, and there are dramatic changes in metabolism and cell surface macromolecules. Crithidia fasciculata, these transcripts peak in abundance in early S-phase, and their regulation is dependent on the presence of one or several octameric motifs with consensus sequence [CAUAGAAG] in the untranslated regions Kinetoplastid parasites undergo cell division in a characteristically well-ordered way. Unlike in many other eukaryotes, subcellular structures and organelles such as the mitochondria, ER, Golgi and flagellum are present in a single copy in G1-phase cells and are replicated at defined times during the cell division cycle T. brucei cells, which we used to identify regulated transcripts over the cell cycle on a transcriptome-wide scale. We cross-validated against starvation-synchronized cells and identified mRNAs that were regulated during the cell cycle: 55 peaking in early G1, 273 in late G1; 98 in S-phase and 120 in G2 phase. Genes functioning in several processes such as DNA metabolism or flagellar formation showed expression peaks at distinctive times in the cell cycle, correlating to a time just prior to the peak demand for the encoded proteins. For several groups of co-regulated transcripts, potential protein binding sites in the untranslated regions were found, which were conserved in different kinetoplastids.So far, investigation of the kinetoplastid cell cycle has involved synchronization of cells by starvation T. brucei cells were synchronized by starvation (starve-synch) and induced to resume the cell cycle by dilution into fresh media as described previously Procyclic  Counterflow centrifugal elutriation is a method for accurately isolating cells and other particles by sedimentation rate. This is achieved by subjecting them to two opposing forces: an outward-directed centrifugal force and an inward-directed flow of the suspension fluid. In an elutriation centrifuge, smaller particles are washed out of the elutriation chamber first, and by gradually increasing the fluid flow rate or decreasing the rotational speed, incrementally larger particles emerge. Relatively small size differences can be resolved, such that log-phase cell cultures can be fractionated into populations of cells at particular phases of the cell cycle In initial experiments, log-phase PC cell cultures were size-separated by conventional counterflow centrifugal elutriation . Cells were elutriated with a gradually increasing flow-rate, and the first and last fractions emerging from the chamber contained cell populations that were mainly in G1 and G2/M phase respectively . HoweverTo improve the separation, we performed a two-step procedure. We first obtained a cell population containing the largest \u223c30% of the cells in a log-phase culture by elutriation at a fixed flow-rate (the cells retained at 24 ml/min) and cultured them for one hour. At this point, they were elutriated a second time at a lower flow-rate to select the small cell population (cells not retained at 21 ml/min) that had undergone cell division within the preceding hour . This yiT. brucei genome.We performed a preparative-scale DCE experiment, this time isolating cell populations from four time-points post-selection: early (0.5 hours) and late (3 hours) G1 phase, S-phase (5.5 hours), and G2 phase (7.25 hours), each population consisting almost entirely of the desired cell cycle stage . Poly-A+in silico predictions . The majority of regulated genes in T. brucei could not have been predicted from homology to regulated genes in humans or yeast. This presumably reflects the large evolutionary distance, and differences in the cell biology between kinetoplastids and opisthokonts. Notably, unlike kinetoplastid organisms, opisthokonts in general do not coordinate the replication of their organelles with the cell cycle.The extent of overlap between our data and data from other organisms was determined. Consensus lists of cell-cycle regulated transcripts were obtained from Cyclebase rganisms . This waT. brucei genes, the 546 putative regulated genes were interrogated for over-represented Gene Ontologies  or four (DCE) expression values for each gene were added together as orthogonal vectors on a 2D plot to appraise the approximate timing of peak expression , and as a group their regulation was strikingly uniform and pervasive , as were other proteins found in the chromosomal passenger complex, in which TbAUK1 is found  over the cell cycle. However, predicted RBPs did not feature prominently on the list of regulated transcripts, which argues against a general model of cell cycle-coupled regulatory cascades in which RBPs bind to each other's transcripts and regulate transcript stability . Perhaps post-translational modifications such as phosphorylation regulate RBP activity, as has been suggested for the CSBP II complex Transcriptional regulation, if present, would be expected to result in co-regulation amongst co-transcribed genes. While a small number of cell cycle-regulated genes were clustered together in a section of chromosome 1 , not allOver-represented sequence motifs among co-regulated genes are good candidates for binding sites of trans-acting regulatory factors that act as post-transcriptional regulators. The [CAUAGA] motif of late G1-upregulated clusters #3 and #6 , homologtrans-acting factors.A short [UAGAU] motif was found to be over-represented in co-regulated cluster #1, which contained highly regulated genes that peaked later in the cell cycle. While this short motif is expected to occur quite often by chance, it occurred significantly more frequently in putative S-phase-specific transcripts in eight kinetoplastid species . The simT. brucei flagellar proteins are diminished during differentiation-associated growth arrest T. brucei flagellum undergoes dramatic elongation during differentiation of the highly motile migratory epimastigote that travels from the foregut to the salivary gland of the tsetse fly, and is also used for attachment to the salivary gland epithelium in the metacyclic forms T. cruzi, the flagellum is even more dramatically regulated, being reduced to a short stub without motile function. Interestingly, an element within the 10 nt sequence [AUGUAUAGUU], which contains a remarkably similar 5\u2032 sequence to the motif from cluster #8 in T. brucei, was found in the UTRs of paraflagellar protein mRNAs in Leishmania mexicanacis-regulatory motifs satisfies all the criteria for kinetoplastid flagellar proteins to be considered as a post-transcriptional regulon.The [AUGUAU*U] motif found in co-regulated cluster #8 contains a likely PUF-family RNA-binding domain cognate core, [UGUA] cis-regulatory sequence motif. Very small co-regulated groups  with respect to overall spot intensity within each of the 48 printing-tip blocks on the array. Outlying replicates for each spot were identified as those replicates for which exclusion from the analysis reduced the standard deviation of the log2-ratio values by at least 0.2 units: a maximum of one outlying replicate was excluded per gene. Raw and normalized microarray data was deposited as a MIAME compliant entry into the ArrayExpress database (accession #E-MTAB-515). Values from replicates were averaged and genes with a weak signal (median intensity of<8 log2 units) were filtered from the dataset.Cell starvation was performed essentially as described 9 procyclic (PC) cells were collected from a log-phase culture by centrifugation, resuspended in 10 ml of elutriation buffer, disaggregated by passing twice through a 20-gauge needle, and injected into the loading chamber of an Avanti J-26 XP elutriation centrifuge equipped with a JE-5.0 rotor (Beckman Coulter). Cells were loaded at<12 ml/min into the elutriation chamber (5 ml capacity) against a constant centrifugal force  at 27\u00b0C. In these pilot experiments, the flow rate was then incrementally increased by 2 ml/min for every 50 ml fraction collected, and most cells were seen to emerge from the chamber at flow-rates of between 15 and 32 ml/min  by elutriation at 4,700\u00d7g against a counter-flow of 24 ml/min at 27\u00b0C. Retained large cells (\u223c30%) were then flushed out of the chamber at 35 ml/min and FCS was immediately added to 20% (v/v). These cells were collected by centrifugation, gently resuspended in complete pre-warmed MEM-pros media and cultured at 27\u00b0C. After \u223c45 minutes, cells were collected and resuspended in elutriation buffer as before, and one hour after the first elutriation, small cells were selected from this population by passing through the elutriator at 4,700\u00d7g against a counter-flow of 21 ml/min. These recently (<1 hr) divided cells  were cultured and time points taken for the next 11 hours (For selection of recently-divided cells by double-cut elutriation (DCE), smaller cells were discarded from log-phase cultures .To compare the p-value cutoff of 0.1 and correction for multiple testing . To extract a list of genes functioning in DNA-related processes, genes containing the word \u201cDNA\u201d in the \u201cProduct description\u201d and \u201cGene ontology\u201d fields (but excluding \u201cDNA-dependent RNA polymerases\u201d) were scored for relevance using the TriTrypDB scoring algorithm. Genes scoring above 40, the chosen cut-off value, usually either contained \u201cDNA\u201d in the product description or at least three times among the Gene Ontology entries, and included many DNA polymerase subunits, topoisomerases, and DNA repair enzymes, but not histones. To generate a list of flagellar proteins, we used the mass-spectrometry results of T. brucei cells lacking a paraflagellar rod Searching for over-represented terms in the automatically annotated GeneDB Gene Ontology fields was performed using the GOstat program T. brucei genome, for which version 1.3 was used. To identify likely orthologues, each annotated CDS sequence in the T. brucei genome was compared to all currently listed CDS sequences in the Trypanosoma congolense, Trypanosoma cruzi  and Leishmania major genomes using one-to-one tBLASTx searches (best hits with score >170). Multiple-copy genes in the T. brucei genome were removed using the list from All sequences were downloaded from the TriTryp database, version 2.3 except for the Transcript extremities were estimated using the observation that individual mRNA sequences on the pre-mRNA  are demarcated by poly-pyrimidine tracts that are recognised by the splicing complex. This complex promotes cutting and polyadenylation of the RNA roughly 70 nt upstream, and also splicing of the invariant 35 nt splice-leader RNA onto the first possible downstream splice acceptor site (AG dinucleotide) de novo. Unregulated (control) transcripts were assigned as those passing minimum expression thresholds and having expression changes of less than 0.3 Log2 units (1.23-fold) in both starvation- and DCE- synchronized cells. Homologous groups of genes (tBLASTx scores >170) from the other trypanosomatids analysed were also collected into clusters, their UTR sequences estimated using Splicemodel and masked using Tandem Repeat Finder The estimated 5\u2032 and 3\u2032 UTR coordinates were used to extract all UTRs from genomic assembly sequences.  and increasing flow-rates. Cells from each fraction were saved for flow cytometry; results for selected fractions are shown. B: Schematic for the DCE procedure showing flow cytometry data for each step, from a pilot experiment. C: Flow cytometry results taken at various times after commencement of culturing of DCE-selected cells.(TIF)Click here for additional data file.Figure S2Averaging cell-cycle regulatory amplitude across genomic regions reveals only one small cluster of regulated genes. All but one copy of tandemly repeated genes were removed prior to analysis, as were pseudogenes, non-protein coding genes, and transcripts with fewer than 300 reads from RNA-seq in any time-point. A: moving average (window size 11 genes) was calculated from log2-regulation amplitudes across all chromosomes. The lower panel represents chromosome 1 only; dashed lines indicate borders of transcription units, as inferred from histone modifications that are characteristic of transcriptional start sites B: Moving averages of regulation amplitudes were calculated across 1000 genomes of randomly shuffled genes and the peak value from each was recorded. The peak value of 0.97 (an average of nearly 2-fold regulation across 11 genes) in the middle of chromosome 1 was higher than the peak value in all but four of the 1000 randomly shuffled genomes (red arrow). This peak region includes genes between Tb927.1.2290 and Tb927.1.2760. There was no other significant cluster of regulation in the genome.(TIF)Click here for additional data file.Table S1T. brucei transcript models from the four chosen time-points after DCE-synchronization of cells. Transcript models assigned to more than 300 reads are shown. Unique reads were used when possible; for transcripts with less than 400 unique reads, multiple-mapping reads were added to the total. Read counts were normalized against the total number of reads per time-point and log2-transformed, and the average value for each gene across the four time-points was set to zero. Microarray data from the starve-synch experiments is also given (right of double line); log2-transformed expression ratios for each time-point from these were also set to an average of zero. Gene names from version 2.3 of the TriTryp database are also listed.Read numbers attributed to (XLS)Click here for additional data file.Table S2Selected over-represented gene ontology terms found associated with transcripts that were cell-cycle regulated (amplitude thresholds as in (XLS)Click here for additional data file.Table S3Categorization of genes in DNA-related processes, flagellar proteins, mitosis/cytokinesis effector proteins and RBPs using TriTrypDB text searches, proteomics studies, literature surveys or protein domain interrogation respectively.(XLS)Click here for additional data file.Table S4Co-regulated gene clusters, identified using K-means clustering in the TM4 software using DCE/RNA-seq data, that were used for sequence motif searches.(XLS)Click here for additional data file.Table S5T. brucei genes in co-regulated cluster #1 Click here for additional data file."}
+{"text": "Dear Editor,I read with great interest the work published by Mogadam et al., entitled \"Comparison of Analgesic Effect between Gabapentin and Diclofenac on Post-Operative Pain in Patients Undergoing Tonsillectomy\" . I found"}
+{"text": "Dear Editor,We have read with great interest the article by Mogadam and colleagues on utilization ofgabapentin and diclofenac for management of post-operative pain in patients undergoingtonsillectomy . Perhaps"}
+{"text": "Hyperglycemia and hypertension impair endothelial function in part through oxidative stress-activated poly (ADP-ribose) polymerase 1 (PARP1). Biguanides and angiotensin II receptor blockers (ARBs) such as metformin and telmisartan have a vascular protective effect. We used cultured vascular endothelial cells (ECs), diabetic and hypertensive rodent models, and AMPK\u03b12-knockout mice to investigate whether metformin and telmisartan have a beneficial effect on the endothelium via AMP-activated protein kinase (AMPK) phosphorylation of PARP1 and thus inhibition of PARP1 activity. The results showed that metformin and telmisartan, but not glipizide and metoprolol, activated AMPK, which phosphorylated PARP1 Ser-177 in cultured ECs and the vascular wall of rodent models. Experiments using phosphorylated/de-phosphorylated PARP1 mutants show that AMPK phosphorylation of PARP1 leads to decreased PARP1 activity and attenuated protein poly(ADP-ribosyl)ation (PARylation), but increased endothelial nitric oxide synthase (eNOS) activity and silent mating type information regulation 2 homolog 1 (SIRT1) expression. Taken together, the data presented here suggest biguanides and ARBs have a beneficial effect on the vasculature by the cascade of AMPK phosphorylation of PARP1 to inhibit PARP1 activity and protein PARylation in ECs, thereby mitigating endothelial dysfunction. Induced by oxidative stress, poly (ADP-ribose) polymerase 1 (PARP1) plays an important role in DNA repair and maintenance of genome stability. Although mild activation of PARP1 can be protective and promote cell survival, excessive and sustained oxidative stress can cause overactivation of PARP1, which escalates the oxidative stress and stimulates pro-inflammatory and necrotic responses [cetylase \u20135. In adcetylase .In vivo, mice with PARP1 ablation are spared from hyperglycemia-induced endothelial dysfunction [-/- mice, led to decreased atherosclerosis [Hyperglycemia, angiotensin II (Ang II), and oxidized low-density lipoprotein activate PARP1 in vascular endothelial cells (ECs), with attendant increase in oxidative and inflammatory stresses . By contfunction . Furtherclerosis \u201312, whicBiguanides  and sulfonylureas  are first-line anti-diabetic drugs. Ample evidence indicates that metformin reduces cardiovascular incidents, possibly by improving vascular functions such as flow-mediated dilation (FMD) . Data frBy phosphorylating substrates such as endothelial nitric oxide synthase (eNOS), peroxisome proliferator-activated receptor-\u03b3 (PPAR\u03b3) coactivator 1 (PGC-1), and sterol regulatory element-binding proteins (SREBPs), AMP-activated protein kinase (AMPK) increases endothelial function via enhanced NO bioavailability and mitochondrial biogenesis and decreased inflammatory and oxidative stresses . AlthougAntibodies against pan-AMPK\u03b1, phospho-AMPK Thr-172, eNOS, and phospho-eNOS Ser-1177 were from Cell Signaling Technology ; anti-AMPK\u03b11 and anti-AMPK\u03b12 antibodies were from Abcam ; anti-PARP1 and anti-PAR monoclonal antibodies were from Trevigen ; anti-\u03b2-actin antibody was from Santa Cruz Biotechnology . Rabbit polyclonal anti-phospho-PARP1 Ser-177 antibody was produced with the sequence \u201cELGFRPEY(pS)ASQ\u201d by AbMax Biotechnology . Metformin, glipizide, telmisartan, metoprolol, atorvastatin, AICAR, Compound C and PJ34 were from Sigma-Aldrich . The PARP assay kit was from Trevigen . NO detection kit was from Beyotime Biotechnology .2 incubator at 37\u00b0C. We used a recombinant adenovirus expressing a constitutively-activated form of AMPK\u03b12, hereafter called Ad-AMPK-CA. The parental adenoviral vector, called Ad-null, was used as an infection control. Confluent ECs were infected with recombinant adenoviruses at the indicated multiplicity of infection (MOI) and incubated for 24 hr before experiments. ECs were transfected with various DNA plasmids with the use of Lipofectamine 2000 RNAi Max (Invitrogen).Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords. With patients\u2019 consensus, the cords were obtained from Department of Obstetrics and Gynecology, the First Affiliated Hospital, Xi\u2019an Jiaotong University. The protocol for the isolation of endothelial cells was reviewed and approved by the Ethic Committee of Xi\u2019an Jiaotong University Health Science Center. HUVECs were cultured in medium M199 supplemented with 20% fetal bovine serum (FBS), 1 ng/mL recombinant human fibroblast growth factor, 90 \u03bcg/mL heparin, 20 mM HEPES (pH 7.4) and 100 U/mL penicillin-streptomycin. Bovine aortic endothelial cells (BAECs) were a gift from Department of Physiology, Peking University. BAECs were cultured in Dulbecco\u2019s modified Eagle\u2019s medium (DMEM) supplemented with 10% FBS and antibiotics. Cells were maintained in a humidified 95% air, 5% CORNA was isolated from cultured cells or tissues by using TRIzol (Invitrogen). For mRNA quantification, total RNA was reverse-transcribed (RT) by use of the iScript cDNA synthesis kit (Invitrogen), followed by quantitative real-time PCR (qPCR) with SYBR Green (Promega) and a 7500 realtime PCR system (Applied Biosystems). The relative level of mRNA was calculated by the \u0394\u0394 Ct method with GAPDH or other appropriate genes as an internal control.Total proteins were extracted by use of RIPA buffer . Bicinchoninic Acid reagents (Thermo Scientific) were used to measure the protein concentration. Equal amounts of proteins were separated by SDS-PAGE and transferred to PVDF membranes. The blots were immunoreacted with primary antibodies and secondary antibodies conjugated with horseradish peroxidase. Protein bands were visualized by enhanced chemiluminescence detection and the intensity was quantified by use of Scion Image software.For the colorimetric PARP1 activity assay, EC nuclear extracts were incubated with histones and PARP cocktail in a strip well format. The PARP1 activity was then assessed by incorporation with biotinylated poly(ADP-ribose), which was quantified by the reading the absorbance at 450 nm. Serially diluted PARP-HSA (high specific activity) standards were used in parallel to generate the standard curve for subsequent calculation. EC-derived NO was measured in condition media by using Griess reagents following the standard protocol.ad libitum. We monitored the mouse and rat body weight, teeth, fur, and behavior on the daily base. According to our close observation, there was no mortality during the experimental period. At the end of experiments, all animals were euthanized by CO2 and tissues were collected afterwards for analyses. All treatments were by oral gavage. C57BL6 mice were purchased from Experimental Animal Center of Xi\u2019an Jiaotong University. Eight- to twelve-week-old male C57BL6 mice were treated with metformin (200 mg/kg/day), glipizide (1.3 mg/kg/day), telmisartan (10 mg/kg/day), metoprolol (30 mg/kg/day), or saline as a mock control for up to 24 hr. AMPK\u03b12-/- mice were a gift from Institute of Vascular Medicine, Peking University Third Hospital. AMPK\u03b12-/- mice and their wild-type littermates (AMPK\u03b12+/+) in a C57BL6 background were treated with metformin (200 mg/kg/day) for 12 hr. Ten-week-old db/db and age-matched db/m mice were purchased from the Model Animal Research Center of Nanjing University. db/db mice were treated with metformin (200 mg/kg/day) or glipizide (1.3 mg/kg/day) for 2 weeks. Fasting blood glucose was measured by use of the Accu-Check Glucose Meter. Spontaneously hypertensive rats (SHRs) and Wistar-Kyoto (WKY) rats were purchased from Vital River Laboratory Animal Technology . Ten-week-old male SHRs and WKY rats were fed standard chow diet and treated with telmisartan (10 mg/kg/day) or metoprolol (30 mg/kg/day) for 8 weeks. Systolic arterial pressure was measured by tail-cuff plethysmography.The animal experiments were approved by the Institutional Animal Care and Use Committee of Xi\u2019an Jiaotong University (No. XJTULAC2014-208). All animals were housed in colony cages  with a 12-hr light/12-hr dark cycle t test between two groups or ANOVA for multiple comparisons. Data were expressed as mean\u00b1SD from at least 3 independent experiments. p<0.05 was considered statistically significant.Significance of variability was determined by unpaired Student\u2019s 2O2, high glucose and Ang II dose- and time-dependently attenuated the phosphorylation of AMPK\u03b1 Thr-172 and PARP1 Ser-177 in HUVECs (2O2 at a lower concentration (1 \u03bcM) slightly induced AMPK and PARP1 phosphorylation . Consistent with results from in vitro experiments, the phosphorylation of PARP1 Ser-177 was lower in metformin-administered AMPK\u03b12-/- than AMPK\u03b12+/+ mice . At the basal level, namely untreated groups, db/db mouse and SHR aortas showed lower phosphorylation of AMPK\u03b1, PARP1, and eNOS and higher protein PARylation than db/m mouse and WKY rat aortas, respectively  have underscored the vasoprotective effect of metformin. In contrast, patients receiving sulfonylurea  did not show a similar extent of cardiovascular protection despite glycemic control ,26,27. VAlthough endothelial dysfunction is prevalent in hypertensive patients, the protective effect of anti-hypertensive drugs on the endothelium is less clear than that of biguanides. In the 2014 evidence-based guideline for management of hypertension in adults, the Eighth Joint National Committee (JNC 8) recommended ARBs as a first-line treatment, with \u03b2-blockers as later-line alternatives in part because of the higher rate of cardiovascular events associated with \u03b2-blockers as compared with ARBs . SeveralMechanistically, AMPK phosphorylation of PARP1 Ser-177 resembled the pharmacological inhibition of PARP1 by PJ-34 , which rBesides AMPK, several kinases have been shown to regulate PARP1. However, these phosphorylation events increase, rather than decrease, PARP1 activity. Extracellular signal-regulated kinase (ERK) can bind to PARP1 and phosphorylate Ser-372 and Thr-373 to enhance PARP1 activity ,46. In tNoticeably, statins (known to activate AMPK)  and otheS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig(PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 Fig(PDF)Click here for additional data file."}
+{"text": "Ecological momentary assessment (EMA) of mental health symptoms may influence the symptoms that it measures, i.e. assessment reactivity. In the field of depression, EMA reactivity has received little attention. We aim to investigate whether EMA of depressive symptoms induces assessment reactivity. Reactivity will be operationalised as an effect of EMA on depressive symptoms measured by a retrospective questionnaire, and, secondly, as a change in response rate and variance of the EMA ratings.This study is a 12-week randomised controlled trial comprising three groups: group 1 carries out EMA of mood and completes a retrospective questionnaire, group 2 carries out EMA of how energetic they feel and completes a retrospective questionnaire, group 3 is the control group, which completes only the retrospective questionnaire. The retrospective questionnaire  assesses depressive symptoms and is administered at baseline, 6\u00a0weeks after baseline and 12\u00a0weeks after baseline. We aim to recruit 160 participants who experience mild to moderate depressive symptoms, defined as a Patient Health Questionnaire (PHQ-9) score of 5 to 15. This study is powered to detect a small between-groups effect, where no clinically relevant effect is defined as the effect size margin \u22120.25<\u2009d\u2009<0.25.To our knowledge, this is the first study to investigate whether self-rated EMA of depressive symptoms could induce assessment reactivity among mildly depressed individuals.http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=5803.Netherlands Trial Register NTR5803. Registered 12 April 2016.  Daily, repeated measurements of mental health symptoms, also known as experience sampling or ecological momentary assessment (EMA), enables clinicians, researchers and patients to monitor psychological processes in real time. EMA is usually operationalised as active monitoring, in which patients respond to prompted or self-initiated items or questions . EMA invActive EMA may not only measure, but also influence mental health symptoms, which is known as assessment reactivity. Studies on alcohol abuse interventions show that repeated assessments draw attention to the monitored behaviour, which can identify problematic behaviour and highlight personal responsibility . In thatIn the field of depression, EMA reactivity has received little attention. Kramer et al.  found thThis study aims to investigate whether EMA of depressive symptoms induces assessment reactivity. First, we will investigate whether EMA of depressive symptoms during a 12-week period has an effect on depressive symptoms measured by a retrospective questionnaire. Secondly, we will investigate whether response fatigue affects the EMA ratings. Response fatigue will be operationalised as response rate over time and correlations with measures of associated constructs. To minimise response burden, participants will monitor only one symptom and 1 to 3 times a day. Because depression is a multidimensional construct  we will This study is a 12-week randomised controlled trial among participants who experience mild to moderate depressive symptoms. The trial consists of three groups: group 1 carries out EMA of mood and completes a retrospective questionnaire; group 2 carries out EMA of how energetic they feel and completes a retrospective questionnaire; group 3 is the control group, which completes only the retrospective questionnaire. The retrospective questionnaire is the Centre for Epidemiologic Studies Depression scale (CES-D) , which aWe aim to recruit 160 adult (18+) participants who experience mild to moderate depressive symptoms among college students and users of mental health websites. We define mild to moderate depressive symptoms as a PHQ-9 score of 5 to 15 [Participants in group 1 and 2 install the MoodMonitor application on their smartphones, which conducts EMA of mood and energy level. This app has been developed by the E-Compared consortium . Every dwww.moodmonitor.nu) that contains more detailed information. Interested individuals can apply to participate in the study via this website by completing the screening questionnaire , after which they can read the study information again and can agree to participate by entering a valid email address (electronic informed consent). Applicants who do not meet the inclusion criteria will be notified instantly. If they are excluded because they score above 15 on the PHQ-9, they are advised to contact their general practitioner. Participants who meet the inclusion criteria will be randomised equally to the three groups, i.e. 1:1:1. An independent researcher performs the allocation using a computerised random number generator. Next, participants are sent a link to the baseline questionnaire , and participants in groups 1 and 2 receive an email with instructions to download and install the MoodMonitor app on their smartphones. Even though we do not notify participants directly as to which group they are randomised, they can find out easily by reading through the study information on the website, and therefore participants cannot be considered to be blinded. After week 6 and after week 12 participants in all three groups are sent an email containing a link to the questionnaires . Participants who complete a measurement are offered 7.50 Euro, i.e. 22.50 for all three measurements, and an additional 10 Euro if they respond to 80\u00a0% or more of the EMA prompts. Participants will receive this incentive in the form of an electronic gift voucher sent to their email address. We will continue recruitment until 160 applicants have been randomised. Data collection runs from April 2016 to November 2016. This trial\u2019s results will be published on the study homepage when the scientific papers concerning the main objectives have been published.We employ three recruitment strategies. First, we distribute flyers on the university campus. Second, we post advertisements on Dutch websites for mental health issues. Third, advertisements are posted on Facebook and Twitter. The flyers and advertisements specifically target people who experience low mood or mood fluctuations and direct them to a website  , which wThe primary outcome measure of this study is retrospectively measured depressive symptoms. We will use the Centre for Epidemiologic Studies Depression scale (CES-D) , 19 whicSeveral demographic variables  will be gathered at baseline to determine the characteristics of the sample. We will also ask the participants how often and for which purposes they use their smartphones. At all measurement points participants are also asked whether they receive any professional help for mental health related problems, because that could influence their depressive symptoms.System Usability Scale (SUS) [The final questionnaire at week 12 contains the le (SUS) , 22 to eAt the end of the study, we will randomly select ten participants in the EMA groups for a semi-structured interview by telephone to obtain qualitative information about: 1) their experience with tracking their mood and energy level; 2) their experience with the app and how it could be improved; 3) participation in this study in general  are also not accessible by the researchers until data gathering has ended. The independent data manager monitors the data flow. There is no (other) data monitoring committee, due to the minimal burden and risk associated with participating in this study. After the publication of this study\u2019s main results, the data obtained by this study will become available on request. Requests should be sent to research@ggzingeest.nl with the topic name MoodMonitor.The primary outcome is the effect of EMA on depressive symptoms as measured by the CES-D at T6 and T12, comparing both EMA groups with the control group. To test this on all available data, we will conduct a mixed models repeated measures regression analysis, with T6 and T12 CES-D data as the dependent variable, and baseline CES-D scores, group , time  and the time*group interaction as independent variables. Effects will be expressed in terms of percentage of variance explained and Cohen\u2019s d .r) with the CES-D scores if the CES-D scores do not decrease. This might indicate a decline in validity.For our secondary analysis, we will examine the EMA response rate over time, which gives an indication of response fatigue. Response fatigue will also be analysed by assessing response accuracy, here defined as a declining correlation over time between theoretically associated measures. Literature has shown that mood swings are associated with higher depressive symptoms . Therefop-value of .05 using the Holm-Bonferroni method [We will maintain a family-wise two-tailed i method . IBM SPSi method  and R [2i method  will be r\u2009=\u2009.65) and variances of the differences between groups (non-sphericity correction epsilon\u2009=\u2009.8). Expecting 25\u00a0% drop-out at T12, we will recruit 160 participants. All participants will be included in the linear mixed model, which is robust for missing data.For the sample size calculation, we assume that a difference between either EMA group and the control group within the margin \u22120.25<\u2009Cohen\u2019s d\u2009<0.25 is clinically negligible. Therefore, a difference between groups of d\u2009=\u20090.25 should reach statistical significance in our analysis. We ran a power calculation in G*Power  for a reThis study aims to investigate whether self-rated EMA of depressive symptoms could induce assessment reactivity among mildly depressed individuals. The linear mixed model analyses can show an effect of Cohen\u2019s d\u2009>0.25 (and\u2009<\u2009\u22120.25) on retrospectively measured depressive symptoms between groups. The 12-week study period and repeated measures design enable us to detect both short-term (6\u00a0weeks) and longer-term (12\u00a0weeks) effects on retrospectively measured depressive symptoms, as well as response fatigue.This study will give a first indication of EMA reactivity on depressive symptoms. An effect of EMA on depressive symptoms would limit EMA as an instrument, because a change in EMA ratings might be attributed to the EMA itself instead of treatment or other factors. This is of immediate interest to research projects in which participants carry out EMA over longer periods, such as the E-COMPARED project . HoweverFirst, a change in retrospectively measured depressive symptoms could be attributed not only to a change in depression severity, but also to a change in response behaviour. If we find an effect, we can explore changes in response behaviour by testing the CES-D outcomes for measurement invariance . Second,To our knowledge, this is the first study to investigate whether self-rated EMA of depressive symptoms could induce assessment reactivity among mildly depressed individuals."}
+{"text": "The pathogenic nature of the variant was confirmed using experimental validation of the effect on mRNA splicing and IL7 pathway function. This case reinforces the need to use additional experimental methods to establish the functional impact of specific mutations, in particular for cases such as SCID where prompt diagnosis can greatly impact on diagnosis, treatment, and survival.Reported synonymous substitutions are generally non-pathogenic, and rare pathogenic synonymous variants may be disregarded unless there is a high index of suspicion. In a case of IL7 receptor deficiency severe combined immunodeficiency (SCID), the relevance of a non-reported synonymous variant was only suspected through the use of additional  This mutation was inherited from his healthy father, but its absence from various mutation repositories  indicates that it is a rare polymorphism. The CADD tool (http://cadd.gs.washington.edu/score), which was developed to evaluate the deleteriousness of various mutation types, indicated a low potential impact of this mutation . However, we also evaluated the potential functional impact of this mutation by evaluating its influence on gene splicing with computational tools developed specifically for this purpose  gel revealed the presence of two bands . He was admitted with a history of high fever, diarrhea, and oral thrush Figure A. PhysicID Table . First, 3) requiring Rituximab, Klebsiella oxytoca, and Enterobacter cloacae sepsis that needed vasoactive drugs and pediatric intensive care unit admission. Immunological reconstitution of the patient was deficient in the T-cell compartment due to reduced recent thymic emigrants, decreased naive CD4 and CD8 T-cells with a senescent T-cell phenotype, reduced CD127 expression, and impaired proliferation to phytohemagglutinin (PHA) stimulation  was made. The patient received HSCT from an unrelated HLA identical donor at the age of 10\u2009months. The conditioning regimen included ATG, fludarabine, and melphalan. He achieved full donor chimerism 2\u2009months after HSCT . The patient is currently 4\u2009years old and although successfully rescued from SCID by HSCT, he has suffered severe complications: hepatic graft-versus-host disease (GvHD), severe malnutrition, and diarrhea caused by norovirus infection, respiratory infections caused by RSV and EBV, steroid-resistant chronic autoimmune thrombocytopenia .2, then the wells were pulsed individually with 1\u2009\u03bcCi of [3H]-thymidine and incubated during the last 18\u2009h. The amount of radioactivity was measured in a scintillation counter with results expressed as counts per minute.Heparinized whole blood from the patients were diluted in complete medium and cultured with the following mitogens: PHA , anti-CD3 antibody, and anti-CD3\u2009+\u2009anti-CD28 antibodies . The samples were cultured in triplicate wells for 3\u2009days at 37\u00b0C in a humidified incubator containing 5% COTotal serum immunoglobulins  were measured by nephelometry .IL7R gene, and their flanking regions were carried out using specific primers (Primers available upon request). Purified PCR products were sequenced using an ABI PRISM 3130 genetic analyzer.Genomic DNA was extracted from EDTA whole blood using a MagNa Pure Compact Nucleic Acid Isolation Kit . PCR to amplify the eight exons of IL7R gene expression quantification was measured by qRT-PCR using a TaqMan probe in a LightCycler 480 instrument (Roche) according to the manufacturer\u2019s protocol. GADPH was used as the endogenous control, and the level of expression of IL7R was quantitatively measured relative to that in 10 different donors.Total RNA was isolated with the RNeasy plus mini kit . One microgram of total cellular RNA was reverse transcribed using a Transcriptor First Strand cDNA Synthesis Kit (Roche). \u2212B+NK+ SCID phenotype is frequently caused by defects in the \u03b1 chain of the interleukin-7 receptor   . More ths CD127) .IL7R gene that results in abnormal splicing. We note that, as expected for a fourfold degenerate site, the site of this mutation appears to have evolved essentially neutrally in the mammalian lineage as suggested by low, negative phyloP, and GERP++ scores . However, the phyloP score computed for the primate lineage (0.505) indicates a high level of conservation of this site in primates, which are the only species that have a GT dinucleotide (specific for a donor splice site) preceding it. Therefore, it is tempting to speculate that conservation of this site in primates was maintained to avoid activation of the cryptic donor splice site in a highly conserved exon (both donor and acceptor consensus dinucleotides are perfectly conserved throughout vertebrates). However, half of all 18 fourfold degenerate sites in exon 3 exhibit similar levels of high conservation in primates, indicating that other mechanisms might be at play concomitantly. This aspect is reinforced by a T>C transition reported at this site (rs199641706) but which is not predicted to activate the preceding cryptic splice site in a manner similar to c.333T>A , inherited from his healthy father, activates a cryptic exonic donor splice site that leads to exon truncation (r.330del49) and premature stop codon. It added to a symptomatic missense mutation  inherited from his healthy mother and together render both IL7R copies non-functional. This case reinforces the need for researchers to functionally evaluate all mutations found in patients. The advent of next-generation sequencing (NGS) provides to researchers with increased genomic coverage, which in turn requires increased efforts for mutation screening. The ever-increasing panel of ever-improving computational tools can facilitate such efforts of functional prioritization, especially in the case of synonymous mutations. Nonetheless, detailed functional reports of specific mutations in patients, such as our report of a causal synonymous mutation in a SCID patient, are ultimately necessary to provide a better understanding of functional impact within the context of specific disease phenotypes.We report the first case of an FG-B performed the laboratory work for this study, computational predictions, and drafted the manuscript. VG performed computational predictions and drafted the manuscript. JR-A, RR-P, JG-H, AS, and LG-G were responsible for the clinical management of the patients. AD, GR, and LE collaborated in computational predictions. LG-G drafted the manuscript. LA designed the research, collaborated in computational predictions, and drafted 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."}
+{"text": "Yersinia pseudotuberculosis; however, the role of CD4+ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4+ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3+ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within na\u00efve CD4+ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of na\u00efve CD4+ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3+ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4+ T cell subsets by altering their TCR downstream signaling.Adaptive immunity critically contributes to control acute infection with enteropathogenic The online version of this article (doi:10.1007/s00018-017-2516-y) contains supplementary material, which is available to authorized users. Yersinia pseudotuberculosis is known to initially infect the terminal ileum and Peyer\u2019s patches, followed by an entering of mesenteric lymph nodes (mLNs). Infections with Y. pseudotuberculosis frequently result in the development of diarrhea, gastroenteritis, and mesenteric lymphadenitis [Yersiniae carry a broad range of virulence factors allowing interaction with immune cells and/or mediating immune evasion. Among others, they encode a type III secretion system (T3SS) on the pYV virulence plasmid, which enables translocation of effector proteins  through a needle-like structure, referred to as injectisome [The intestinal immune system requires an extremely tight control as it is constantly exposed to high loads of harmless foreign antigens such as microbiota and food, while at the same time it has to be ready to mount rapid and efficient immune responses against invading pathogens. Among these pathogens, enteropathogenic adenitis , 2. Yers system TSS on theectisome , 5. Receectisome .Y. pseudotuberculosis, involving neutrophils, macrophages, dendritic cells (DCs), and natural killer cells [Yersiniae is only incompletely understood. Besides studies underpinning the importance of CD8+ T cells in control of Yersinia infection [+ T helper cell responses. These studies suggest the involvement of IFN\u03b3-producing proinflammatory Th1 cells in protection against Yersinia [+ T cells in responding to Y. pseudotuberculosis superantigens in an MHCII-dependent manner [+ regulatory T cells (Tregs) towards IL-17-producing proinflammatory Th17 cells has been reported for several enteropathogenic infections [Yersiniae can directly modulate differentiation of CD4+ T cells, thereby favoring the establishment of infection [While innate immunity represents a well-characterized part of the immune response against er cells \u201310, the nfection , 12, theYersinia , and rept manner . A shiftfections \u201318. Howenfection , 19.Y. pseudotuberculosis directly interacts with CD4+ T cells during the acute phase of infection and exemplify an involvement of Th17 cells and Tregs in the pathomechanism of disease. Using both de novo and in vitro T cell differentiation assays, we could demonstrate that T3SS-dependent modulation of T cells by Y. pseudotuberculosis results in a strongly impaired induction of Foxp3+ Tregs, while differentiation towards Th17 cells is highly supported. This immunological skewing of T cell differentiation is potentially mediated through the direct modulation of T cell receptor (TCR) downstream signaling pathways by the pathogen.Here, we show that hCD2 \u00d7 Rag2\u2212/\u2212xDO11.10 (BALB/c), Foxp3hCD2 \u00d7 CD90.1 (BALB/c), and Foxp3hCD2 (BALB/c) mice were bred and housed under specific pathogen-free conditions at the Helmholtz Centre for Infection Research . BALB/c mice were purchased from Janvier. Gender- and age-matched mice were used in all experiments. Mice were housed and handled in accordance with recommendations of FELASA and the national animal welfare body GV-SOLAS guidelines. Experimental protocols were approved by the Lower Saxony Committee on the Ethics of Animal Experiments as well as the responsible state office (Lower Saxony State Office of Consumer Protection and Food Safety) under permit number 33.9-42502-04-13/1240.Foxp3Fluorochrome-conjugated anti-B220 (RA3-6B2), anti-hCD2 (RPA-2.10), anti-CD3 (17A2), anti-CD4 (RM4-5), anti-CD8 (53-7.3), anti-CD11b (M1/70), anti-CD11c (N418), anti-CD19 (6D5), anti-CD25 (PC61.5), anti-CD49b (DX5), anti-CD86 (GL1), anti-CD103 (2E7), anti-F4/80 (BM8), anti-Foxp3 (FJK-16S), anti-IFN\u03b3 (XMG1.2), anti-IL-10 (JES5-16E3), anti-IL-17 (TC11-18H10), anti-Ly6G (1A8), anti-MHCII (M5/114.15.2), anti-pERK1/2 (20\u00a0A), anti-ROR\u03b3t (AFKJS-9), and anti-Ova-TCR (KJ1-26) antibodies were purchased from BioLegend, eBioscience, and BD. Intracellular Foxp3/ROR\u03b3t and pERK1/2 stainings were performed according to the manufacturer\u2019s instructions . To determine the absolute number of living cells prior to flow cytometry analysis, propidium iodide (Sigma-Aldrich) was added, and cell number was determined using Accuri C6 Cytometer (BD). Dead cells were excluded based on the staining with the LIVE/DEAD Fixable Blue Dead Cell Stain (Thermo Fisher Scientific) and scatter properties. Cells were analyzed on LSRFortessa (BD) with Diva software v8.0.1 (BD), and data analysis was performed with FlowJo software v9.9.3 (TreeStar).Y. pseudotuberculosis strain (Yptb-WT) [Yersiniae strains were grown at 25\u2009\u00b0C in Luria\u2013Bertani broth medium (BD), washed, and diluted in PBS prior to infection. For in vitro co-culture experiments, bacteria were diluted 1:50 after overnight incubation, followed by incubation for 2\u00a0h at 25\u2009\u00b0C and additional cultivation for 3\u00a0h at 37\u2009\u00b0C. 50\u00a0\u03bcg/ml kanamycin (Sigma-Aldrich) was used for bacterial selection.The YPIII wild-type Yptb-WT)  carryingYptb-WT) . Overnig8 Yptb-WT using a ball-tipped gavaging needle. 2\u00a0days p.i., the frequency of neutrophils in the peripheral blood of infected and non-infected mice was analyzed by flow cytometry, and significantly increased mobilization of neutrophils into peripheral blood of infected mice was taken as an indicator of a successful infection (data not shown). In general, body-weight loss and signs of severe illness of mice infected with 2\u2009\u00d7\u2009108 Yptb-WT peaked at day 5\u20136. At indicated time points p.i., infected mice were analyzed or subjected to further experimental procedures.Female BALB/c mice (Janvier) aged between 6 and 7\u00a0weeks were subjected to fasting for 16\u00a0h prior to infection. Subsequently, mice were orally infected with 2\u2009\u00d7\u200910hCD2xRag2\u2212/\u2212xDO11.10 mice. Before transfer, cells were labeled with the proliferation dye Cell Trace\u2122 Violet , and 4\u2009\u00d7\u2009106 cells were injected in 100\u00a0\u00b5l PBS i.v. per recipient mouse. For induction of T cell differentiation, 20\u00a0\u00b5g Ova323\u2013339 peptide was injected i.v. on two consecutive days, starting 1\u00a0day after adoptive T cell transfer. At day 3 after the first antigen application, cells were isolated from mLNs and stained for flow cytometric analysis. Intracellular cytokine staining was performed after restimulation with phorbol 12-myristate 13-acetate (PMA)/ionomycin for 2\u00a0h  and with 10\u00a0\u03bcg/ml Brefeldin A for additional 2\u00a0h at 37\u2009\u00b0C , followed by fixation and standard staining of surface markers.For adoptive transfer, single cell suspensions were generated from spleens and LNs of Foxp3+ T cells were enriched from spleens and LNs of BALB/c mice using CD4 (L3T4) MicroBeads and autoMACS separation (Miltenyi Biotec). CD4+ T cells were co-cultured with Yptb-WT for 1\u00a0h at MOI (multiplicity of infection) 100, followed by washing. Fixation was performed by 1\u00a0h incubation and washing with cacodylat buffer. Cells were placed on poly-l-lysine-coated cover slips, followed by fixation with 3% glutaraldehyde and washed with TE-buffer , dehydrated with a graded series of acetone , and critical-point dried. After sputter coating with a gold film (appr. 10\u00a0nm), samples were analyzed using a Zeiss DSM 982 Gemini FESEM.Total CD49 Yptb-WT-Bla. At day 3, single cell suspensions of mLNs were stained for cell surface markers and subsequently labeled with CCF4-AM, using the LiveBLAzer-FRET B/G Loading Kit (Thermo Fisher Scientific) for 1\u00a0h at room temperature in the presence of 1.5\u00a0mM probenecid (Sigma-Aldrich) and 50\u00a0\u03bcg/ml gentamicin (Sigma-Aldrich). To study Yop translocation in vitro, na\u00efve CD4+ T cells were isolated from spleen and LNs of Foxp3hCD2 mice. Briefly, cells were stained with anti-CD25-APC and anti-hCD2-APC, followed by a depletion of APC+ cells using anti-APC MicroBeads (Miltenyi Biotec) and autoMACS separation system. Subsequently, CD4+ T cells were magnetically sorted as described before. The resulting na\u00efve Foxp3hCD2\u2212CD62LhiCD44loCD25\u2212 CD4+ T cells were co-cultured with Yptb-WT-Bla or \u0394T3SS-Bla at an MOI of 10 for 1\u00a0h at 37\u2009\u00b0C, washed twice with RPMI supplemented with 50\u00a0\u03bcg/ml gentamicin to eliminate bacteria. Subsequently, 2\u2009\u00d7\u2009106 cells were labeled with CCF4-AM and analyzed by flow cytometry.For in vivo analysis of Yop translocation, BALB/c mice were infected intragastrically with 2\u2009\u00d7\u200910+ T cells were enriched from spleens and LNs of Foxp3hCD2 mice via autoMACS separation. MACS-separated CD4+ T cells were co-cultured with Yptb-WT-Bla and \u0394T3SS-Bla strains at MOI of 50 for 1\u00a0h at 37\u2009\u00b0C and washed twice with gentamicin-containing RPMI. Subsequently, cells were stained for CD4 and Foxp3hCD2, loaded with 4\u00a0\u03bcg/ml Indo-1 AM cell permanent dye (Thermo Fisher Scientific) at 37\u2009\u00b0C for 45\u00a0min in dark, followed by pre-decoration with 18\u00a0\u03bcg/ml anti-CD3-Biotin and 1\u00a0\u03bcg/ml anti-CD28-Biotin (both from BD). TCR crosslinking was induced by the addition of 40\u00a0\u03bcg/ml Streptavidin (Dianova) to the pre-warmed cell suspension. Ionomycin at 4\u00a0\u03bcg/ml concentration served as positive control providing maximum Ca2+ influx. The Ca2+ signal was measured in the respective gates of Tregs and na\u00efve T cells by flow cytometry.Total CD4+ T cells were enriched from spleens and LNs of Foxp3hCD2 mice by autoMACS separation. MACS-separated CD4+ T cells were co-cultured with Yptb-WT-Bla and \u0394T3SS-Bla strains at MOI of 50 for 1\u00a0h at 37\u2009\u00b0C and washed twice with gentamicin-containing RPMI. Subsequently, cells were first stained with LIVE/DEAD Fixable Blue Dead Cell Stain and anti-CD4 antibody, followed by decoration with 10\u00a0\u03bcg/ml anti-CD3-Biotin and 5\u00a0\u03bcg/ml anti-CD28-Biotin antibodies for 15\u00a0min on ice. Crosslinking was induced by the addition of 10\u00a0\u03bcg/ml Streptavidin to the pre-warmed cell suspensions. At indicated time points, cells were fixed and permeabilized, followed by intracellular staining with anti-pERK1/2 and anti-Foxp3 antibodies overnight at 4\u2009\u00b0C. Next, ERK phosphorylation was determined in the respective gates of live CD4+Foxp3 na\u00efve T cells and CD4+Foxp3+ Tregs.Total CD4+ T cells were isolated from spleen and LNs of Foxp3hCD2 mice as described before. The resulting na\u00efve Foxp3hCD2\u2212CD62LhiCD44loCD25\u2212 CD4+ T cells were co-cultured with Yptb-WT-Bla and \u0394T3SS-Bla at an MOI of 50 for 1\u00a0h at 37\u2009\u00b0C. After the removal of bacteria by washing with gentamicin-containing RPMI, cells were cultured under Th0, Th1, Th17, or Treg-polarizing conditions. For Treg cultures, 5\u2009\u00d7\u2009105 cells/well were cultured on 96-well round-bottom plates in RPMI supplemented with 10\u00a0ng/ml IL-2 (R&D), 5\u00a0ng/ml TGF-\u03b21 (R&D), 50\u00a0\u03bcg/ml gentamicin, and anti-CD3/CD28 Dynabeads Mouse T Activator (Thermo Fisher Scientific) at 1:1 ratio. Frequency of Foxp3hCD2+ cells was determined 4\u00a0days later. For Th17 culture conditions, 2\u2009\u00d7\u2009106 cells/well were cultured in 24-well plates coated with 3\u00a0\u03bcg/ml anti-CD3 (BioLegend) and 5\u00a0\u03bcg/ml anti-CD28 (eBioscience) in IMDM supplemented with 2\u00a0ng/ml TGF-\u03b21, 30\u00a0ng/ml IL-6, 10\u00a0ng/ml IL-1\u03b2, 5\u00a0\u03bcg/ml anti-IL-2, 20\u00a0ng/ml TNF-\u03b1 , 10\u00a0\u03bcg/ml anti-IFN-\u03b3 (BioXCell), and 50\u00a0\u03bcg/ml gentamicin for 4\u00a0days. Then, cells were replated in fresh medium and cultured without TCR stimulation for two additional days. On day 6, frequency of IL-17+ cells was determined after restimulation and fixation as described before. For Th0 and Th1 cultures conditions, 2\u2009\u00d7\u2009106 cells/well were cultured in 24-well plates coated with 2\u00a0\u03bcg/ml anti-CD3 and anti-CD28 in IMDM containing 50\u00a0\u03bcg/ml gentamicin. The Th1 culture medium was supplemented with 20\u00a0ng/ml IL-12 (PeproTech) and 10\u00a0\u03bcg/ml anti-IL-4 (BioLegend), whereas cells cultured under Th0 conditions did not receive any cytokines or neutralizing antibodies. Cells were replated in fresh medium on day 2 and cultured without TCR stimulation for three additional days. On day 5, frequency of IFN-\u03b3+ cells was determined after restimulation and fixation, as described before.Na\u00efve CD4p values\u2009<\u20090.05 are considered as significant . Prism software  was applied for all statistical analyses and graphs.Group sizes were estimated according to a presumed standard deviation (SD) and an expected type I error of <0.05. The sample size was adjusted, if required, based on initial results. For all figures, each data point represents a single mouse if not stated otherwise. For comparison of unmatched groups, two-tailed Mann\u2013Whitney statistical test was applied. The comparison of more than two groups was performed by one-way ANOVA followed by Bonferroni\u2019s post-test. All data are presented as mean or mean\u2009\u00b1\u2009SD, and + Tregs and proinflammatory Th17 cells in combatting acute Yersiniae infections is only incompletely understood [Y. pseudotuberculosis has an effect on the peripheral de novo generation of these two opposing T cell subsets. Since gut-draining mLNs are among the first target organs of Y. pseudotuberculosis, we decided to focus on T cell differentiation events taking place within mLNs. Thereto, BALB/c mice were infected sublethally with wild-type Y. pseudotuberculosis (Yptb-WT). At day 2 post infection (p.i.), shortly before the infection reaches its peak, TCR-transgenic, ovalbumin (Ova)-specific na\u00efve Foxp3\u2212CD4+ T cells were labeled with a cell proliferation dye and adoptively transferred into infected mice, while uninfected recipient mice served as controls . In contrast to this increase in IL-17-producing T cells, the frequency of IFN-\u03b3- and IL-10-producing endogenous CD4+ T cells remained unchanged during the first 12\u00a0days p.i. , further supporting the important contribution of Th17 cells to the control of Y. pseudotuberculosis infection [Y. pseudotuberculosis infection shifts the immunological balance from immunoregulatory towards proinflammatory T cells, thereby favoring the establishment of a protective, inflammatory environment in the intestine.The role of immunoregulatory Foxp3derstood , 19. Thueviously . At day eviously , a high nfection . Togethe+ Tregs [Y. pseudotuberculosis, and might contribute to the observed shift from Tregs to Th17 cells. At day 5 p.i., the overall frequency of MHCIIhiCD11chi conventional DCs (cDCs) was strongly reduced in mLNs of mice infected with Yptb-WT . However, absolute numbers of cDCs within mLNs did not change significantly when comparing infected mice to uninfected controls . Nevertheless, we could observe a substantial alteration among specialized subsets within the cDC compartment. Tolerogenic CD103+CD8+ cDCs, known to be involved in de novo Treg induction [\u2212CD8\u2212 cDCs, which have been described to be responsible for priming of Th1 and/or Th17 cells [\u2212CD8\u2212 cDCs displayed a strong increase in CD86 expression upon infection with Yptb-WT . To monitor in vivo Yop translocation and to identify CD4+ T cell subsets being modulated by Y. pseudotuberculosis, BALB/c mice were infected sublethally with Yptb-WT-Bla. At day 3 p.i., the frequency of modulated cells among na\u00efve CD4+ T cells and CD25hiCD4+ Tregs from mLNs was analyzed by flow cytometry. The \u03b2-lactamase reporter assay revealed that the frequency of in vivo-modulated Tregs, albeit being at a low level, was significantly higher when compared to na\u00efve CD4+ T cells  revealed that the peak of ERK phosphorylation occurred after 1.5\u00a0min (data not shown), and this time point was chosen for all further analyses. In accordance with published data [+ T cells showed a higher frequency of pERK+ cells as compared to unmodulated Foxp3+ Tregs, while both cell types showed strong pERK activation upon stimulation with PMA and ionomycin -free system under polarizing conditions. Stimulation of Yptb-WT-Bla-modulated na\u00efve CD4+ T cells under Th17-polarizing conditions resulted in a significantly enhanced frequency of IL-17+ cells at day 6 of the culture when compared to differentiation of unmodulated na\u00efve CD4+ T cells .The opposite was observed when Yptb-WT-Bla-modulated na\u00efve CD4lls Fig.\u00a0b. Also tBla Fig.\u00a0b. Import+ T cells alters their differentiation potential, resulting in a skewing from suppressive Foxp3+ Tregs to proinflammatory Th17 cells.Together, these in vitro data suggest that the direct T3SS-dependent modulation of TCR downstream signaling within na\u00efve CD4+ T cells to change their phenotype and to acquire special functional properties might be critical during acute infection with enteropathogenic Y. pseudotuberculosis. Here, we report that Yersiniae selectively disrupt the balance between Tregs and Th17 cells, towards an increased differentiation of proinflammatory Th17 cells and a reduction in the generation of immunosuppressive Tregs, whereas the frequency of IFN-\u03b3-producing Th1 cells or IL-10-producing T cells remained largely unaltered. The Yersinia-mediated support of proinflammatory responses is surprising as pathogenic bacteria usually promote Treg expansion, enabling their long-term survival in the host and establishment of chronic infection [Yersiniae rather impair efficient de novo Treg induction within gut-draining mLNs, potentially favoring systemic dissemination of the pathogen [Y. pseudotuberculosis, similar to changes instigated by Citrobacter rodentium and Salmonella infections [Yersiniae disrupt the Th17-Treg balance, which might be a critical strategy of the pathogen in establishing acute infection.In the intestine, the balance between effector and regulatory pathways needs to be tightly controlled in order to maintain immune homeostasis and efficiently combat infections. Therefore, the ability of CD4nfection , 32. Howfections , 34. On fections , but ratYersiniae fine-tune the CD4+ T cell compartment, we characterized DC subsets within mLNs during acute infection. It had been shown before that inflammatory conditions within the gut not only alter the distribution of DCs [+ subset [+ Tregs [Y. pseudotuberculosis indirectly affects T cell differentiation via modulation of DC subsets within mLNs. Indeed, the strongly decreased frequency of tolerogenic CD103+CD8+ DCs could account for reduced Treg induction [\u2212CD8+ DCs, which most probably belong to resident DCs [Y. pseudotuberculosis only resulted in a decreased frequency of CD103+CD11b+ DCs, without affecting any other DC subsets [+ and CD103\u2212 DCs were altered by enteropathogenic Yersiniae, and most likely these changes jointly contribute to the establishment of a strong inflammatory response. Moreover, increased CD86 expression levels of the latter DC subset demonstrate their highly activated status and suggest the possible involvement of Toll-like receptors (TLRs) in creating a shift towards Th17-dominated immune responses [Y. pseudotuberculosis contribute to the increased Th17 differentiation.To better understand how n of DCs , 37, but+ subset , which i[+ Tregs , 26. Thenduction , 24, 38,dent DCs  and for dent DCs , are alsdent DCs . Interes subsets . This isesponses \u201343. HoweYersiniae cannot only affect the DC compartment. Using the \u03b2-lactamase reporter system, we could demonstrate that upon infection via the natural route Yersiniae can directly modulate CD4+ T cells within infected mLNs, extending previous findings from a systemic infection model [+ T cell population, Tregs were preferentially modulated when compared to na\u00efve T cells. The increased translocation rate of Yops into Tregs can be explained by the binding of Y. pseudotuberculosis invasin to \u03b21 integrin VLA4 [Nonetheless, on model . Interesrin VLA4 , which irin VLA4 .Yersiniae-dependent Treg modulation and alteration of na\u00efve T cell differentiation, we observed a stark decline of Ca2+ mobilization after TCR triggering for na\u00efve CD4+ T cells, while Ca2+ signaling within Foxp3+ Tregs was even fully abrogated. In line with reduced Ca2+ mobilization, phosphorylation of ERK was strongly abolished in both na\u00efve CD4+ T cells and Foxp3+ Tregs. Since \u0394T3SS-Bla affected neither Ca2+ signaling nor ERK phosphorylation within both cell types, our findings indicate that direct modulation of T cells requires the presence of the T3SS. The importance of Yop translocation was also suggested from results of the APC-independent, in vitro T cell differentiation assay, where increased frequencies of Th17 cells and reduced de novo Treg induction were observed only upon co-culture of na\u00efve T cells with Yptb-WT and not with T3SS-deficient Yersiniae. Among Yops, the tyrosine phosphatase YopH is known to interfere with early T cell receptor signaling events, either via direct phosphorylation of signaling molecules [2+ mobilization and ERK phosphorylation we observed in CD4+ T cell subsets being modulated by Y. pseudotuberculosis. Moreover, ERK activity in Jurkat T cells had been restored by addition of a YopH inhibitor [+ T cells and Foxp3+ Tregs. Additionally, the interference of YopH with TCR signaling could also lead to reduced secretion of IL2 [Y. pseudotuberculosismediated alterations in IL-2 production also contribute to the skewing from Tregs to Th17 cells, and the lower frequency of de novo-induced Tregs might enable enhanced generation of Th17 cells. Yet, whether YopH is the master regulator of Y. pseudotuberculosis-mediated modulation of T cell differentiation or other mechanisms contribute to the fine-tuning of the closely related transcriptional regulation of Tregs and Th17 cells [+ T cells with Yptb-WT-Bla did only affect expression of the cytokine IL-17 in Th17-polarizing cultures, but not the expression of the Th17 lineage specification factor ROR\u03b3t, although under Treg-inducing conditions a severe impact on the expression of the Treg lineage specification factor Foxp3 could be observed.When dissecting the mechanism of olecules \u201348, or bolecules . Thus, Ynhibitor , furthern of IL2 , 51. Sinn of IL2 , 53, it 17 cells , 55 rema+ T cell subsets in the pathomechanism of acute Y. pseudotuberculosis infection, showing that this enteropathogen favors the generation of Th17 cells, and in parallel leads to a decline in Treg induction. Our data provide evidence that Y. pseudotuberculosis interferes with TCR signaling in both Foxp3+ Tregs and na\u00efve CD4+ T cells, thereby directly modulating T cell-mediated immune responses. Efforts to understand the precise pathomechanism of gastric infections could permit the development of future therapeutics for an efficient modulation of the immune system.Taken together, the present study implicates a critical role of CD4Below is the link to the electronic supplementary material.8 Yptb-WT, and the frequency of cytokine-producing endogenous CD4+ T cells was analyzed at indicated time points p.i. by flow cytometry. Graphs depict frequencies of (a) IFN-\u03b3-, (b) IL-10- and (c) IL-17-producing CD4+CD3+ T cells in mLNs of mice infected with Yptb-WT. PBS-treated mice were taken as uninfected controls. Data are pooled from two independent experiments with 2\u20138 mice per group. *p\u2009<\u00a00.05. (EPS 614 KB)Supplementary material 1: Endogenous T helper cell subsets in mLNs of Yptb-WT-infected mice. BALB/c mice were infected with 2\u2009\u00d7\u200910-WT-infected mice. BALB/c mice were infected with 2\u2009\u00d7\u2009108 Yptb-WT, and at day 5 p.i. the frequency and absolute number of cDCs within mLNs was determined by flow cytometry. (a) Representative dot plots depict MHCIIhiCD11chi cDCs among Lin-(CD3\u2212CD19\u2212CD49b\u2212B220\u2212) cells in mLNs of uninfected control mice or in mLNs of mice infected with Yptb-WT. Numbers indicate frequencies of cells in gates. (b) Scatterplot summarizes frequencies of MHCIIhiCD11chi cDCs among Lin\u2212 cells within mLNs of indicated groups. Data are pooled from two independent experiments with 3\u20137 mice per group. ***p\u2009<\u00a00.001. (c) Scatterplot depicts absolute number of MHCIIhiCD11chi cDCs within mLNs of indicated groups. Data are pooled from two independent experiments with 3\u20137 mice per group. Ns, not significant. (EPS 747 KB)Supplementary Figure S2: Characterization of cDC compartment in mLNs of Yptb+ T cells with Y. pseudotuberculosis and culture under Th17 polarizing conditions does not affect ROR\u03b3t expression. Na\u00efve CD4+ T cells were enriched from secondary lymphoid organs of Foxp3hCD2 mice and co-cultured with Yptb-WT-Bla and \u0394T3SS-Bla for one hour, or were left unmodulated. Subsequently, modulated T cells were cultured under Th17-polarizing conditions for six days, and ROR\u03b3t expression was assessed by flow cytometry at the end of the cultures. Representative dot plots from two independent experiments show expression of ROR\u03b3t in cells from indicated cultures. Numbers indicate frequencies of cells in gates. (EPS 624 KB)Supplementary Figure S3: In vitro modulation of na\u00efve CD4+ T cells with Y. pseudotuberculosis does not affect IFN-\u03b3 production of cells cultured under Th0 or Th1 conditions. Na\u00efve CD4+ T cells were enriched from secondary lymphoid organs of Foxp3hCD2 mice and co-cultured with Yptb-WT-Bla for one hour, or were left unmodulated as control. Subsequently, modulated T cells were cultured under Th0-polarizing or Th1-inducing conditions for five days, and IFN-\u03b3 expression was assessed by flow cytometry. Scatterplot summarizes frequencies of IFN-\u03b3+ cells from indicated cultures. Data are pooled from two independent experiments, and means of technical replicates are depicted. Ns, not significant. (EPS 518 KB)Supplementary Figure S4: In vitro modulation of na\u00efve CD4"}
+{"text": "With at least 1-year follow up, a progression of renal deterioration was demonstrated in 29.70% of the control group and 25.09% of the study group. Patients in the study group had significantly reduced progression of CKD with adjusted odds ratio 0.79 . However, when ACEI monotherapy and ARB monotherapy were analyzed separately, none of their associations with CKD progression was statistically significant. In conclusion, ACEI or ARB monotherapy may retard the deterioration of renal function among patients with CKD and hypertension.It remains unclear how different uses of angiotensin-converting inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) influence the progression of chronic kidney disease (CKD). This study explored CKD progression in a multicentre, longitudinal cohort study that included 2639 patients with CKD stage 1\u20135 and hypertension. Patients treated with ACEI or ARB for \u226590 days during a 6-mo period comprised the study group, or no treatment, comprised the control group. The study group was subdivided on the basis of treatment: ACEI monotherapy or ARB monotherapy. Progression of renal deterioration was defined by an average eGFR decline of more than 5\u2009mL/min/1.73\u2009m Patients with CKD generally exhibit progressive deterioration in kidney function that concludes with end-stage renal disease (ESRD). Identifying effective measures to prevent and retard its progression is challenging but necessary4. For most types of renal diseases, effectively controlling blood pressure and minimizing proteinuria significantly attenuate kidney function deterioration. The MDRD Study 5 discovered that a reduction of proteinuria independently slowed the rate of GFR decline and that the renoprotective effect from lowering blood pressure depended on the level of proteinuria. Among antihypertensive agents, both angiotensin-converting enzyme (ACE) inhibitors (ACEI) and angiotensin II receptor blockers (ARBs) demonstrated a renoprotective effect attributable to both antihypertensive and antiproteinuric effects7. Further, these drugs interrupt the renal\u2013angiotensin\u2013aldosterone system (RAS)11, which plays a critical role in renal disease progression. Many clinical trials have demonstrated the value of ACEIs or ARBs for both patients with diabetes12 and those without13. Theoretically, the combination of an ACEI and an ARB might achieve a more complete inhibition of the RAS, and thereby achieve a stronger renoprotective effect. However, most published clinical trials and meta-analyses on combination therapy for renal protection have been inconclusive. A meta-analysis by Kunz et al. that examined 49 randomized trials (6181 patients) concluded that the combination of ACEIs and ARBs more effectively reduced proteinuria; however, most of the studies examined were small and did not provide details concerning adverse drug reactions14. Two recent clinical trials17 identified a decrease of albuminuria as a result of combination therapy with ACEIs and ARBs, but without slowing long-term renal deterioration. More adverse events, including acute kidney injury and hyperkalaemia, were associated with combination therapy17. We defined the progression of renal deterioration by an average eGFR decline of more than 5\u2009mL/min/1.73\u2009m2/yr or the commencement of dialysis. Given the uncertainties concerning the efficacy of ACEI or ARB treatment to slow the rapid progression of renal function, we conducted a study on a large multi-center cohort comprised of a Taiwanese population using the National Health Insurance Database in Taiwan, and examined the influence of ACEI monotherapy or ARB monotherapy on renal disease progression among patients with CKD and hypertension.Chronic kidney disease (CKD) is a highly prevalent and concerning public health issue in the Taiwanese populationAfter excluding patients without hypertension, with less than 1-year follow up, receiving dialysis or renal transplant before enrolment, receiving dialysis or renal transplant within the first six months of observational period, with missing risk factor data, 2639 patients with CKD and hypertension were enrolled in this study  of CKD progression in Table\u00a0We further analyzed the beneficial effect of ACEI-ARB in CKD stage 1\u20133a and CKD stage 3b-5 as noted in Tables\u00a0Regarding patients with glomerulonephritis and immunosuppresants prescription, we analyzed the beneficial effect of study group and control group. We defined patients who had used immunosuppressants for more than one month within one year prior to the recruitment of this study as immunosuppressants user. The adjusted ORs were 0.55  and 0.82  among patients with and without immunosuppressants, respectively. We did not analyze ACEI monotherapy and ARB monotherapy separately because of small sample size.Figure\u00a018. In contrast to definitions used in previous studies, we identified CKD progression events as either an annual average eGFR decline >5\u2009mL/min/1.73\u2009m2 or advancement to the dialysis stage.In this prospective cohort study, we investigated the relationship between ACEI or ARB therapy and the risk of eGFR decline in patients with CKD and hypertension. The multi-center cohort study enrolled patients with different stages of CKD to compare the influence of ACEI or ARB therapy and linked to the National Health Insurance database with corresponding dataAt baseline, our control group was older than the study group, and had other comorbid conditions including CAD, stroke, and cancer; the Charlson comorbidity index scores were not significantly different. Significantly more patients in the study group had DM and more ACEI or ARB use within the year before the index date compared with the control group Table\u00a0. Unexpec21, and whether to use ACEIs or ARBs clinically in patients with advanced CKD remains a debatable topic22. Studies have detected the Ang-II escape phenomenon24 and poor local Ang-II inhibition with ACEI monotherapy19.Both ACEIs or ARBs have been noted to have antihypertensive and antiproteinuria effects because of different RAS-inhibition mechanisms2 or advancement to the dialysis stage events was significantly lower among ACEI or ARB users than in the nonuser group (29.7% in the nonuser group vs. 25.09% in the user group)  trial, ramipril retarded eGFR decline and the risk of end-stage kidney disease in patients with CKD with proteinuria of >3\u2009g/day31. In the REIN-2 trial, no additional benefit was demonstrated from further BP reduction32. Further, Jafar et al. determined that the antiproteinuric effects of ACEIs are greater in patients with a high baseline urine protein excretion33. All of these studies indicated that ACEIs and ARBs are renoprotective independent of their antihypertensive effects.Consistent with other studies, the number of eGFR decline >5\u2009mL/min/1.73\u2009mWe further analyzed the beneficial effects of ACEI or ARB in CKD stage 1\u20133a and CKD stage 3b-5 as noted in Tables\u00a0We concluded that ACEI or ARB monotherapy is associated with a lower proportion of eGFR decline events compared with the nonuser group  to include biochemical data when analysing the results to ensure the quality of study outcomes. Detailed biochemistry data was available to define the stage and severity of CKD.However, some limitations were encountered when conducting this study. First, because patients voluntarily enrolled in the study, a potential selection bias was unavoidable. Second, variables of clinical disease were collected using a structured questionnaire, introducing the potential for underestimation of certain test results. Third, this study did not contain drug-use details concerning dosage, which might influence data analysis. In conclusion, this study determined the influence of ACEI or ARB uses on progression of renal deteriorationa mong patients at various stages of CKD and hypertension. We determined that ACEI or ARB use significantly retarded renal function deterioration through all stages of CKD. Moreover, a significant renoprotective effect was noted with medication use in later CKD stages (eGFR\u2009\u2264\u200945\u2009mL/min). Thus, ACEI or ARB monotherapy may considered in patients with CKD and hypertension and close monitored about side effects.This study was reviewed and approved by the institutional ethical committee of Taipei Medical University - Shuang Ho Hospital (TMU-JIRB 201204036), Tri-Service General Hospital (TSGHIRB100-05-197), Cardinal Tien Hospital (TMU-JIRB 201204035), Changhua Christian Hospital (CCHIRB 20405), Kaohsiung Medical University Chung-Ho Memorial Hospital (KMUHIRB 20120019), Kaohsiung Chang Gung Memorial Hospital (101\u20131096B), National Cheng Kung University Hospital (A-ER-101-117), and China Medical University Hospital (DMR101-IRB2-273(CR-1)). After a complete explanation of the study, written informed consent was obtained from all participants. All clinical and biological samples were collected after patient consent, and all experiments were performed in accordance with relevant guidelines and regulations.We conducted a multicentre, longitudinal cohort study based on the Epidemiology and Risk Factors Surveillance of CKD database from 2008 to 2013; the database is maintained separately by the Bureau of Health Promotion, Ministry of Health and Welfare, Taiwan. Epidemiology and Risk Factors Surveillance of CKD database is including 7956 patients with CKD and ages younger than 20 years old from 14 hospitals. The same medical laboratory criteria and protocol were used in our study hospitals, and the value of serum creatinine was derived from a different hospital, but could be compared and standardized. In addition, we linked the biochemical laboratory data to the health insurance database in Taiwan from 2004 to 2013. All registrations and claim data of participants were available to this study . In this study, patients were continually followed from the baseline date to the end of the study period , and patients were re-examined in the same hospital to control for individual variation. Patients who were without previous diagnose of hypertension, less than 1-year follow up, receiving dialysis treatment or renal replacement therapy before including in this study, receiving dialysis treatment or renal replacement therapy within the first six months after enrolled in this study, with missing data for BMI or UPCR, receiving ACEI\u2013ARB switch therapy or dual therapy were excluded.Patients were grouped according to the use of ACEI and ARB drugs during the first six months of observational period; if the patients were treated with ACEIs and ARBs for at least 90 days within a 6-mo period, they were categorized into the medication group, and the others were classified as the control group. The study group was subdivided according to the nature of treatment, such as ACEI monotherapy and ARB monotherapy. We defined that the ACEI monotherapy group used only ACEI for more than 90 days during the first six months of observational period, and we defined that the ARB monotherapy group used only ARB for more than 90 days during the first six months of observational period.2 per year or progression into the dialysis stage40. CKD was defined according to the Kidney Disease Outcomes Quality Initiative guidelines41, and was evaluated using eGFR, which was calculated using the Chronic Kidney Disease\u2013Epidemiology Collaboration equation, as recommended by KDIGO guidelines. CKD was classified as follows: CKD stage 1, eGFR \u226590\u2009mL/min/1.73\u2009m2 and the presence of kidney damage ; CKD stage 2, eGFR\u2009=\u200960\u201389\u2009mL/min/1.73\u2009m2 and the presence of kidney damage ; CKD stage 3a, eGFR\u2009=\u200945\u201359\u2009mL/min/1.73\u2009m2; CKD stage 3b, eGFR\u2009=\u200930\u201344\u2009mL/min/1.73\u2009m2; CKD stage 4, eGFR\u2009=\u200915\u201329\u2009mL/min/1.73\u2009m2; and CKD stage 5, eGFR <15\u2009mL/min/1.73\u2009m2\u200942.Renal deterioration progression was defined as an average eGFR decline of more than 5\u2009mL/min/1.73\u2009m2. Cigarette smoking was dichotomized as smoking (smoking \u2265100 cigarettes during the patient\u2019s lifetime) and never smoking. Alcohol consumption was dichotomized as current and noncurrent drinking.Baseline variables included demographic characteristics, namely age and sex; clinical variables were DM, CAD, stroke, and cancer; physical examination variables were waist circumference, BMI, systolic blood pressure (SBP), and diastolic blood pressure (DBP); laboratory test variables were levels of serum creatinine, blood urea nitrogen, uric acid, glycated haemoglobin (HbA1C), triglyceride, total cholesterol, and proteinuria; and health-related behaviours included cigarette smoking, alcohol consumption, betel nut chewing, and exercise. The demographic and health-related behaviour data were collected using a structured questionnaire. The physical examination and laboratory variables were obtained through medical chart reviews, and the clinical variables were obtained from the health insurance database. BMI was classified as <18.5, 18.5 to 24.9, and \u226525\u2009kg/mThe characteristics of treatment groups and reference group were compared using the chi-squared test for categorical variables, ANOVA test for continuous variables with normal distribution, and Kruskal-Wallis test for continuous variables with non-normal distribution. We used logistic regression models, including all potential confounders, to evaluate the association between ACEI and ARB use and eGFR decline. We first estimated the crude ORs, and then we estimated the adjusted ORs by including age, sex, previous comorbid conditions, such as DM, stroke, and cancer, Charlson comorbidity index scores, use of ACEI and ARB medication within the previous 1 year, cigarette smoking, alcohol consumption, betel nut chewing, BMI, baseline UPCR, and baseline eGFR in the model. Next, we did several subgroup analyses stratified by age, sex, DM, Stroke, cancer, Charlson comorbidity index, baseline CKD stage, prevalence ACEI user, prevalence ARB user, and immunosuppressants prescription. All analyses and calculations were performed using SAS Version 9.4 ."}
+{"text": "Mycobacterium houses some of the most deadly human pathogens; however, the impact of HGT in Mycobacterium has never been addressed in a systematic way. Previous initiatives to explore the genomic imprints of HGTs in Mycobacterium were focused on few selected species, specifically among the members of Mycobacterium tuberculosis complex. Considering the recent availability of a large number of genomes, the current study was initiated to decipher the probable events of HGTs among 109 completely sequenced Mycobacterium species. Our comprehensive phylogenetic analysis with more than 9,000 families of Mycobacterium proteins allowed us to list several instances of gene transfers spread across the Mycobacterium phylogeny. Moreover, by examining the topology of gene phylogenies here, we identified the species most likely to donate and receive these genes and provided a detailed overview of the putative functions these genes may be involved in. Our study suggested that horizontally acquired foreign genes had played an enduring role in the evolution of Mycobacterium genomes and have contributed to their metabolic versatility and pathogenicity.Horizontal gene transfer (HGT) was attributed as a major driving force for the innovation and evolution of prokaryotic genomes. Previously, multiple research endeavors were undertaken to decipher HGT in different bacterial lineages. The genus  This mode of gene exchange between reproductively isolated species, commonly known as horizontal gene transfer (HGT) or lateral gene transfer, was attributed as a major evolutionary force in several prokaryotic lineages10. Previous studies have implicated horizontally transferred genes in various important traits including novel metabolic pathways13, oxygenic photosynthesis14, antibiotic resistance15, pathogenesis16 and microbial translation efficiency17 and various other features10. Moreover, foreign genes were shown to assist microbes in colonizing new niches and in sustaining environmental changes18. Considering their implications in bacterial genome evolution, here in this study, an initiative has been undertaken to trace the probable HGT events among all currently available completely sequenced Mycobacterium genomes.A significant fraction of genes in all living species was considered to be acquired from genealogically distant speciesMycobacterium comprises more than 160 species of which about 15 deadly pathogens are commonly encountered in human and other animals19. Among the pathogenic Mycobacterium species, M. tuberculosis alone was estimated to have infected one-third of the human population causing more than 2 million annual deaths globally20. Pathogenic strains were suggested to originate from their free-living ancestors driven by independent or combined influence of genome reduction, gene duplication, gene rearrangement and HGT evolutionary processes. Horizontal gene transfer, among these, was attributed as a major factor contributing to Mycobacterium pathogenesis. Although there are controversies regarding the intensity and extent of HGT among different Mycobacterium species, however, it is clear from recent studies that Mycobacterium genomes had undergone many episodes of intra and interspecies HGTs acquiring genes from diverse origins including some members of eukaryotic families25. Along with substantial evidence of HGTs in several Mycobacterium genomes, previous studies provided important insights regarding their function and evolutionary importance25. For instance, foreign genes were shown to play important roles in the evolution of M. ulcerans (from M. marinum)26, M. avium subsp. paratuberculosis27 and in shaping pathogenic potential of M. abscessus28 and M. tuberculosis23. However, the contribution of HGT in Mycobacterium genome evolution has never been investigated in a systematic way. Earlier studies were mainly focused to find the genomic imprints of HGT in few selected genomes, specifically among the members of M. tuberculosis complex. Further, those studies were conducted either on selected genomic regions25 or considered only intra-genus gene exchanges between Mycobacterium species24. To this end, in the present analysis, we considered all available completely sequenced Mycobacterium genomes (109 genomes at time of data collection during February 2016) and set-out for a systematic analysis of all probable HGTs that these species may have undergone with any non-Mycobacterium species.The genus i) parametric methods which are based upon atypical sequence composition such as unusual codon usage or GC content and (ii) phylogenetic methods which infer HGT by contrasting well-supported gene ancestry with established species phylogeny31. Among these, phylogenetic methods were considered to be superior to any parametric methods7 and have been widely used in the recent analysis.To date, various methods have been proposed in order to detect genes acquired through HGT. These approaches can be grouped mainly into two categories  dedicated for in-silico identification of horizontally acquired genes. The main difference between the two approaches is that while phyletic pattern analysis depends on human expertise, T-REX infers probable HGTs based on statistical reconciliation of gene and species trees33. Considering both these two approaches here we identified several instances of horizontal gene exchanges in Mycobacterium genomes which are described in subsequent sections with emphasis on their functional implications. Our comprehensive study suggested that although intra-domain (bacteria) gene exchange is more frequent among Mycobacterium genomes, however, Mycobacterium species have occasionally received genes from other domains of life. On the functional level, our study suggested that horizontally acquired foreign are integral to several biochemical pathways important for the survival of some Mycobacterium as pathogens.To identify the putative HGTs in the selected 34 (ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/bacteria/) and retrieved 109 completely sequenced Mycobacterium proteomes  based on Markov gene clustering method35. Inflation index is an important parameter that determines the size of the cluster35. A lower inflation value (stringent clustering i.e. fewer clusters with more proteins) may place true orthologs in separate clusters, while higher inflation value (lenient clustering i.e. more clusters with fewer proteins) may cluster sequences of different functions together35. Here, we considered inflation index value of 1.5 which was suggested to balance sensitivity and specificity while including the maximum number of sequences in the clusters35. OrthoMCL resulted in 17,215 families of orthologous proteins (Cluster of Orthologous Groups or COGs) of which 523 groups of short proteins (less than 50 amino acids) and 252 probable species-specific groups containing members from same species  were discarded. Remaining 16,440 groups were considered for further analysis.To collect a comprehensive dataset, we considered the largest bacterial genome repertoire at NCBI GenebankMycobacterium genus, the longest member of each remaining COG  was searched against the NCBI non-redundant (NR) protein database with the BlastP (v-2.3.0+) algorithm. For each COG, Blast hits were complemented with other members of the group and filtered with a combination of three cutoffs, minimum e-value 1\u2009\u00d7\u200910\u22126, sequence identity 50% and query coverage 70%. COGs were then classified into two categories based upon the distribution of their significant blast hits, (a) groups showing hits within Mycobacterium genus and (b) groups showing hits outside of Mycobacterium genus. If a group was found to have multiple significant hits in same species (indicated by same taxonomic identifier), we retained the hit with the highest degree of identity. Here we considered the groups  database using Blastdbcmd utility and their full taxonomic lineages were obtained from NCBI taxonomy database using ETE toolkit36 with the help of their NCBI Gene Identifier (GI) numbers. For each group, multiple sequence alignment was generated by Muscle (v-3.6) multiple sequence alignment tool under default settings37. Phylogenetic trees were constructed in two phases. First, we constructed maximum likelihood trees from each such alignments using double-precision version of FastTree38 algorithm with parameters -spr 4, -mlacc 2, \u2013slownni for slower and more exhaustive search and \u2013gamma option which accounts for the uncertainties in rates of evolution at different sites38. For each group, the best fitting amino acid substitution model optimized for maximum likelihood trees was detected by ProtTest339 and implemented in FastTree. Currently, FastTree supports three amino acid substation models JTT, WAG, and LG38. When ProtTest suggested (for 183 of 9104 groups) any other model we used the best of these three models. For the initial screening here we used FastTree because FastTree was shown to be 100\u20131,000 times faster than many of other standard maximum likelihood-based phylogenetic tree construction algorithms such as PhyML 3.0 or RAxML40. However, the maximum likelihood topology of FastTree was suggested to be less accurate than that of RAxML which uses more intensive tree search algorithm38. Therefore, to reduce false detection of HGTs all the groups where we found signals for probable HGTs (next section) in FastTree phylogeny were again subjected to phylogenetic tree construction using RAxML40 with 100 bootstrap replicates and option for automatic detection of the best substitution model parameters. Finally, HGTs were inferred in RAxML phylogeny when we found similar signals for HGT as in FastTree phylogeny. All these phylogenetic trees have been deposited at http://www.mediterranee-infection.com/article.php?laref=981.In order to identify probable orthologs outside of BlastP v-.3.0+ alg31. The pattern that we considered as probable HGT with Mycobacterium species is described in Fig.\u00a0Mycobacterium groups), a donor clade (the sister group of the receiver clade) and an external clade (the sister group of the donor clade). Mycobacterium species were considered to receive genes from other bacterial species, when one or more Mycobacterium genes (receiver clade) branched within some unrelated bacteria  such that there is no Mycobacterium gene in up to five neighboring sister clades and there is no member from Corynebacterium and Nocardia genera (closest relatives of Mycobacterium41) in the donor clade. To detect gene exchanges with non-bacterial species, we assumed that acquired genes must be present in Mycobacterium species but will be absent from other closely related bacterial genomes. Here, we considered similar patterns, however, no bacterial homolog was allowed either in the donor or in the external clade . For Mycobacterium species tree needed for this analysis, we first listed all the Mycobacterium genomes involved in gene transfer (receiver clade of trees where we found HGT). A representative phylogenetic tree  of the members of each donor clade. Donor groups were inferred according to the lowest taxonomic rank  of the common ancestor. Lowest taxonomic rank of the species in donor groups was fetched from ETE toolkit36 using their taxonomic identifier.Although, our aim was to identify probable HGTs at the species level, however, in most of the cases we found that the donor and receiver groups consisted of genes from multiple genomes. To identify the point of acquisition of foreign genes we reconstructed their ancestral states along the in-silico detection of HGT33. Given a test and a reference tree, T-REX calculates their proximity by several distance-based measures and predicts minimum-cost scenario HGTs by the progressive reconciliation of those trees. T-REX was optimized by taking into account the evolutionary events including gene duplication and deletion and was shown to be faster and more accurate than most of the other currently available tree discordance methods like LatTrans and RIATA-HGT32. The trees generated by the FastTree38 algorithm were used as gene trees for this analysis. Species trees were constructed for each gene tree separately. For this, we retrieved the taxonomic ids of all the members of each alignment and fetched their common tree from NCBI taxonomy browser using those ids. Each pair of species and gene tree was then subjected to the T-REX algorithm for inferring probable HGTs. We discarded the \u201cTrivial\u201d (low confidence) HGTs from the prediction and considered the cases where one or more Mycobacterium species were listed in the acceptor field with no Mycobacterium in the donor field.T-REX is a suite of phylogenetic tools dedicated for several analyses including v-66) functional annotation tool44. InterPro provides comprehensive information about protein families, domains, and functional sites by integrating signatures from several protein annotation servers including Pfam, PRINTS, PROSITE, SMART, ProDom, SUPERFAMILY, PANTHER, CATH-Gene3D, TIGRFAMs and HAMAP44. COGs are clusters of homologous genes and supposed to consist of proteins sharing the same function. Therefore, we choose one representative protein (longest member) from each COG and used it for functional prediction. However, we first tested the utility of our representative protein in retrieving the functional annotation of groups affected by HGT. We retrieved all members of receiver groups for more than 100 families and compared their functional annotations with the functional annotation of the corresponding representative proteins. As expected, we noticed that all the members of each receiver group were composed of similar types of functional domains and were reflected in the composition of the representative proteins. By this way, we could annotate more than 68% of the groups involved in gene transfer with their Gene Ontology (GO) terms and Pfam domains. To get a relative idea, we compared the distribution of Pfam domains and three ontologies  among the candidate HGTs in reference to their distribution in the whole proteome of 15 Mycobacterium species  functional categories using BlastKOALA46 genome annotation tool. BlastKOALA assigns KEGG Orthology functional categories (designated by \u201cK\u201d numbers) based upon homology to precompiled databases. Here, the representative sequence (longest) of each HGT COG was searched against \u201cspecies prokaryotes\u201d database of BlastKOALA using default settings. Enzyme Commission (EC) numbers and pathway information were retrieved following the functional description of the best \u201cK\u201d number assigned to each input sequence. KEGG metabolic pathway map was generated using iPATH Interactive Pathways Explorer (v-3)47 with the help of \u2018K\u2019 numbers.Genes involved in HGT were mapped to the KEGGi) ARDB-Antibiotic Resistance Genes Database (v-1.1)48, (ii) The Comprehensive Antibiotic Resistance Database (CARD) (v-1.2.1)49, (iii) Antibiotic Resistance Gene-ANNOTation database (v-3)50, and (iv) MEGARes: an Antimicrobial Database for High-Throughput Sequencing (v-1.0.1)51. These databases are repositories of putative antibiotic resistance genes and related information collected from various resources. Currently, these databases contain 7828, 2311, 1808, and 3824 putative antibiotic resistance gene/protein sequences respectively. To identify the sequences with significant similarity we considered cut-off values of 50% identity over half (50%) of the protein length and e-value less than 10\u22125. HGT proteins were classed according to their putative antibiotic resistance potentiality based upon the functional description of their significant Blast hits. For virulence information we retrieved the protein coding sequences of bacterial virulence factor determinants from the Virulence Factors of Bacterial Pathogens database52 and treated in a similar manner. Currently (last updated 24th October 2017), this database contains sequences of 2,595 experimentally verified and 26,524 known and predicted virulence factors. Here, our query sequences were annotated against the experimentally verified dataset.Candidate HGT proteins were annotated with antibiotic resistance information based upon their sequence similarity with known antibiotic resistance genes. Representative sequences from the candidate HGT COGs were searched against the following databases with BlastP algorithm  and then the groups showing signals for probable HGTs were further screened with more robust tree construction algorithm RAxML . In our primary screen (FastTree phylogeny) in ~72%  of trees we did not find any pattern that could be considered as a probable case of HGT and were discarded. In 5.60%  of trees HGT pattern was found in multiple branches, suggesting several instances of gene transfer. Because the direction of transfer can\u2019t be detected with confidence we discarded these groups, however considering the possibilities of HGTs a detailed list of these trees with most probable donor and receiver groups are listed in Supplementary File\u00a0Mycobacterium genes seem to be acquired from non-Mycobacterium origin , Hoyosella subflava DQS3-9A1 (14 events), Tomitella biformata (29 events), Rhodococcus fascians (10 events) and Segniliparus rotundus DSM 44985 (12 events). According the characteristics of the recipient Mycobacterium genomes we categorized the candidate HGTs into following classes (i) where donor group comprised only pathogenic mycobacteria (host associated), (ii) donor group comprised only non-pathogenic mycobacteria (iii) donor group comprised only opportunistic mycobacteria and (iv) HGTs in mixed categories (Supplementary File\u00a0Rhodococcus (4 events) and Streptomyces (3 events), family Thermomonosporaceae (2 events) and several individual species like Actinomyces gerencseriae, Amycolatopsis methanolica and Brevibacterium senegalense (each 1 event). In 93 COGs at least one significant hit was detected in the viral domain of which our phylogenetic pattern analysis suggested gene transfer in 7 COGs  which infers ancestral states without any restriction on character evolution either in the reversibility of changes or in the number of character transitions (gains or losses). By this way, we could detect the relative time frame of the evolution of horizontally acquired genes. For any group, if candidate HGT genes were suggested (by Count42) to be present at the root node of Mycobacterium tree with (i.e. lost in some branches and regained in the other branches) or without subsequent gains then they were considered to be most ancestral HGTs acquired by the last common ancestor (LCA) of Mycobacterium clade. If candidate HGT genes were suggested to be absent at root node however one or more gains were predicted at internal nodes (with or without any further gain) then they were assumed to be not ancient or not recent (in-between), acquired during the evolution Mycobacterium sub-lineages. On the other hand, if gains were predicted only at individual species (affecting only species and no node) then they were assumed to relatively recent HGTs acquired during the evolution of individual species. The results suggested that 641 HGTs were possibly present at the root node of Mycobacterium phylogenetic tree (ancient), 678 HGTs were possibly gained at different internal nodes (in-between) and 347 HGTs were gained by different individual Mycobacterium species (recent HGTs)  were found to occur with similar but at a low frequency. However, we found significant (P\u2009<\u20090.05) difference for several functional classes (Supplementary File\u00a0etc. were found to be significantly (P\u2009<\u20090.05) over-represented among the candidate HGTs and DNA replication (GO:0006260), translation (GO:0006412), transposition (GO:0006313) etc. were found to be significantly (P\u2009<\u20090.05) under-represented , transposase activity (GO:0004803), etc. were found to be underrepresented  over-represented among candidate HGTs with underrepresentation of ribosome (GO:0005840)  terms and protein domain composition. Considering three types of GO ontologies  we found 572 unique GO terms among the candidate HGTs and 1512 unique GO terms in the reference set of 15 ted Fig.\u00a0. Among m40) Fig.\u00a0. The vas13. Considering the general enrichment of several metabolic and enzymatic processes among the candidate HGTs here we decided to explore the issue in more details. When candidate HGTs were annotated against KEGG pathways (see section 2.7), functional annotation could be retrieved for 42% . Finalses Fig.\u00a0.Figure 425. Here, we tested whether the candidate HGT genes had played any role in the origin of antibiotic resistance among Mycobacterium species. Considering homology with pre-compiled antibiotic resistance genes from several databases, we found evidence of putative antibiotic resistance like properties in 11 groups out of 1,683 groups with detected gene transfer events  where mycobacterium genes branched with other bacteria with strong statistical support and without any close relative in the neighboring sister clades. Moreover, we considered the possibility of sequence contamination extensively and filtered-out the groups where our analysis indicated that the pattern may arise due to such artifacts. Finally, we tested the HGTs detected from phylogenetic trees with an algorithm that identifies putative HGTs through statistical reconciliation of gene and species trees. Different methods were suggested to results in non-overlapping sets of transferred genes54. However, here we noticed a general agreement between the two approaches which suggested that the HGTs that we detected by pattern searching are the highly confident set of Mycobacterium horizontally acquired genes.The genus Mycobacterium genomes27. Considering their strict niche specificity initially it was proposed that Mycobacterium genomes rarely exchanged their genetic material with any other species27. M. tuberculosis was considered clonal, untouched by HGT and evolving only by random genetic drift and selection55. However, recent genomic analyses have provided strong evidence for extensive HGTs in and between different Mycobacterium species including M. tuberculosis which raised question about the validity of the clonal paradigm55. Considering phylogenetic signals Becq et al. and Rosas-Magallanes et al. independently speculated that extent of HGTs among Mycobacterium species is comparable to any other species23. However, their studies were limited to few genomic regions from selected M. tuberculosis strains. Current availability of a large number of completely sequenced Mycobacterium genomes has provided us with an opportunity to test their hypothesis in more detail. Our gene phylogeny-based analyses indicated that Mycobacterium species have indeed undergone many more events of HGTs than previously anticipated. The topologies of the phylogenetic trees allowed us to identify the species/groups most likely to constitute the donor and recipient clades. To identify the taxonomic lineages of the donor groups we determined the lowest taxonomic level that describes all the members of donor clade. Genomic signature analyses previously suggested three major donor groups, environmental Actinobacteria followed by Proteobacteria and viruses23. Here we refined these results in a broader context with more confidence, showing that the phylum Actinobacteria and genera Rhodococcus, Gordonia, Streptomyces and Tsukamurella mainly contributed as sources for HGT events. Among the potential donors at the species level, our data suggested that different Mycobacterium species have received genes mostly from Smaragdicoccus niigatensis, Hoyosella subflava DQS3-9A1 and Tomitella biformata. Noteworthy, these genera along with Mycobacterium mainly comprise environmental organisms residing in soil56. Thus our observations agree with the earlier speculation that soil is a major ecosystem for genetic material exchanges between living species.Previously, a number of initiatives have been undertaken to quantify the extent of HGTs among Mycobacterium phages. Bacteriophages act as a vehicle of gene transfers, however, their identification was considered to be difficult due to rapid evolution10. Here we detected traces of phage genes in Mycobacterium suggesting their recent evolution.Along with numerous cases of inter-domain gene exchanges, our analysis indicated a significant fraction of genes has been contributed from other domains of life. Here we noticed several instances of gene exchanges with Viruses. Notably, all the common partners of gene exchange among viruses are 7. Mycobacterium genomes are not naturally competent for transformation while there is limited experimental evidence for bacteriophage-mediated transduction55. Therefore, mycobacterial species were suggested to be refractory to transduction and transformation but to favor conjugation23. Presence of plasmids and phages, the main vehicles of conjugation in different Mycobacterium genomes further suggested that conjugation is the most active mechanism of gene transfer in mycobacteria55. Based on this evidence here we speculate that a significant fraction of HGTs detected in our analysis has been mediated by conjugative processes.One important goal of molecular biology studies is to understand the mechanisms by which organisms can exchange their genetic material. Horizontal gene transfer was shown to be mediated by three general mechanisms: transduction, transformation, and conjugationMycobacterium and other bacterial genera, we found it interesting to explore their functional relevance. Thus we noticed that the genes involved in energy production and transformation are more likely to be exchanged than the genes involved in basic housekeeping functions which is in accordance with earlier literature suggesting that genes involved in core biological functions are reluctant to HGT57. Specifically, here we noticed a general enrichment of several functional classes such as methylation, and transport among the candidate HGTs. DNA methylation, the only mechanism of epigenetic inherence among prokaryotes is an important component of their gene regulatory system. In the context of mycobacteria, DNA methylation was suggested to promote their persistence under stress full conditions aiding human infection58. Transporters are integral to all prokaryotic genomes irrespective of their host association and lifestyle. While their primary purpose is to actively transport different types of metabolites, several types of Mycobacterium transporters are implicated in resistance to antibiotics and are used as potential drug targets60. Although essential for both extra and intracellular lifestyle, Mycobacterium genomes encode a comparatively lesser number of transporters than other bacteria such as Escherichia coli or Bacillus subtilis60. To date, there is no clear understanding about their origin, however, the role of HGT in shaping their evolution is suggested by previous studies showing that the exportin Rv0986-Rv0987 (important for the host association of M. tuberculosis) including several others are acquired foreign genes23. Considering the general enrichment of different transporters in our HGT dataset here we hypothesized that a significant fraction of Mycobacterium transporters have been acquired through HGT.Given the list of genes that were exchanged between Mycobacterium species employ a large fraction of their coding capacity to encode different types of metabolic genes by virtue of which they can synthesize all the amino acids, vitamins, and the enzymes necessary for the production of their primary metabolites61. It appears from recent studies that interplay of different factors including HGT has contributed to their immense metabolic versatility12. Mycobacterium genomes were suggested to be shaped by a biphasic evolution of gene acquisition and duplication followed by loss leading to vast expansion of their metabolic genes specifically genes related to lipid metabolism and PE/PPE family62. Here our analysis extends these previous observations showing a wide presence of genes related to different metabolic processes among our candidate HGTs. A significant fraction of candidate HGTs detected in this analysis was mapped to different predictive enzymatic functions broadly belonging to oxidoreductase, transferase and hydrolase categories. Being strict aerobes, mycobacteria are dependent on the extensive use of enzymes related to different oxidative processes required for the generation of ATP. Therefore, mycobacteria are predicted to encode a large number (200) of oxidoreductases, with a significant number of enzymes dedicated for the hydrolysis of ATP and electron transport system61. Based on our analysis here we propose that frequent HGTs may have played a key role behind the expansion of their enzyme repository, crucial for the metabolic flexibility which in turn facilitates these bacteria to adapt with different environmental conditions.Metabolic capacity is another important feature with direct impact on bacterial survival. 15. HGTs within the microbial community were considered as one of the primary reason for bacterial antibiotic resistance and evolution, maintenance, and transmission of virulence15. Due to their thick lipid-rich cell wall, mycobacteria are inherently recalcitrant to several antibiotics63. In addition, mycobacteria employ a host of innate and adaptive strategies. Acquired drug resistance in pathogenic Mycobacterium species specifically in M. tuberculosis is suggested to arise from chromosomal mutations rather than through the acquisition of foreign genes63. In this study, we noticed putative antibiotic resistance like properties among some of our candidate HGTs. Identification of antibiotic resistance genes among the candidate HGTs will increase our knowledge about their implications in human health and diseases. Despite widespread research, there is little understanding about the molecular mechanisms of Mycobacterium pathogenesis. Possible involvement of HGT in Mycobacterium virulence has been anticipated in a number of earlier studies64. It is believed that a number of Mycobacterium operons with potential virulence activity have originated from the substantial invasion of foreign genes. Here, our detailed gene by gene screen identified several virulence factors among the candidate HGTs suggesting that foreign genes have possibly helped these bacteria to acquire pathogenesis.Bacterial infections are becoming increasingly difficult to treat due to widespread antibiotic resistance which was co-related with higher frequencies of HGTs among microbial pathogensMycobacterium. Through systematic phylogenetic analysis here we first identified the genes that Mycobacterium may have acquired from any non-Mycobacterium species then characterize those genes in view of their evolutionary importance. Our observations indicated to their crucial roles in Mycobacterium genomes suggesting their potential involvement in different biological functions and metabolic pathways. In addition, our results pointed to some unexplored roles of those genes that have been never been addressed so far. Our computational analyses need biological validations to help to decipher the virulence and resistance mechanisms of pathogenic Mycobacterium species and will facilitate the development of new therapies.In this work, we reported the first large-scale investigation of putative horizontally acquired genes in the widely diverse genus Supplementary InformationDataset 1"}
+{"text": "NFTsim, is presented to facilitate numerical simulations of a wide range of brain systems using continuum neural field modeling. NFTsim enables users to simulate key aspects of brain activity at multiple scales. At the microscopic scale, it incorporates characteristics of local interactions between cells, neurotransmitter effects, synaptodendritic delays and feedbacks. At the mesoscopic scale, it incorporates information about medium to large scale axonal ranges of fibers, which are essential to model dissipative wave transmission and to produce synchronous oscillations and associated cross-correlation patterns as observed in local field potential recordings of active tissue. At the scale of the whole brain, NFTsim allows for the inclusion of long range pathways, such as thalamocortical projections, when generating macroscopic activity fields. The multiscale nature of the neural activity produced by NFTsim has the potential to enable the modeling of resulting quantities measurable via various neuroimaging techniques. In this work, we give a comprehensive description of the design and implementation of the software. Due to its modularity and flexibility, NFTsim enables the systematic study of an unlimited number of neural systems with multiple neural populations under a unified framework and allows for direct comparison with analytic and experimental predictions. The code is written in C++ and bundled with Matlab routines for a rapid quantitative analysis and visualization of the outputs. The output of NFTsim is stored in plain text file enabling users to select from a broad range of tools for offline analysis. This software enables a wide and convenient use of powerful physiologically-based neural field approaches to brain modeling. NFTsim is distributed under the Apache 2.0 license.A user ready, portable, documented software package,  PLoS Computational Biology Software paper.This is a NFTsim, that can simulate scales from a few tenths of a millimeter and a few milliseconds upward, thereby making contact with experiments ,[2089:2105]};2 these_traces = {'Propagator.1.phi', 'Propagator.1.phi'};3 nf.plot_timeseriesx-direction. In these plots, the distance between the stimulation sites and neighboring sites increases vertically from the center to the top and bottom edges. The vertical dashed lines are not automatically produced by nf.plot_timseries, but have been added to mark the onset time of the positive (red dashed) and negative (blue dashed) inputs, respectively.\u22121) and incorporate volume conduction or hemodynamic effects  requested in the configuration file.In O, the runtime is dominated by writing operations. This overhead is expected for two reasons: (i) a simulated data sample is written to disk every \u0394tout, which takes additional time; and, (ii) writing the output to a text file requires conversion of numbers to text. Despite the runtime overhead this last point entails, text files are a convenient format to store the output because they are easier to debug than binary files.For large M, used by a NFTsim process is dominated by the number of grid points N and the history arrays of P internal populations with delay depth Da, which is the number of integration steps for a signal to propagate to the target population from the source population. So,The required memory, NFTsim process, including those which are not directly specified in a configuration file.NFTsim\u2019s performance, we select the corticothalamic model presented in earlier sections, with the parameters taken from previously published work [To assess hed work  and thust = 2\u221214 s \u2248 10\u22124 s, respectively. So, the only varying parameter that affects the runtime and storage is Nodes (N). The choice of this integration time step size is such that is sufficiently small to resolve high frequency oscillations and to satisfy the Courant condition for numerical stability for a range of discretization values between 3 mm < \u0394x < 50 mm. The Courant number ranges between 0.014 < p < 0.15 for a fixed velocity vab = 10 m s\u22121.The simulation length and integration time step are held constant at 16 s and \u0394Gno, runs the simulation and only writes a copy of the configuration file to the output file. The subscript no means no output. In this case the runtime represents the effective time spent executing a simulation without the time overhead due to writing operations. From Gno has kout \u2248 0. The second group of simulations Gwo consists of identical simulations to those of Gno, except that all the model variables , for all the nodes, sampled at 512 Hz, are written to a file in the hard disk.Two groups of simulations were run. The first group, Approximate runtimes and memory usage are measured using tools available on Linux systems. The computer used for the benchmarks has Red Hat Enterprise Linux (RHEL) 6.9 as operating system, GNU Compiler collection (gcc) 4.9.2 as the default compiler, a 3.50 GHz Intel i5-4690 processor and 8GB of RAM.ksim \u2248 0.15 s for the simulation group Gno and and ksim \u2248 kout \u2248 0.15 s for group Gwo. From these results, we conclude that in order to produce one minute worth of data sampled at a rate typically used in clinical EEG recordings, NFTsim takes about four minutes to run the simulation and write the output to disk. Thus, NFTsim\u2019s simulation length to real-time data length ratio  for EEG-compatible outputs is approximately 4. To reduce this ratio users can decrease the size of the output O, by writing only a few relevant variables to disk.NFTsim\u2019s performance, they are a valuable practical tool for users and provide: (i) estimates of resources required to run simulations; and, (ii) a guide to make informed decisions between the execution runtimes and accuracy .While these benchmarks offer a narrow view of NFTsim, a user-ready, extensible and portable suite for numerical simulations of neural activity based on neural field models expressed in differential form. NFTsim is based on the well established framework of neural field theory [We have introduced d theory  and has NFTsim has been tested on a range of Linux distributions . Current users have reported compatibility with OS X 10.11 and mac OS 10.12 in conjunction with the CLANG compiler, provided that the C++11 standard is supported. NFTsim has not been tested under Microsoft Windows.Written in C++, NFTsim is written to a plain text file and ancillary modules written in Matlab contain functions to assist in simulation execution, quick analysis and visualization of the results. NFTsim thus provides an efficient solution to simulating various continuum spatiotemporal models including spatially uniform (homogeneous) and nonuniform (inhomogeneous) neural field models [NFTsim follows essential practices of modern open-source scientific software development [The output of d models ; systemsd models ; and, thelopment  such as:The code is licensed under the Apache 2.0 license.https://github.com/BrainDynamicsUSYD/nftsim.Our code sources are hosted on Github: We use pull requests to review new features and bug fixes.Our users can open issues reporting bugs and/or other problems they encounter.Doxygen [The developer documentation is produced using Doxygen .A separate manual is provided for end-users.Releases are tagged, so users can refer to and download continuously improved versions of the code that are considered stable and tested. For instance, for this paper, we have used v1.1.0.+nf.Most notably, the activity from neural populations can be used to calculate biophysical signals such as LFP, ECoG, or EEG signals, the latter being the most commonly found in previous studies. Other forms of biophysical observables, such as fMRI and VSDI may also be implemented, but require additional modeling work to define how the electrical activity relates to the corresponding measurements . Further physical effects can be implemented as a part of postprocessing modules like NFTsim allows for a systematic study of both healthy and unhealthy brain function. For instance, in [NFTsim\u2019s flexibility allowed for the investigation of nonlinearities, introducing them one at the time in different neural populations. This enabled the authors to determine which anatomical structures and physiological mechanisms were responsible for the dynamics observed in experiments.Due to its flexibility and generality, ance, in  the authNFTsim is extensible and can accommodate new features presented in theoretical work on neural fields. In fact, a tool like NFTsim is essential for the study of nonlinearities and connectivities configurations that do not necessarily follow the random connectivity approximation [NFTsim. However, further investigation and development work is required before implementing a general mechanism of parameter modulation, which would allow for the study different types and functional forms of neural feedbacks [Due to its modularity, ximation , 50, 99 ximation  exploredximation  incorporeedbacks , 100.NFTsim, as any scientific software, should not be used blindly. As a minimal requirement, users should check that:We remind potential users that (i)The integration time step is small enough to resolve the simulated dynamics correctly, especially if the system exhibits chaotic and bursting dynamics;(ii)Interval, which effectively subsamples the timeseries written to disk, is an integer multiple of the time step; and, is small enough so as to avoid temporal aliasing if there are signals with high-frequency content  is sufficient to respect Nyquist\u2019s sampling theorem.The parameter (iii)Deltat would by running the simulation with increased or decreased time steps to check for stability and convergence of the solutions to a limiting case.The integration time step is small enough to respect the Courant conditions. If this condition is not met the code throws an error. A way to select an appropriate value of (iv)Deltat, Interval and Nodes as multiples or submultiples of powers of two , minimizes numerical errors due to the inherent limitations of representing floating point numbers on a computer. In addition, other advantages of using powers of two are (i) achieving optimal performance of FFT algorithms when applied to the output timeseries (1 second of data will have a power-of-two number of samples); and, (ii) avoiding zero-padding which is a frequent default behavior of FFT algorithms. However, users are not obliged to use NFTsim\u2019s default values. They can select any value and simulations will be executed.Setting parameters such as (v)Artifacts of periodicity introduced by PBCs as illustrated in Note that PBCs may be preferable over absorbing BCs in scenarios when one is interested in studying wave-wave interactions. However, in neural field models that approximate a small patch of cortex such as the primary visual cortex V1, in which waves of activity propagate away from a source point of stimulation , then abNFTsim already accepts spatial variations in many parameters, although more development work needs to be done to provide general mechanisms of parameter variation.In the present work we have concentrated mainly on a high-level description of the software and presented examples for which model parameters are assumed to be spatially uniform. NFTsim solves differential equations. It does not solve integrodifferential equations like those of Nichols and Hutt\u2019s Neural Field Simulator [NFTsim.Note that imulator . It is iClasses and their interactions, NFTsim currently has Gaussian white noise in its collection of external driving signals because in the literature [As mentioned in terature , 101\u2013104However, there are several limitations that make this type of signal a poor choice. The first limitation is that idealized continuous noise is not physically realistic because it has an infinite bandwidth and infinite power. The second limitation is that in computer simulations, where continuous models are inevitably discretized, the bandwidth of a white noise signal depends on the size of the discretization. This dependence implies that if either the time step or the spatial step are reduced, the bandwidth increases and as a result a white noise signal has additional modes . One can use a scaling parameter to adjust the overall power of the discretized driving signal , 81. ThiNFTsim scientific features by including (i) a new bandlimited noise generation to render the inputs even more biologically realistic; (ii) generalized mechanisms of spatiotemporal variations for different model parameters and variables; (iii) generalized mechanisms of neuromodulation; (iv) absorbing boundary conditions; and, (v) spherical topology. In addition, a number of technical enhancements will be made such as (i) implement support for output binary files; and (ii) extend and automate unit test coverage to ensure that new additions to the code do not break previous functionality.Future work will extend S1 Appendix(PDF)Click here for additional data file.S2 Appendix(PDF)Click here for additional data file."}
+{"text": "In the original article, there were some minor errors in Tables 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."}
+{"text": "Due to a production error, the first sentence of the conflict of interest statement was inadvertently not included. The complete statement should read:The handling editor declared a past co-authorship with one of the authors SA.The remaining 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.The publisher apologizes for this mistake.The original article has been updated."}
+{"text": "In the original article, there was a mistake in Table The original article has been updated.Now one can read: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."}
+{"text": "The complement system plays critical roles in development, homeostasis, and regeneration in the central nervous system (CNS) throughout life; however, complement dysregulation in the CNS can lead to damage and disease. Complement proteins, regulators, and receptors are widely expressed throughout the CNS and, in many cases, are upregulated in disease. Genetic and epidemiological studies, cerebrospinal fluid (CSF) and plasma biomarker measurements and pathological analysis of post-mortem tissues have all implicated complement in multiple CNS diseases including multiple sclerosis (MS), neuromyelitis optica (NMO), neurotrauma, stroke, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD). Given this body of evidence implicating complement in diverse brain diseases, manipulating complement in the brain is an attractive prospect; however, the blood-brain barrier (BBB), critical to protect the brain from potentially harmful agents in the circulation, is also impermeable to current complement-targeting therapeutics, making drug design much more challenging. For example, antibody therapeutics administered systemically are essentially excluded from the brain. Recent protocols have utilized \u201cTrojan horse\u201d techniques to transport therapeutics across the BBB or used osmotic shock or ultrasound to temporarily disrupt the BBB. Most research to date exploring the impact of complement inhibition on CNS diseases has been in animal models, and some of these studies have generated convincing data; for example, in models of MS, NMO, and stroke. There have been a few recent clinical trials of available anti-complement drugs in CNS diseases associated with BBB impairment, for example the use of the anti-C5 monoclonal antibody (mAb) eculizumab in NMO, but for most CNS diseases there have been no human trials of anti-complement therapies. Here we will review the evidence implicating complement in diverse CNS disorders, from acute, such as traumatic brain or spine injury, to chronic, including demyelinating, neuroinflammatory, and neurodegenerative diseases. We will discuss the particular problems of drug access into the CNS and explore ways in which anti-complement therapies might be tailored for CNS disease. The CNS was, for a long time, considered an immunologically privileged organ because the brain and spinal cord are protected from circulating inflammagens by the BBB. The BBB is a specialized membrane comprised of endothelial cells with tight junctions, vascular pericytes and perivascular glia , which cThe healthy BBB forms early in development and restricts the infiltration of circulating immune cells into the brain parenchyma; hence, the dominant immune cells of the brain are the resident macrophage population\u2014microglia . This seImportantly for the subject matter of this review, there is compelling evidence that, during inflammation, not only microglia and astrocytes, but also neurons, oligodendrocytes, and endothelial cells in the brain, can express complement components, receptors, and regulators.Complement is recognized as an important branch of the innate immune system, providing the first line of defense against microorganisms. As complement is the subject of this issue, we will confine ourselves to a brief summary  or blocking antibody against C3a administered during neural tube formation cause loss of neural crest cell organization, demonstrating a role for C3a and its receptor in the migration of neural crest cells . Central\u2212/\u2212) mice had improved hippocampal-dependent learning and memory . In traumatic SCI the primary pathology is caused by a mechanical force directly damaging the neural tissue\u2014this primary insult is difficult to protect against. However, post-injury inflammation, with infiltration of immune cells and production of pro-inflammatory mediators, results in secondary pathology in adjacent areas characterized by oedema, ischemia, and excitotoxicity ; however, comparison of C1q KO and C1q WT mice in a peripheral conditioning lesion model of SCI showed no differences in axon length, lesion volume or scarring, although C1q deficiency was associated with increased axonal turning . AD is characterized by two hallmark pathologies; amyloid-\u03b2 (A\u03b2) plaques and neurofibrillary tangles comprising hyperphosphorylated tau. Recent studies have implicated complement in AD pathogenesis. Genome wide association studies identified single nucleotide polymorphisms (SNPs) associated with risk of late-onset AD in genes encoding complement proteins Clusterin (CLU) and CR1 (CR1)  expression decreased in AD  , 113. BiR1 (CR1) \u2013117. ImmR1 (CR1) \u2013120. TheR1 (CR1) . Cells eR1 (CR1) . A weakned in AD . Increased in AD , 125.in vitro and in animal models. A\u03b2 fibrils activate and consume complement classical and alternative pathways in vitro and generate C3a, C5a, and TCC , TAR DNA binding protein (TDP-43), fused-in-sarcoma-protein (FUS), and chromosome 9 open reading frame 72 (C9orf72). The only currently available treatment for ALS is Riluzole, an ion channel blocker and inhibitor of glutamate release which modestly increases survival  followed by neuronal loss in these areas which spreads to the hippocampus  repeat  in exon 1 of the pocampus , 160. Nepocampus  dependenpocampus ; indeed,pocampus .HD post-mortem tissue showed abundant reactive astrogliosis and microgliosis and intranuclear ubiquitin positive inclusions in the caudate and temporal lobes . IHC shoper se rather than neuroinflammation in general , an inhibitor of the mitochondrial citric acid cycle, developed striatal lesions, weight loss, gait disturbances, dystonia and ataxia, thus reproducing some of the pathological and clinical characteristics of HD . Oral ad general . 3-NP trhuntingtin gene, including a pathological trinucleotide repeat; they develop progressive behavioral and neuropathological deficits, including synaptic loss, but do not develop neuronal loss and fail to demonstrate upregulation of complement proteins  fused to a fAb fragment of an anti-insulin receptor mAb bound the insulin receptor at the BBB, was transcytosed across the barrier enabling it to access and recognized A\u03b2 within the brain and was then shuttled out again with its A\u03b2 cargo for disposal . Anti-coSmall, hydrophobic molecules can cross the BBB via lipid-mediated diffusion. As an example, oral administration of the small molecule NLRP3 inhibitor MCC950 in PD mouse models reduced nigrostriatal dopaminergic degeneration, motor deficits and accumulation of \u03b1-synuclein through inhibiting inflammasome activation . Severalwww.clinicaltrials.gov NCT00904826), treatment reduced the number of neurological episodes  and atypical hemolytic uremic syndrome (aHUS) and recently for Myasthenia Gravis. In a recent small trial of eculizumab in NMO, a demyelinating disease characterized by BBB disruption and inflammation/degeneration of the optic nerve and spinal cord (episodes . This stand encourage resolution of inflammation. Evidence from studies of the impact of NSAIDs on neurodegeneration support the idea that early and long-term treatment is protective but treatment post-onset fails (reviewed by 106). Early intervention requires ways of identifying those at high risk; genetic studies have identified polygenic signals that include many inflammatory genes and are highly predictive of risk of AD . For AD, models that better reflect the human disease are now available and may help overcome this issue. Switching off complement systemically will impact immune defense; while this may not be an issue for short-term therapy in acute conditions, in chronic diseases requiring life-long treatment it is a major consideration. Despite all these problems, inflammation and complement activation present tractable targets in neuroinjury and neurodegenerative disease and deserve investment into basic understanding and the development of CNS-targeting anti-inflammatory and anti-complement drugs.Therapy of acute neurological injury and neurodegenerative diseases represent a major therapeutic challenge. Most of the diseases described above currently have no effective therapies and new approaches are desperately needed. Although there are some common features, notably inflammation and complement activation, the described diseases are very heterogeneous, even within disease labels\u2014AD is not a single disease! Patient stratification, for example, selecting patients with high inflammatory markers and evidence of ongoing complement activation for anti-complement drug therapy, will be necessary for successful trails in the future; this requires better biomarkers. For most of the diseases, proof of concept for new approaches to therapy is stymied by the lack of good models; critically, agents that are effective in current models usually fail in human trails (All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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."}
+{"text": "M\u00fcller et al. provide the first full 3D reconstructions of microtubule networks in a mammalian cell, the islet \u03b2 cell. Microtubules are mostly disconnected from centrioles and endomembranes, while associated with cortical insulin granules, highlighting their importance for regulated secretion. Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet \u03b2 cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven \u03b2 cells, and generate a comprehensive spatial map of microtubule\u2013organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion. Caenorhabditis elegans  and kept in liquid nitrogen until freeze substitution. They were substituted as previously published  or accorPrior to FIB milling, small vertical posts were trimmed to the region of interest guided by x-ray tomography data obtained by a Zeiss Versa XRM-510 and optical inspection under a microtome. A thin layer of conductive material of 10 nm gold followed by 100 nm carbon was coated on the trimmed samples using a Gatan 682 High-Resolution Ion Beam Coater. The coating parameters were 6 keV, 200 nA on both argon gas plasma sources, 10 rpm sample rotation with 45\u00b0 tilt. The coated samples were imaged using a customized FIB-SEM with a Zeiss Capella FIB column mounted at 90\u00b0 onto a Zeiss Merlin SEM. Details of the enhanced FIB-SEM systems were previously described , 2020a,cDue to the differences in organelle morphology and involved length scales, segmentation of the different organelles poses specific challenges that we approached with a dedicated method for each organelle class: large and contrast-rich organelles such as plasma membrane, nucleus, and mitochondria allowed fast semi-manual tracing or admitted relatively uncomplicated pixel-wise classification methods. The crowded distribution of insulin SGs and the convoluted structure of the Golgi complex required a two-stage process to first generate and curate training data for a subsequent final machine learning\u2013based segmentation step (see Golgi apparatus and SGs paragraphs below). Microtubules posed the biggest segmentation challenge due to their small diameter (25 nm) and relatively low electron density. We thus opted in this case to perform laborious yet accurate manual tracing.Microtubules were traced manually by creating a skeleton with the KNOSSOS software . The oriPlasma membrane, nucleus, and centrioles were segmented manually with the Microscopy Image Browser  using inWe used ilastik to create a preliminary foreground mask of 10 crops (128 \u00d7 128 \u00d7 128 pixels each) from different cells. After manual curation, these labeled crops were used to train a three-class U-Net , which wMitochondria were segmented with ilastik , which eDeep learning has been successfully applied to detect insulin SGs in 2D images . Due to Segmentation masks were first exported as hdf5 or Amira files, then imported into Fiji, and finally saved as TIFF files . MicrotuBased on the segmentation and labeling data, we calculated distance maps of organelle pixels and distance and connectivity relations between individual labels with ImageJ2  and ImgLX where X signifies the organelle with respect to which the distances were calculated .We used Python-based tools . The viewer utilizes a preliminary version of LabelEditor (https://github.com/juglab/LabelEditor), a novel layer on top of BigDataViewer (http://www.jfree.org/jfreechart/).The plugins available in Fiji via our update site, taViewer  for disphttps://sites.imagej.net/BetaSeg via the Help>Update... in the menu. The data folder of at least one cell needs to be downloaded from https://betaseg.github.io/. Afterwards, one can start the viewer from the menu by clicking Analysis>BetaSeg and then pointing to the directory of one cell in the following popup. A new window with a BigDataViewer area on the left and a list of data components on the right will open, and the EM source of the cell will be automatically displayed. The list of data components allows one to interactively show or hide segmentations as well as analysis results, e.g., the length or tortuosity of microtubules. These properties are displayed via lookup tables in the viewer on top of the EM source. Colors can be adjusted. Individual values of, e.g., single microtubules can be displayed by clicking the respective object. The analysis data can also be displayed as a histogram or table, available via the (...) button on the right of the specific data item.Fiji/ImageJ users can install the BetaSeg Viewer by adding the update site https://theobjects.com/dragonfly) or blender (https://www.blender.org).Overlays of raw data and segmentations were visualized with 3Dscript . 3D rendReview HistoryClick here for additional data file."}
+{"text": "The short-term effects of anti-vascular endothelial growth factor (anti-VEGF) treatment on macular neovascularization (MNV) morphology is well described, but long-term studies on morphologic changes and correlation of such changes to the type of MNV have not been conducted. This study aims to determine if different types of MNVs in neovascular AMD (nAMD) behave differently with anti-VEGF treatment as visualized on optical coherence tomography angiography (OCTA).Treatment-na\u00efve nAMD patients were retrospectively screened for baseline and follow-up OCTA imaging 10 or more months after initial treatment. Images were graded for MNV type, area, activity, mature versus immature vessels, vessel density, presence of atrophy, atrophy location and area. Growth rate was calculated as the percent change in lesion area from baseline over the years of follow-up. In addition, the occurrence of complete regression and the percent of lesions that grew, remained stable, and shrunk per type was also evaluated.Forty-three eyes from 43 patients with a mean follow-up of 2\u00a0years were evaluated. On structural OCT, 26 lesions were classified as pure type 1 MNVs, 12 MNVs had a type 2 component, and 5 MNVs had a type 3 component. Of these cases, 2 mixed-type MNVs were considered to have completely regressed. There was no significant differences in MNV area and growth rate between type 1 and type 2 lesions, but all cases of type 3 lesions shrunk in the follow-up period. There was no correlation between the number of injections per year and growth rate, endpoint MNV area or endpoint activity status for any MNV type. There was no significant association between the development of atrophy and the number of injections, baseline MNV area, baseline vessel density, or lesion growth rate.In nAMD, complete regression of an MNV network exposed to anti-VEGF is rare. This work emphasizes the role of anti-VEGF as anti-leakage rather than vascular regression agents in nAMD. Age-related macular degeneration (AMD) is a major cause of blindness in older adults. Neovascular AMD (nAMD) is an advanced stage of the disease that can lead to vision loss due to macular damage from abnormal blood vessel exudation. There are three main forms of neovascularization that can occur in nAMD based on where the new blood vessels originate and grow . Type 1 Anti-vascular endothelial growth factor (anti-VEGF) therapy is currently the mainstay treatment for a wide range of retinal pathologies, including MNVs associated with nAMD. Anti-VEGF treatment is considered to have two main beneficial effects on retinal pathologies: reduction in vascular permeability and decreased neovascular growth . Anti-peConversely, MNVs in nAMD respond more variably to anti-VEGF treatment. Some studies have shown decreased neovascular membrane area, pruning of small capillaries, and expansion of vessel caliber with anti-VEGF treatment for MNVs in nAMD \u201317. HoweOCT angiography (OCTA) offers a non-invasive and depth-resolved alternative to gold-standard dye-based angiography, yielding high-resolution images that can localize vascular complexes to specific retinal and choroidal layers, which allows for the detailed study of MNV morphology , 21. ConThis was a retrospective cohort study of eyes with treatment na\u00efve nAMD that were treated with anti-VEGF injections and imaged at the New England Eye Center, Boston, MA or at the Centre Ophtalmologique Rabelais, Lyon, France between December 2014 and December 2018. Both institutions received institutional review board approval and research was performed in accordance with the Declaration of Helsinki and the Health Insurance Portability and Accountability Act.Patients were considered for inclusion in this study if they met the following criteria: (1) the patient had a treatment na\u00efve MNV, (2) OCTA imaging was performed at baseline prior to the initiation of anti-VEGF treatment, (3) OCTA imaging was available at least 10\u00a0months after the baseline visit, and (4) the patient received any number of anti-VEGF injections over the follow-up period either per a treat and extend, pro re nata, or a monthly injection protocol. Exclusion criteria were (1) evidence of other retinal vascular disorders  as noted in the patient chart and (2) an inability to interpret the baseline or endpoint OCTA image due to significant image artifact or hemorrhage blocking the signal. Patients were not excluded if they had received anti-VEGF injections in the fellow eye. Best corrected visual acuity (BCVA) at the baseline and endpoint visits were converted to logMAR and recorded for each patient. The number of anti-VEGF injections received in the follow-up period, as well as the type of agent given , was determined by chart review and recorded. If a patient was noted to have had treatment with more than one anti-VEGF agent, the agent type attribute was assigned as \u201ccombination\u201d.Images were acquired on one of three OCTA devices and qualitatively analyzed in each devices\u2019 respective review software. The three devices used in this study were: the spectral domain RTVue XR Avanti with Angiovue , the spectral domain Cirrus HD-OCT 5000 , and the swept-source PLEX Elite 9000 . The baseline image was defined as the first OCTA image available for a newly diagnosed MNV prior to anti-VEGF treatment. The endpoint image was selected as the most recent OCTA image on file that met image quality standards. The 6\u2009\u00d7\u20096\u00a0mm macular scan pattern was the default for analysis, but the 3\u2009\u00d7\u20093\u00a0mm macular scan pattern was used if the 6\u2009\u00d7\u20096\u00a0mm image was of poor quality or unavailable.Images were graded independently by 2 of 3 qualified graders . Adjudication was performed if graders were in disagreement over a categorical metric or if there was greater than a 10% difference between quantitative measurements. If there was still disagreement between graders, a retina specialist reviewed the case and made a final judgement .The following qualitative outcomes were assessed at both the baseline and endpoint visits: the presence of immature or mature vessels, lesion activity status, and the presence and location of macular atrophy . Immature vessels were defined as fine, branching vessels . This software permits tracing over en face OCTA images and custom image thresholding for each device and scan size. The OAT software was used to allow for uniform analysis across the different imaging platforms utilized in this study. The outcomes measured in the OAT software included MNV area, MNV vessel density, and atrophy area; these outcomes are defined below.2 encompassed by the MNV tracing. Vessel density was defined as the ratio of white pixels (representing flow) to total pixels within MNV tracing after binarization. An optimal global threshold for binarization, which allows separation of vessel pixels from non-vessel pixels, was empirically selected and applied to all images during analysis.To analyze MNV area and vessel density, images were first manually segmented on the OCTA device to obtain the best possible en face view of the neovascularization. En face images were then imported into the OAT software, where the MNV lesion was manually traced. If MNV vessels were not well visualized on the en face image, the tracing was performed while scrolling through the corresponding OCTA B-scans in the device review software where the pixels representing flow could be followed. For such images, MNV vessel density measurements were excluded from analysis. Lesion area was defined as the total area in mm2 encompassed within all traced areas.Atrophy was defined as the presence of signal hypertransmission on the B-scan and accompanying RPE attenuation. Because the extent of these two features were not measured, incomplete RPE and outer retinal atrophy (iRORA) and complete RPE and outer retinal atrophy (cRORA) were not distinguished . AtrophyThe growth rate was calculated as the percent change in lesion area (baseline area minus endpoint area divided by the baseline area) divided by the follow-up duration in years. This normalized growth to initial lesion size. Furthermore, lesions were subdivided into three growth categories including those that remained stable (\u2264\u200910% change from baseline to endpoint), shrunk (>\u200910% reduction from baseline) or grew (>\u200910% increase from baseline). The percentage of lesions per growth category was calculated for each MNV type.A Kruskal\u2013Wallis test was employed to compare continuous variables among MNV types, including the number of injections, follow up duration, growth rate, baseline and endpoint MNV areas, baseline and endpoint vessel densities, and baseline and endpoint visual acuities. If the Kruskal\u2013Wallis test revealed a significant difference across the three MNV types for a given variable, a Mann\u2013Whitney U test for pairwise comparison was performed to inform where a significant difference occurred. A Wilcoxon signed rank test was also performed to compare the baseline and endpoint MNV areas and vessel densities within a given MNV type. Spearman\u2019s rank correlation was used to assess the relationship between endpoint MNV area and number of injections, baseline vessel density and growth rate, and growth rate and number of injections. A Chi-square test was used to assess categorical variables per MNV type including vessel maturity at baseline, complete regression at the endpoint, endpoint activity status, and growth category. Logistic regression modeling was performed to assess the correlation between development of endpoint atrophy and various continuous variables including baseline MNV area, baseline vessel density, number of injections, follow-up duration, and growth rate. This logistic regression analysis was performed after excluding patients with baseline atrophy and after controlling for the number of anti-VEGF injections per subject.Finally, a Kruskal\u2013Wallis test was utilized to assess potential differences across anti-VEGF agent type. Growth rate, endpoint MNV area, and endpoint activity status across agent type  were assessed in the overall cohort. MNV type was not accounted for in this sub-analysis to avoid further sample size reduction.A p value of \u2264\u20090.05 was considered significant. All statistical analyses were performed using RStudio version 1.1.463.Of the 966 nAMD patients screened for this study, 52 met inclusion criteria. The most common reason for exclusion was lack of treatment-na\u00efve status at the time of initial OCTA imaging. Forty-three eyes 26 OD, 17 OS) from 43 patients with at least 10\u00a0months of follow-up were successfully evaluated  Fig.\u00a0, 5. Thesen face for both baseline and endpoint images. For this reason, growth rate and growth category were only assessed for lesions that could be traced at both baseline and endpoint visits. The number of lesions used for growth rate and category calculations are noted in Table\u00a0In an attempt to further quantify the growth patterns of lesion types, the percent of lesions per type that grew, remained stable, or shrunk over time was calculated. It is important to note that not all MNV areas could be accurately measured Results for Spearman correlation between various growth parameters are shown in Table\u00a0Lastly, anti-VEGF agent analysis showed no difference in average growth rate (p\u2009=\u20090.967), endpoint MNV area (p\u2009=\u20090.3438), or endpoint activity status (p\u2009=\u20090.1106) across agent categories.A 66-year-old Caucasian woman with a history of anti-VEGF injections for nAMD and macular atrophy in the left eye presented with worsening vision in the right eye. Her visual acuity was 20/70 in the right eye, down from a baseline of 20/30. Her visual acuity in the left eye was count fingers. She was found to have a submacular mixed type 1, type 2 MNV with overlying fluid in the right eye on OCTA Fig.\u00a0a\u2013c and dA 74-year-old Caucasian woman with a history of hypertension presented to retina clinic for worsening vision. Her visual acuity was 20/50 in the right eye (baseline vision and vision in the left eye unknown). She was found to have a chorioretinal anastomosis, consistent with a mixed type 1 and type 3 MNV in the right eye on OCTA .We postulate that low MNV regression rates in response to anti-VEGF treatment are due, in part, to the effects of extracellular matrix composition and cell\u2013cell interactions involved in angiogenic signaling mechanisms . Within These observations may be due to differences in crosstalk and activation of signaling mechanisms involved in the two different angiogenic processes. Microscopy in epiretinal membranes associated with PDR shows narrow vessels surrounded solely by fibroblasts and occasional macrophages. In contrast, vessels in the subretinal membranes of nAMD patients are surrounded by a rich cellular environment abundant in proteoglycans and cellular components such as pericytes, fibroblasts, RPE cells and numerous macrophages and leukocytes .The finding of immune cells around MNV is consistent with work identifying inflammation and choroidal endothelial cell (CEC) activation in type 1 and type 2 MNV formation . AccordiPrevious theories have proposed that mature MNV vessels, surrounded by pericytes that supply local VEGF, are preferentially protected from anti-VEGF treatment when compared to their immature counterparts . It is pDespite similarities in the developmental pathway of type 1 and type 2 MNVs, the two types cannot be uniformly grouped together . Recent The current study saw more type 2 MNVs grow during the follow-up period compared to type 1 MNVs (50% versus 28.57%), however this result did not reach statistical significance. Overall, the frequencies of growth categories across MNV type are not statistically significant. Similarly, there was no significant difference between the baseline and endpoint MNV areas within a given MNV type nor was there a significant difference in the magnitude of growth rate across types. This means that even though lesions generally shrunk or grew, there was no significant change in size from the baseline measure overall. Trends noted in this study suggest that the presence of a type 2 component might lend to the risk for expansion, though future, larger studies are required to evaluate this idea.Finally, this study did not find a correlation between the number of anti-VEGF injections and the final MNV area or the growth rate in type 1 or type 2 MNVs, a phenomenon that was also observed by Xu et al. . There wIn the present study, the few cases of type 3 MNVs that were included shrunk in the follow-up period with anti-VEGF treatment. Several groups using OCTA have reported complete regression of type 3 MNVs after anti-VEGF treatment in 29\u201348.7% of cases , 24, 42.Along with assessing the regression and growth rates across MNV types, this study also sought to correlate quantitative OCTA metrics, such as vessel density, with growth rate. There was no correlation between baseline vessel density and growth rate in the type 1 and type 2 lesions. This is consistent with the report by Kim et al. that who found no significant changes in vessel density for both type 1 and type 2 MNVs after treatment and no correlation between vessel density and the rate of change of MNV size . Xu et aThis study also attempted to investigate the effects of anti-VEGF exposure on vessel maturity. The process of maturation with anti-VEGF exposure is attributed to cycles of low VEGF levels leading to the regression of new vascular sprouts and the resulting high flow stimulus for vessel dilation, or arteriogenesis, which is not VEGF dependent . Though Finally, the association with the development of atrophy and anti-VEGF use has been hotly debated in recent years, but this study found no significant association between the development of atrophy and anti-VEGF exposure or MNV growth variables . Though The two cases of regression posit an interesting discussion about the relationship between atrophy and MNVs. In Case 1, the endpoint image revealed a large area of atrophy Fig.\u00a0d\u2013f, and There are several limitations to this investigation. First and foremost, the retrospective nature of this study lends itself to faults. As mentioned previously, the small sample size, which was dictated by the inclusion criteria of the retrospective screen, is not large enough to obtain robust data on expansion or regression rates for the different types of MNV lesions. Furthermore, the small number of qualifying cases meant that only a few cases of pure type 2 and type 3 lesions were studied. This necessitated the combination of mixed lesions with a type 1 component into the aforementioned categories, which could confound findings. Despite possible confounding, the number of mixed-type lesions found in this screen suggests these lesions are common and should not be excluded from further study simply due to their varied components. In fact, type 2 MNVs rarely exist in a pure state. Choroidal vasculature must break through the RPE and proliferate under the retina to form these lesions. This suggests that type 2 lesions likely proliferate from underlying type 1 lesions. It is, therefore, unclear if a type 2 lesion can truly be pure at all, or if the type 1 component is simply not visible in seemingly pure cases. For these reasons, we felt it necessary to group lesions by components.en face images with their corresponding B-scans. This method increases the risk of biased evaluation since all images are listed by acquisition date on device platforms. Finally, patients were imaged on a variety of OCTA devices, which further contributes to variability. MNV area, while reproducible across different spectral-domain OCTA devices, is variable\u00a0between spectral domain and swept-source OCTA technology [Treatment regimen and follow-up duration were not controlled for in this study. This can be seen as a study strength since our cohort reflects a more generalizable clinical sample. However, not controlling for treatment regimen and follow-up duration introduces variability and limits our ability to find significant differences across MNV types. Another limitation to this study is the fact that grading was not independent of examination date. Graders were required to search for specific images within the device software to assess chnology , 49. Furchnology . Larger Anti-VEGF treatment is not associated with vascular regression on OCTA in nAMD. This work emphasizes the clinical role of anti-VEGF as an anti-leakage agent rather than a vascular regression agent. This study also suggests that the regression pattern of neovascularization that originates in the choroid is different to that which originates in the retinal vasculature. Future studies are required to determine if OCTA metrics can predict how an individual MNV will behave with anti-VEGF exposure."}
+{"text": "The structures were determined through a combination of MS and 1D/2D NMR spectroscopic techniques. Likely monomeric precursors, identified on the basis of HRMS analysis, allow a plausible biosynthetic pathway to be proposed for the biosynthesis of 1 and 2, involving the dimerisation of the monomer through a hetero-Diels-Alder mechanism. A gene cluster, including a putative sesquiterpene 1\u201311 cyclase, was identified through phylogenetic and RNA-seq analysis, and is proposed to be responsible for the biosynthesis of 1 and 2.Basidiomycete fungi are a rich source of natural products with a diverse array of potentially exploitable bioactivities. Two dimeric sesquiterpenes, bovistol B ( The basidiomycete fungi are a phylum of organisms with a hugely diverse range of biological capabilities . AlthougCoprinopsis strossmayeri [Coprinus quadrifidus [Coprinopsis, Coprinus, Coprinellus, and Parasola genera in the Psathyrellaceae family. Generally favouring a habitat in herbivore dung, these organisms have evolved to live in highly competitive environments. Notable bioactive metabolites previously reported from coprinoid species include: the antimicrobial sesquiterpene illudins, illudin I, I2, J and J2, from Coprinopsis episcopalis [Coprinus heptemerus [Coprinus sp. [We have recently re-evaluated the coprinoid basidiomycete ssmayeri , previoudrifidus ; a specidrifidus . The copscopalis ,8; the dptemerus , as wellinus sp. . Basidioinus sp. \u2013a group inus sp. .C. strossmayeri were carried out. This led to the isolation of two novel compounds, 2 and 7, as well as the identification of 1 previously isolated from Cyclocybe aegerita [Bacillus subtilis ATCC 6633, Escherichia coli DH5\u03b1 and Saccharomyces cerevisiae Y10000. RNA-seq data led to the identification of a proposed gene cluster responsible for the biosynthesis of 1 and 2.In our ongoing search for novel antimicrobials, investigations of the culture filtrate of aegerita , the strCoprinus quadrifidus, was obtained from The CBS Fungal Biodiversity Centre and its internal transcribed spacer region . The fungus was maintained on potato dextrose agar  at 20\u00b0C.Isolate CBS 177.39, listed as C. strossmayeri was grown in potato dextrose broth (PDB) (potato dextrose broth 24 g/L) for 14 days at 20\u00b0C at 170 rpm. Fungal material was removed via filtration through Miracloth, culture filtrate was acidified to pH 3.0 with HCl, EtOAc was added to the culture filtrate at a 1:1 (v:v) ratio and mixed for 30 minutes then vacuum filtered to remove remaining mycelial fragments. The filtrate was separated using a separation funnel and the organic phase collected. The aqueous phase was extracted two further times and the extracts combined. Water was removed from the pooled solvent fraction using anhydrous MgSO4 before drying using a rotary evaporator. This yielded 400 mg total crude extract.A 1 L culture of 18, 100 \u00c5, 10 \u00d7 250 mm) was used for reverse-phase chromatography, with Phenomenex Security Guard precolumn (Luna C5 300 \u00c5). UV absorbance was detected between 200\u2013400 nm with Waters 2998 diode array detector; mass spectrometry with Waters Quattro Micro; and approximate target compound abundance evaluated with Waters 2424 for ELSD. Mobile phases consisted of A: water with 0.05% formic acid; and B: acetonitrile with 0.05% formic acid. A gradient of 20% B to 90% B in 30 minutes was employed with flow rate of 5 mL/min. Compounds from the five major peaks were collected across five fractions (A-E) giving the following yields: A = 1.6 mg, B = 0.5 mg, C = 1.3 mg, D = 2.8 mg, E = 3 mg.A 90 mg sample of crude extract was initially assessed and fractionated using preparative HPLC. A Waters 2767 Sample Manager with Waters 2545 pump system, Phenomenex LUNA column  at 1.4 bar; dry gas (N2) at 8 L/min; dry temperature at 180\u00b0C. Ion transfer conditions as, ion funnel 1 RF at 200 Vpp; ion funnel 2 RF at 200 Vpp, hexapole RF at 200 Vpp; quadruple ion energy at 5 ev, quadrupole low mass set at 55 m/z; collision energy at 5.0 ev; collision RF ramping from 800 to 1500 Vpp; transfer time set at from 100 to 155 \u03bcs; pre-Pulse storage time set at 5 \u03bcs. Calibration was done with sodium formate (10 mM) through a loop injection of 20 \u03bcL of standard solution at the beginning of each run.LC-MS analysis was performed on the fractions with Dionex 3000RS UHPLC coupled with Bruker MaXis Impact Q-TOF mass spectrometer. An Agilent Zorbax Eclipse plus column  was used. Mobile phases consisted of A: water with 0.1% formic acid; and B: acetonitrile with 0.1% formic acid. After 5 minutes of isocratic run at 5% B, a gradient of 5% B to 100% B in 15 minutes was employed with flow rate at 0.2 mL/min, UV was set at 210 nm. Mass spectrometer was operated in electrospray positive ion mode with a scan range 50\u20133,000 1 and 2 and 1 mg of 7. Samples were analysed immediately by NMR spectroscopy.Each compound was further purified using an Agilent Zorbax C18 column  connected to an Agilent 1100 HPLC at a flow rate of 5 mL/min, monitoring absorbance at 210 nm. Mobile phases consisted of A: water containing 0.1% formic acid; and B: acetonitrile containing 0.1% formic acid. The following program was used to elute the column: 0 min, 80% B; 5 min, 80% B; 25 min, 100% B; 30 min, 100% B; 33 min 80% B; 38 min 80% B. Fractions containing target compounds were identified using ESI-HR-Q-TOF-MS and pooled. Organic solvent was removed using a rotary evaporator and the resulting aqueous solutions freeze dried yielding 2 mg each of 1 H, COSY, HSQC and HMBC NMR spectra were acquired in d4- MeOH (180 \u03bcL in 3 mm tube) on a Bruker Avance II 700 MHz spectrometer equipped with a TCI cryoprobe at 298 K. The solvent peak was used to calibrate the spectra.B. subtilis ATCC 6633 ; E. coli DH5\u03b1 ; S. cerevisiae Y10000 . 50 \u03bcl of each fraction was aliquoted into centrally bored wells in each assay plate and the plates incubated appropriately . Antimicrobial activity was determined by the presence of a zone of inhibition surrounding the central well.Chemical fractions obtained by preparative HPLC were dissolved to a concentration of 1 mg/mL in dimethyl sulfoxide. Microbial plates were prepared in 90 mm Petri dishes by resuspending microbial cells in the appropriate growth medium supplemented with 2, 3, 5-triphenyl-2H-tetrazolium chloride (200 \u03bcg/mL)\u2013C. strossmayeri mycelium, harvested from a two-week culture grown in PDB, using the E.Z.N.A\u00ae Fungal RNA Kit (OMEGA bio-tek). Isolated RNA was quality checked using RNA Analysis ScreenTape\u00ae (Agilent). Approximately 500 ng of total RNA was prepared for sequencing using the Illumina TruSeq Total RNA LT Kit (Illumina). The data were processed using RTA version 1.18.64, with default filter and quality settings. The reads were demultiplexed with CASAVA 1.8.4, allowing no mismatches. This was carried out at the Bristol Genomics Facility. RNA-seq data were processed using Galaxy QC and manipulation tools to trim the sequences followed by the TopHat RNA analysis tool to map the RNA-seq reads to the assembled genomes. These data were then viewed in Artemis to evaluate relevant expression levels of genes of interest. Partek\u00ae Genomics Suite was also used to map RNA-seq reads to assembled genomes and to genes of interest. RNA-seq data are available on the NCBI SRA database under the accessions: STUDY: PRJNA604530; SAMPLE: CBS 177.39 (SAMN13973684); EXPERIMENT: C.s (SRX7684385); RUN: AB_C_ACAGTG_L001_R1_001.fastq (SRR11032120).RNA was extracted from C. strossmayeri was fractionated using preparative HPLC. Five fractions were obtained, A-E, each containing one of the major peaks. These were examined for inhibitory activity against B. subtilis ATCC 6633, E. coli DH5\u03b1 and S. cerevisiae Y10000 using plate-based bioassays. Weak antimicrobial activity was detected in fractions A and E against B. subtilis  and fraction D the related bovistol D (2), both yellowish-white solids. Fraction C was identified as a sesquiterpene monomer strossmayerin (7). Compounds present in fractions A and B were produced at too low titre to yield sufficient product for structural elucidation.Initial preparative HPLC fractions were analysed using LC-MS and major components were further purified with semi-preparative HPLC. Structural elucidation was carried out with a combination of HRMS and 1D/2D NMR spectroscopic techniques; this led to the identification of 1 with the molecular formula C30H38O5, + 479.2792) with 12 degrees of unsaturation. The UV \u03bbmax at 290 nm also indicates the presence of an aromatic ring or conjugated multiple double bonds. 1 H, 13 C, COSY, HSQC and HMBC NMR spectra were acquired in d4- MeOH  instead of a methyl group connected to C4\u2019, HMBC correlation from H12\u2019 to C3\u2019 and C5\u2019confirmed the presence of the isolated double bond. The relatively high field methylene signal at 0.26/1.38 ppm (H13\u2019) and 0.80/1.13 ppm (H14\u2019) suggest the presence of a cyclopropane group. HMBC correlation from H13\u2019 and H14\u2019 to C4\u2019, C3\u2019 and C2\u2019 established the left-hand side of the structure. Key HMBC correlation observed from H15\u2019 to C1\u2019, C2\u2019 and C3\u2019 from the left side and C15, C2 from right-hand side established the connectivity between the two monomers.Bovistol B: High resolution MS data established 1 is proposed as a dimeric sesquiterpene; the structure of 2 was elucidated as the oxidised form of 1, where the C10 hydroxymethyl group is oxidised to a carboxyl group. 1, previously isolated from C. aegerita, has been characterised by MS and NMR spectroscopy and the relative configurations assigned by ROSEY correlations [8) has also undergone extensive structural characterisation [7 showed high similarity with the right-hand side of compound 1 and appears to be derived from delta 6-protoilludene 3 , this is then oxidised to illudin C (4), then illudin C3 (5). A dimerisation then takes place to yield prebovistol (6), this asymmetric dimerisation is predicted to proceed via an inverse-electron demand hetero-Diels-Alder (DA) mechanism.C. strossmayeri genome [1 -dependent enzyme, LepI from Aspergillus nidulans, can catalyse intramolecular DA and hetero-DA reactions involved in the biosynthesis of leporin B [C. strossmayeri genome when a tblastn search was performed. There are increasing reports of characterised intramolecular Diels-Alderases of natural origin [We are aware that this is not the first report of dimeric sesquiterpenes from basidiomycete fungi (e.g. spirodienone) , howevericicolin , howevereporin B , but siml origin , howeverS1 File(PDF)Click here for additional data file."}
+{"text": "Traditionally, chromosomal polymorphisms (CPMs) are normal genetic variants in individuals with no phenotypic variations. However, some studies have shown that CPM is related to reproductive diseases. We explored the influence of CPM on embryonic development and molecular karyotype in chromosomal translocation (CT) patients undergoing preimplantation genetic testing (PGT) between February 2013 and May 2019. Twenty-six cases with CPM and 56 controls with normal chromosomes were included. Furthermore, a 1:4 match pair analysis by female age included 39 cases with CTCPM and 185 controls with CT. There was no statistical difference in fertilization rate (78.48% vs. 78.33%), cleavage rate on Day 3 (90.32% vs. 89.16%), blastocyst rate (60.00% vs. 60.80%), and the high-quality blastocyst rate (36.31% vs. 35.22%) between CPM and normal chromosomes. The high-quality blastocyst rate of CTCPM was significantly lower than that for CT (26.78% vs. 38.89%). Moreover, there was no statistical difference in fertilization rate (70.65% vs. 70.37%), cleavage rate on Day 3 (88.67% vs. 89.53%), and blastocyst rate (48.48% vs. 53.17%) between CTCPM and CT. In addition, one CTCPM spouse had a lower high-quality blastocyst rate, especially of males with CTCPM. Abnormal embryo rates of CTCPM were significantly higher than those for CT (78.64% vs. 68.93%). Abnormal embryo rates were higher in both CTCPM and CPM paternal carriers with CT partners, respectively. For CT, CTCPM may have an impact on the high-quality blastocyst rate and embryonic molecular karyotype, especially in male patients. Patients with CTCPM are relatively rare, but this population would benefit from being explored using a larger sample size. Chromosomal polymorphism (CPM) is known as some minor variation of banding karyotype in normal people, and it is constant in an individual. Most variations consist of heterochromatic regions, which are enriched with highly repetitive satellite DNA sequences. These are located in the non-coding sequences of DNA and often occur in the long or short arms of chromosomes 1, 9, and 16. Polymorphisms of D and G group chromosomes  are included in the short arms, the satellites, and stalks of acrocentric chromosomes. Pericentric inversions on chromosomes 1, 2, and 9, and pericentric inversions and the size of long or short arms of chromosome Y are also included.in vitro fertilization (IVF)  . Howeveron (IVF) .In the current study on chromosomal translocation (CT) carriers, clinical and laboratory data of preimplantation genetic testing (PGT) were analyzed for CTCPM carriers and CT carriers, in order to explore the influence of CPM on embryo development and embryonic molecular karyotype in CT carriers.We retrospectively analyzed the clinical and laboratory data of PGT for CPMs, CTCPMs, CTs, and normal chromosomes at the Reproductive Medicine Center of the First Affiliated Hospital of Zhengzhou University from February 2013 to May 2019. All study methods were approved by the Institutional Review Board and Ethics Committee of the First Affiliated Hospital of Zhengzhou University and were conducted in accordance with relevant guidelines and regulations. All subjects enrolled in the study gave written formal consent for their participation.2 at 37\u00b0C for culture. After the above cells were cultured for 3 days, 60 \u03bcg/mL of colchicine with a concentration of 20 \u03bcg/mL was added. Colchicine was transferred to a 37\u00b0C constant temperature incubator for further culture for 1 h, and then conventional cell harvesting was performed, prefixation by hypo-osmosis, dropping tablet at 56\u00b0C, and baking tablet at 65\u00b0C, and G banding was used to capture the karyotype by automatic chromosome scanning system GSL-120 . At least five karyotypes were analyzed in each case, and 20 metaphase fission phases were counted. Twenty-six cases with CPM (30 cycles) and 56 controls with normal chromosomes (66 cycles) were investigated. Because the CTCPM carriers were far fewer than the CT carriers, a 1:4 match pair analysis ultimately identified 39 cases with CTCPM (47 cycles) and 185 controls with CT (188 cycles). Pairs were matched based on female age. The CTCPM carriers were divided into two subgroups: one spouse with CPM and a partner with CT, or one spouse with CTCPM and a partner with no abnormalities.The chromosome karyotype analysis was assessed in peripheral blood lymphocytes of parents. Peripheral blood samples 3 to 5 mL were extracted; 0.5 mL was inoculated into peripheral blood lymphocyte culture medium, mixed upside down, and placed in an incubator without COOne female underwent an early follicular phase long-acting regimen for controlled ovarian stimulation , and six2 and 5% O2 incubator.Mature oocytes (MII) obtained after oocytes retrieval were fertilized by ICSI. Meanwhile, the obtained sperm by masturbation was enriched by gradient centrifugation. And then, it was added into the ICSI dish, and an alive and well-formed sperm was selected and pressed vertically at the lower part of the sperm tail to immobilize. The sperm was absorbed by the injection needle, and the sperm was injected into the ooplasm after the oocyte was fixed. After ICSI, either G1-Plus  or K-SICM  microdroplets were used in the embryo culture medium, and the embryos were transferred to G2-Plus  or K-SIBM  microdroplets on the 3rd day. All embryos were cultured at 37\u00b0C in a 6% COOn the 3rd day of operation, the zona pellucida of embryo was drilled with the laser and then continued to be cultured in the blastocyst culture incubator. The blastocyst formed on the 5th or 6th day could be seen that trophoblast ectoderm cells were hatched from the pores. Two to six trophoblast ectoderm cells were absorbed by biopsy needle in the biopsy dish and placed at 37\u00b0C in an aerated incubator for PGT.2. The blastocysts were evaluated on Day 5/6 according to Gardener score: those scoring \u2265 3 BB were considered high-quality blastocysts. After embryo biopsy, vitrification was performed, and embryos identified as balanced or normal underwent frozen embryo transfer.Subsequently, single-nucleotide polymorphism (SNP) microarray or multiple annealing and looping-based amplification cycle (MALBAC) next-generation sequencing technology were used to assess these biopsied cells. Whole-genome amplification for trophectoderm (TE) cells obtained via biopsy was conducted using the QIAGEN REPLI-g Single Cell kit. After cell lysis, the samples were incubated in the polymerase chain reaction (PCR) machine and then placed at room temperature. The amplifications were microarray by HumanCytoSNP-12 microarray (Illumina) for SNP microarrays. On another way, the samples were preamplified in the PCR machine in the MALBAC preamplification mixture and then placed at room temperature and then incubated in the PCR machine in the MALBAC amplification mixture. The MALBAC amplification products were purified, and the data were sequenced by Illumina Hiseq 2500 using an ultrahigh-throughput sequencing system. After biopsied cell collection, the blastocyst was quickly transferred to a four-hole plate and put into an incubator at 37\u00b0C and 6% COt testing. The rates were compared by \u03c72 test and Fisher exact probabilities, and P < 0.05 was considered statistically significant. The test level of pairwise comparisons among three groups was corrected by Bonferroni tests, and P < 0.02 was considered statistically significant.Baseline materials were analyzed by independent All patients recruited were diagnosed by karyotype analysis of peripheral blood. First, the baseline data of 26 cases with CPM, including 30 cycles, and 56 controls with normal chromosomes, including 66 cycles, were not significantly different. Thirty-nine couples including CTCPM carriers and 186 CT carriers performed 47 and 188 PGT cycles, respectively. With the exception of body mass index (BMI), there were no significant differences between the two groups within the baseline information .CPM in humans is usually located in secondary constrictions, centromeres, satellites, and the distal long arms of Y chromosomes. Variants of the long arm of chromosome 1, 9, and 16 included 11 cases in couples with CTCPM and 5 cases in patients with CPM. Polymorphisms of D and G group chromosomes  included 5 cases in couples with CTCPM and 6 cases in patients with CPM. There were 16 cases with pericentric inversions on chromosomes 1 and 2, and 9 in couples with CTCPM, and 7 cases in patients with CPM. In addition, eight cases in couples with CTCPM and six cases in patients with CPM were recruited with polymorphism of chromosome Y.P = 0.95, P = 0.59, P = 0.82, and P = 0.81, respectively; In patients with CPM, 467 oocytes were collected, 395 of them were mature metaphase II stage (MII), and 310 (78.48%) fertilized normally. After fertilization, 280 embryos (90.32%) developed to Day 3, and 168 (60.00%) embryos developed to blastocysts on Day 6, of which 61 (36.31%) were evaluated as high-quality blastocysts. For patients with normal chromosomes, 910 oocytes were retrieved; 789 were MII, and 618 (78.33%) were fertilized normally. The number of embryos on Day 3 was 551 (89.16%), and 335 (60.80%) of them reached blastocyst stage, of which 118 (35.22%) were scored as high-quality blastocysts. When comparing the two groups, there were no statistical differences in fertilization rate, cleavage rate on Day 3, blastocyst rate, and high-quality blastocyst rate between the two groups . Furthermore, there was no statistical difference in fertilization rate, cleavage rate on Day 3, and blastocyst rate between the two groups  fertilized normally. After fertilization, 493 (88.67%) embryos developed to Day 3, and 239 (48.48%) embryos developed to blastocysts on Day 6, of which 64 (26.78%) were evaluated as high-quality blastocysts. For patients with CT, 3,132 oocytes were collected; 2,755 were MII, and 2,204 (70.37%) were fertilized normally. The number of embryos on Day 3 was 1,973 (89.53%), and 1,049 (53.17%) of them reached blastocyst stage, of which 408 (38.89%) were scored as high-quality blastocysts. However, when the two groups were compared, the high-quality blastocyst rate of patients with CTCPM was significantly lower , especially for male embryos  blastocysts were normal or balanced, and 86 (61.87%) had an abnormal molecular karyotype. For patients with normal chromosomes, 2 were amplified abortively, 86 (29.66%) blastocysts were normal or balanced, and 204 (70.34%) had an abnormal molecular karyotype. When comparing the two groups, there was no statistical difference in abnormal embryos rates .2 = 7.62 P = 0.01; The numbers of blastocysts biopsied in patients with CTCPM and patients with CT were 215 and 957, respectively. For patients with CTCPM, 9 were not diagnosed successfully, 44 blastocysts (21.34%) were normal or balanced, and 162 (78.64%) had an abnormal molecular karyotype. For patients with CT, 72 of a total of 957 were amplified abortively, 275 (31.07%) blastocysts were normal or balanced, and 610 (68.93%) had an abnormal molecular karyotype. When comparing the two groups, abnormal embryo rates were significantly higher in patients with CTCPM . However, when compared with females with CT, there were no significant differences in maternal carriers  and embryo euploidy , pairs wEmbryo development suggests that CPM is associated with multinucleated embryo formation  and has Our data showed that although the abnormal embryo rates of CPM were higher than normal, there was no statistical difference. However, compared with the CT group, abnormal embryos rates were significantly higher in patients with CTCPM, which was mainly attributed to one spouse having CTCPM, particularly the male partner. This may indicate that in patients with CT, CPM amplified its effects, rather than remaining inactive. In colorectal cancer, chromosomal instability is also proposed to shape aneuploidy landscapes . MoralesThere were some limitations to our study. Because of the relative rarity of patients with CTCPM, the sample size of the study group was small; in future studies, it would be important to expand the sample size.https://figshare.com/articles/dataset/For_Frontiers_in_Physiology/13128011/1).The genotype and sequencing data has been deposited into Figshare  for the publication of any potentially identifiable images or data included in this article.GL conceived and designed the experiments. YG and YCS selected and supervised suitable patients. GL performed comprehensive chromosome screening. WN and JX performed next-generation sequencing and sequencing data analysis. YCS and YG recruited the patients, retrieved oocytes, and transferred embryos. YPS provided overall supervision. GL and WS drafted the manuscript. All authors reviewed the final 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."}
+{"text": "The elevated TM (>13.3 TU/mL) had a predictive value of 96.0% for excluding VTE, and the normal TM had a predictive value of 90.4% for excluding sepsis. The overall 28-day mortality of these patients with D-dimer >5.0 ug/mL was 14.2%, the TAT level on admission was independently associated with 28-day mortality . Conclusions: The medical emergencies associated with markedly elevated D-dimer levels were revealed, specific markers of endothelial dysfunction and thrombin generation measured by automatic analyzer have the potential to distinguish diagnoses and predict outcomes in these patients.Background: Markedly elevated D-dimer levels can occur in emergency patients with various clinical situations, and is likely to indicate the presence of coagulopathy, rapid differential diagnosis was crucial for them. Methods: D-dimer was detected in consecutive 813 patients entering the emergency department of our hospital, for the patients with D-dimer levels above 5.0\u2009\u00b5g/mL, the final diagnoses and 28-day mortality were confirmed, and the levels of thrombomodulin (TM), thrombin-antithrombin complex (TAT) and plasmin-antiplasmin complex (PAP) on admission were detected. Results: There were 148 emergency patients with D-dimer levels higher than 5.0\u2009\u00b5g/mL mainly due to sepsis, malignancy, trauma, venous thromboembolism (VTE), cerebrovascular accident, and so on. Both of the TM and TAT levels among these diagnoses were significantly different ( Emergency patients who present with markedly elevated D-dimer levels are a concern, as they were more likely to have associated coagulopathy3. However, markedly elevated D-dimer levels can occur in various clinical situations and is commonly hard to distinguish rapidly1, this may due to that as an indirect marker of activation of coagulation as well as fibrinolysis, D-dimer alone can\u2019t indicate the specific state. For instance, with similarly high D-dimer, hyperfibrinolysis is considered to be the major manifestation and therapeutic target of trauma-induced coagulopathy, meanwhile, endothelial dysfunction, excess thrombin generation and fibrinolysis shutdown are the feature of sepsis-induced coagulopathy5. Hence, more specific coagulation markers may be needed for distinction of these conditions.D-dimer is a biomarker of fibrin formation and degradation and has been used routinely for variety of diagnostic purposes, including excluding venous thromboembolism (VTE), determining the optimal duration of anticoagulation in VTE patients, and diagnosing disseminated intravascular coagulation (DIC) In this study, we aimed to investigate the clinical features, diagnoses and outcomes of emergency patients with markedly elevated D-dimer levels on admission; Meanwhile, markers of endothelium, thrombin and plasmin in these patients were detected and evaluated, we assumed that some of these markers might has discriminatory or prognosis value in patients with markedly elevated D-dimer level, i.e. a high possibility of coagulopathy.7, we selected the average value of 5.0\u2009\u00b5g/mL to define a markedly elevated of D-dimer. A total of 166 (20.4%) patients had D-dimer levels above 5.0\u2009\u00b5g/mL , within 24\u2009hours after admission, and the samples were reserved for further detection.D-dimer was detected in consecutive 813 patients entering the emergency department of our hospital from July 2019 to October 2019. As 3.0\u20137.0 ug/mL of D-dimer levels had been used as the cutoff of item in several DIC criterias, or to define high risk of VTEThis study was approved by the Ethics Committee of Tongji Hospital , and written informed consent was obtained from all patients or their family members. All methods were carried out in accordance with relevant guidelines and regulations.8. In addition, in the other 647 patients with D-dimer \u22645.0 ug/mL, 338 (52.2%) were hospitalized, and there were no further investigation on them due to the high rate of lost to follow-up.Eighteen of the 166 patients were lost to follow-up or died before a diagnosis was confirmed, and were excluded from the study. The remaining 148 patients were all hospitalized, and a retrospective review of their characteristics and outcomes was performed using the hospital\u2019s electronic medical records system, the results of breathing rate, mean arterial pressure, bilirubin and creatinine on admission were recorded to describe multiple organ dysfunctions of each enrolled patientg for 10\u2009minutes to obtain platelet-poor plasma, and D-dimer was detected using a STA-R analyzer and STA-Liatest D-Di reagent  based on the latex immune turbidimetric assay . As markers of endothelium, thrombin and plasmin, the levels of thrombomodulin (TM), thrombin-antithrombin complex (TAT) and plasmin-antiplasmin complex (PAP) were detected using a HISCL 5000 analyser and original chemiluminescence reagents , the reference intervals of these three markers were suggested by the manufacturer and had been validated before application in our hospital.Blood samples were collected from each patient within 24\u2009hours after admission. Samples were collected into vacuum tubes  with 3.2% sodium citrate for coagulation tests. The samples for coagulation tests were centrifuged at 2700 \u00d7 9, the presence of infection was confirmed by pathogen detection or comprehensive judgment of our clinician (Dengju Li). Cases of malignancy were confirmed by necessary pathological and imaging examinations.All cases of VTE, aortic dissection, and cerebrovascular accident were confirmed by Doppler ultrasound, computed tomography or magnetic resonance imaging. The diagnosis of sepsis was based on the sepsis 3.0 definitionP-value of <0.05 was considered statistically significant. Data were analyzed using SPSS 21.0 for Windows .Quantitative variables are presented as median (interquartile range). Multiple comparisons among biomarkers of each diagnosises were performed using Kruskal-Wallis H test. Between survivors and non-survivors, comparisons of continuous variables were performed using the Mann-Whitney test, and categorical variables were evaluated using Fisher\u2019s exact test or Pearson\u2019s Chi-squared test, where appropriate. Categorical and consecutive variables were evaluated by logistic regression analysis for their ability to predict 28-day mortality. A p\u2009<\u20090.001).The 148 consecutive emergency patients with D-dimer levels >5.0\u2009\u00b5g/mL included 89 men and 59 women, with a mean age of 56.5 years (range 12\u201388 years). The primary diagnoses and levels of coagulation biomarkers on admission of these patients are shown in Table\u00a0P\u2009=\u20090.012).Due to the patients with diagnoses of sepsis and VTE had the highest and lowest TM levels, respectively, diagnostic values of TM for sepsis and VTE were evaluated Table\u00a0. In thesP\u2009=\u20090.004) and PAP (P\u2009<\u20090.001) levels in these emergency patients, the correlation coefficients were 0.236 and 0.558, respectively. No significant correlation was found between D-dimer and TM levels (P\u2009=\u20090.570).Correlation analysis showed that D-dimer levels had significant correlations to TAT (P\u2009<\u20090.05), these parameters were further evaluated by the multivariable logistic regression analysis . The comparison of age, sex ratio, biomarkers of coagulation and organ function between survivals and non-survivals were showed in Table\u00a010, and the ISTH has also endorsed the role of D-dimer testing in the diagnostic algorithm for DIC11. In our study, the emergency patients with D-dimer levels higher than 10 times of the upper normal limit mainly due to sepsis, malignancy, trauma, venous thromboembolism, cerebrovascular accident, and so on. A higher hospitalization rate in these patients were found, comparing to patients with D-dimer \u22645.0 ug/mL (89.2% vs 52.2%). The good correlation between D-dimer and TAT or PAP in our study confirmed the large generations of thrombin and plasmin in these patients, and indicated the existence of coagulopathy.Clinicians routinely use D-dimer levels as part of a diagnostic algorithm to exclude a diagnosis of VTE12. In sepsis, release of TM from the surface of vascular endothelial cells into the blood circulation partly through proteolytic cleavage by neutrophil elastase14. Hence, it\u2019s considered to be a biomarker for endothelial cell damage, and that plasma TM levels are elevated in patients with sepsis has been described previously15. On the other hand, no association of TM with venous thromboembolism has been found in a former study16, also as shown in our study, this may be explained as when VTE is the primary or only diagnosis of a patient rather than being secondary to trauma or infection et al., endothelial dysfunction commonly is not the major factor. Hence, it\u2019s reasonable that in our emergency patients with markedly elevated D-dimer, a low or high TM level could be helpful to exclude sepsis or VTE, respectively. In addition, TM had higher on-admission levels in non-survivors compared with survivors, which is consistent with previous studies18, however, it\u2019s not independently associated with the outcome according to the logistic regression analysis.TM, also called CD141, is an anticoagulant protein constitutively distributed on the surface of vascular endothelial cells. TM binds to thrombin, then this complex activates protein C and forms the major physiological anticoagulant pathway20. Although it\u2019s not needed to distinguish trauma patient from others by blood testing, TAT might be used for prognosis as it was associated with the outcome and independent of other organ dysfunctions in this study. In addition, as both of VTE and artery dissection could present markedly elevated D-dimer and similar symptoms21, patients with artery dissection showed higher TAT level, this indicates more obvious coagulation activation in artery dissection than in VTE and the potential of differential diagnose, and also can be used to explain that why patients with artery dissection often show a DIC-like coagulopathy22. However, a study with larger sample size is needed to confirm it. Relatively, as a biomarker of plasmin generation, PAP seemed to be of little differential or prognosis value in patients with markedly elevated D-dimer.As a biomarker related to thrombin generation, TAT had the highest level in trauma patients of our study, that has also been described in previous reportsAccording to these results, it seemed that detection of these specific coagulation markers could support a further explanation of elevated D-dimer mechanistically, moreover, unlike most previous studies, the TM, TAT, and PAP levels were all measured by an automatic analyzer in our study, the shorter turn-around time and simpler methodology ensure that these biomarkers can be used for emergency case.7, this definition might still be arbitrary.This study had some limitations. The TM, TAT and PAP levels of patients with D-dimer less than 5.0 ug/mL had not been detected and evaluated. In addition, we defined the markedly elevated D-dimer as higher than 10 times of the upper normal limit (5.0 ug/mL) according to previous studiesNevertheless, the current results revealed that medical emergencies commonly associated with markedly elevated D-dimer levels, which were correlated with more productions of thrombin and plasmin, and indicated the risk of coagulopathy. Plasma TM and TAT levels measured by automatic analyzer have the potential to distinguish diagnoses and predict outcomes in these emergency patients."}
+{"text": "Blastocystis is one of the most common protozoa found in the human gut and are genetically diverse and widely distributed around the world. Nonspecific and inconsistent symptoms have been associated with this protozoon; thus, its clinical importance remains controversial. Our aim was to estimate the relative frequency of Blastocystis subtypes 1, 2, and 3, which are the predominant subtypes reported in South America, based on conserved regions of SSU rDNA sequences and determine the factors associated with them. A total of 116 Blastocystis-positive stool samples were processed using conventional PCR with Blastocystis-specific primers. We identified subtype 1 (10.3%), subtype 2 (7.8%), subtype 3 (25.0%), and mixed subtype infections (8.7%). However, we could not identify any Blastocystis subtypes in 48.3% of the samples; therefore, it is likely that other subtypes were present in the area. No association was found between any gastrointestinal symptom and single or mixed Blastocystis subtypes. We found a statistically significant association between Blastocystis subtype 2 and irritable bowel syndrome ; however, the number of samples with IBS was small (n= 4). There was no association between the Blastocystis subtypes and any epidemiological variable studied. In rural populations, we only identified subtype 1, while in urban and periurban populations, we identified subtypes 1, 2, and 3. Blastocystis is a protozoon found in the gut of humans and animals [ animals . This mi animals  distribu animals . Subtype animals , and sub animals . Subtype animals ,6 and th animals ; subtypeBlastocystis has been studied widely, but it remains unclear whether it is pathogenic [Blastocystis and gastrointestinal symptoms, such as abdominal pain, diarrhea, vomiting, constipation, and irritable bowel syndrome (IBS) [Blastocystis and health issues [Blastocystis could be considered normal or beneficial for the microbiota [Blastocystis transmission is generally associated with poor access to healthcare and unsanitary living conditions around the world [Over the last decade, thogenic . Some stme (IBS) ,9,10, whh issues ,12 or sucrobiota ,14. Blashe world ,15.Blastocystis subtypes in Peru [Blastocystis infection has been reported in some Peruvian cities [Blastocystis  in urban and periurban human populations in Arequipa city, Peru. We also assessed the association of these subtypes with gastrointestinal symptoms and sanitary living conditions.In South America, there have been some studies focused on this protozoon ,16,17,18n cities ,20,21 ann cities ,23. Our The protocol for this study was reviewed and approved by the Institutional Review Boards of the Universidad Peruana Cayetano Heredia (reference number 18006). Written informed consent was provided by all participants prior to the study. Written assent and parents\u2019 informed consent was provided for minors under the age of 18.Arequipa city is the second-most populous city in Peru, with approximately a million inhabitants, located in the country\u2019s southern highlands at 2400 meters above sea level . ArequipBlastocystis-infected participants from different districts of Arequipa, both with and without gastrointestinal symptoms, both adults and children, and from both sexes. The recruitment method was described elsewhere [We obtained stool samples from 116 lsewhere . All parEach participant\u2019s sample was collected in a sterilized plastic mouth flask without any additive. All participants were instructed about the correct procedure to collect their stool sample. Those instructions were given in a short letter to each participant. At that point, we answered any questions that the participants had. Blastocystis-positive samples were aliquoted in cryovials and stored at \u221280 \u00b0C.All stool samples were analyzed using a rapid concentration saline solution. We examined the pellet under light microscopy at 400\u00d7 magnification and confirmed with blue methylene-stained stool smear under 1000\u00d7 magnification, as was explained previously . BlastocBlastocystis-specific primers based on the conserved regions of SSU rDNA sequences: F:5\u2032-GAAGGACTCTCTGACGATGA-3\u2019/R:5\u2032-GTCCAAATGAAAGGCAGC-3\u2019 (351 bp) for subtype 1 [Blastocystis subtypes 1 and 3 were one cycle of initial denaturation at 94 \u00b0C for 3 min, followed by 35 cycles for denaturation at 94 \u00b0C for 30 s, annealing at 54 \u00b0C for subtype 1 and 57 \u00b0C for subtype 3, extension at 72 \u00b0C for 60 s, and an additional cycle for the final extension at 72 \u00b0C for 5 min. For subtype 2, the PCR conditions were one cycle of initial denaturation at 94 \u00b0C for 5 min, followed by 35 cycles for denaturation at 94 \u00b0C for 30 s, annealing at 58 \u00b0C for 50 s, extension at 73 \u00b0C for 90 s, and an additional cycle for the final extension at 73 \u00b0C for 7 min. Electrophoresis was performed by adding 8 \u00b5L of the PCR products to a 1.7% agarose gel and staining with GelRed\u00ae Nucleic Acid Gel Stain, 10,000\u00d7 in Water  for 30 min at 100 V. Blastocystis samples were considered positive for a subtype when a clear visible band of the correct size for the primer pairs were observed in the gel under UV light.We used 200 mg of stool samples stored at \u221280\u00b0C to extract DNA using the Norgen Stool DNA Isolation Kit . PCR was performed with 25 \u03bcL of 2X PCR Taq Plus MasterMix , 100 ng (~2 ng/\u03bcL) of a DNA template, and 200 nM of ubtype 1 , and F:5ubtype 1  and adjuBlastocystis subtypes frequencies per study area, the composition of subtypes within households, and other variables of interest, such as household sanitary conditions, demographics, and food consumption behaviors. The association of Blastocystis subtypes with gastrointestinal symptoms and sanitary living conditions were analyzed using chi-square and Fisher\u2019s exact tests. The association between Blastocystis subtypes and the patients\u2019 ages was analyzed using the Kruskal\u2013Wallis test. For the epidemiological analysis, we selected variables that could explain the possible source of Blastocystis subtypes, as were suggested in previous studies [Blastocystis subtypes and potential risk factors. We also explored the spatial distribution of subtypes in the city. All analyses were conducted using R 3.6.2 [Descriptive analysis was used to explore  studies ,29. Odds R 3.6.2 .Blastocystis-positive individuals, 50 of which were co-infected with other protozoa. The participants\u2019 ages ranged from 2 to 82 years old and 50.4% were female. Out of the 116 stool samples analyzed, 61.2% came from periurban areas, 33.6% came from urban areas, and 5.2% from rural areas.We obtained DNA from 116 Blastocystis subtype 3 was the most prevalent subtype, was found across the city, and was the only one subtype identified in many districts. Blastocystis subtype 2 was the only subtype identified in two districts. Blastocystis subtypes other than 1, 2, or 3 (unknown subtype) were present in all areas across the city. Tiabaya district, located in the southwestern area of the city where sewage water is disposed into the river, showed the presence of Blastocystis subtypes 1, 2, and 3, including unknown subtypes. The map also shows a concentration of unknown subtypes in the northeastern part of the city. Participants were enrolled from urban, periurban, and rural areas across the city . The spaBlastocystis subtype 3 was the most prevalent in the study sample (25.0%), followed by subtypes 1 (10.3%) and subtype 2 (7.8%). The most common mixed subtypes identified were subtypes 1 and 3 (7.8%), followed by subtypes 1, 2, and 3 (0.9%) . The median age with single-subtype infections was 36.5 years old, whereas the median age with mixed infections was 16.5. Blastocystis subtypes in each study area, with Blastocystis subtype 3 being the most frequent in both urban and periurban areas . Subtype 2 was the least frequent in periurban areas (4.2%) and subtype 1 was the least frequent in urban areas (2.6%); only subtype 1 was identified in participants from rural localities (We identified 43.1% of samples with single subtype infections and 8.7% samples with mixed subtype infections. 3 (0.9%) . We coulcalities .Blastocystis subtypes 1, 2, and 3, as well as unknown Blastocystis subtypes in each household (n = 78 households), where the most common composition involved subtypes 1 and 3 with 10.3% (8/78 households). Twenty households presented with only one subtype , where subtype 3 was most prevalent in urban and periurban areas (18/42 and 14/33 respectively). A total of 61.5% of households had an unknown subtype: urban 15/33, periurban 29/42, and rural 4/5. Additionally, we observed the presence of the three Blastocystis subtypes  in one participant from an urban household, and the presence of the three subtypes and unknown subtype(s) in one periurban household but in four different household members. We also identified other intestinal protozoa in the stool samples. The different coinfection combinations between Blastocystis subtypes and other protozoa are detailed in Blastocystis-positive participants who were negative to other microorganisms. Fisher\u2019s exact test showed no association between single-subtype infections and individual gastrointestinal symptoms. However, there was a statistically significant association between Blastocystis subtype 2 and IBS . In order to exclude intestinal symptoms and disorders caused by other protozoa, the statistical analysis of clinical manifestations was performed only with n = 10) as an additional subtype group to compare, though we excluded the rural area from the analysis because we had only one participant in that category. We did not find any statistical association between the participants\u2019 age and infection with specific Blastocystis subtypes (p = 0.993). We also did not find any statistically significant association between Blastocystis subtypes, either single or mixed, and sanitary living conditions  were more prevalent in this area of Peru [Blastocystis subtypes circulating in human Peruvian populations increased the chances of different combinations of mixed subtype infection between subtypes 1, 2, 3, and others. These mixed infections could lead to low accuracy in the amplification process for primer competition or inhibition for the presence of the whole stool DNA in the sample [Blastocystis subtype; however, these hypotheses should be studied in detail.Regarding the high number of non-identified  of Peru . Furthere sample ,27, whicBlastocystis subtypes 1, 2, and 3 were reported in Brazil, Turkey, and Iran [Varying prevalence of dual-mixed subtype infections between and Iran ,32,34. Hand Iran ,39,40,41and Iran , or evenBlastocystis subtypes across communities and districts remain unknown.We assumed that human migration in Arequipa city established a complex mixing of subtypes between periurban and urban areas across the city. Unfortunately, we could not extend this finding to rural areas because we had few participants from this area. Our findings also showed clustering in the distribution of subtypes between rural and urban/periurban areas. However, the wide spatial distribution of subtypes all over Arequipa city contrasted with the distribution of subtypes in Turkey, where subtypes 1, 2, and 3 were restricted to cities and subtypes 5, 6, and 7 occurred in less urban areas . Other fBlastocystis sources. Only subtype 3 has been associated with drinking unpurified water [Blastocystis. These findings were similar to those from Spain, which showed no association with drinking water or hand- or food-washing behavior [Blastocystis infection and untreated water consumption, feces disposal practices, and inadequate hand-washing habits [Blastocystis in the human gut. However, this factor should be studied because it has previously been found to be an important factor that affects Blastocystis prevalence, subtypes, and the pathogenicity linked to gut microbiota [There is limited available research about the role of sanitary conditions on transmission routes and ed water . Other ved water . We did behavior . Howeverbehavior . Generalg habits ,45. Thercrobiota ,47. Our Blastocystis subtypes. A study from Rio de Janeiro, Brazil, reported statistical differences between the prevalence of Blastocystis in men and women, and proposed that economic activities could explain this finding [Blastocystis subtypes and sex, despite many adult women working in the household in our study population, which could increase the risk of infection due to food contamination [Demographic characteristics were explored as another factor that could be linked to the distribution of  finding . Contrarmination ,48, lackmination , and dommination . On the mination ,42,49. WBlastocystis pathogenicity was suggested in 1986 [Blastocystis subtypes 1, 2, and 4 are related to IBS [ in 1986 . Nowaday in 1986 ,9, which in 1986 ,52,53. Ad to IBS .Blastocystis subtypes that are less common in this population. We also recommend exploring the likely association between subtypes 2 and 3 with IBS in murine models to discard this pseudo-association or conducting a case-control study with IBS patients. Our study contributes to a better understanding of the molecular epidemiology of this protozoon and its potential impact on clinical manifestations. Our findings also form the basis for future research and developing new hypotheses about the distribution and clinical manifestations of Blastocystis subtypes in Peru and Latin America.An important limitation of our study was not considering the potential presence of subtypes reported at lower proportions in South America and expecting similar subtype frequencies as those previously reported in Peru . Future Blastocystis subtypes 1, 2, and 3 in rural, urban, and periurban areas of Arequipa city in one of the few molecular studies of this protozoon conducted in South America. Even though subtypes 1, 2, and 3 are the most common worldwide and in South America, in our study population in Arequipa, they only accounted for 51.8% of the Blastocystis-positive samples, suggesting the presence of other highly prevalent subtypes that should be studied. No significant association was found between sanitary living conditions and any specific Blastocystis subtypes. We report the presence of"}
+{"text": "Complementary actions of the neocortex and the hippocampus enable encoding and long-term storage of experience dependent memories. Standard models for memory storage assume that sensory signals reach the hippocampus from superficial layers of the entorhinal cortex (EC). Deep layers of the EC on the other hand relay hippocampal outputs to the telencephalic structures including many parts of the neocortex. Here, we show that cells in layer 5a of the medial EC send a copy of their telencephalic outputs back to the CA1 region of the hippocampus. Combining cell-type-specific anatomical tracing with high-throughput RNA-sequencing based projection mapping and optogenetics aided circuit mapping, we show that in the mouse brain these projections have a unique topography and target hippocampal pyramidal cells and interneurons. Our results suggest that projections of deep medial EC neurons are anatomically configured to influence the hippocampus and neocortex simultaneously and therefore lead to novel hypotheses on the functional role of the deep EC. Interplay between the hippocampus and the neocortex is a central tenet of theories for systems memory consolidation . The entRecent discoveries revealed complex and distinct input\u2013output interactions of molecularly defined subtypes of neurons in the deep layers of medial EC (MEC), suggesting their functions extend beyond passing on hippocampal outputs. Layers 5a (L5a) and 5b (L5b) of the EC are genetically distinct with differential input\u2013output organizations. L5a but not L5b is the sole origin of the long-range telencephalic outputs of the EC , whereasAn intriguing possibility, noted in experiments with classic retrograde tracers, is that deep layers of MEC might also project to the hippocampal CA1 region, which provides the deep EC with hippocampal output . Such reHere we reveal that, neurons in L5a, but not L5b, of the MEC project to hippocampal CA1. We establish that the telencephalon-projecting neurons copy their outputs directly to pyramidal and subclasses of interneuron populations in CA1. Our data define a novel anatomical framework for layer 5a of the MEC to coordinate neocortical and hippocampal networks.To find out whether neurons in either L5a or L5b project to the hippocampus we injected retrograde adeno-associated viral vectors (AAVs) expressing green fluorescent protein (GFP) or mCherry into intermediate hippocampus . As wellRbp4-Cre; Rbp4-Cre mice, fluorescent signal was detected mainly in L5a  were found to be fluorescently labelled  and stratum moleculare (SM) of CA1 , axons fRbp4-Cre mouse line  or nucleus accumbens (NucAcb), and a Cre-dependent fluorescent reporter virus into the MEC . With thuse line . These rEC outputs have diverse targets, including the entire cortical mantle as well as parts of basal ganglia and amygdala . To testWe injected the MAPseq barcode RNA virus library into the full dorso-ventral extent of the deep MEC  and quanWhich cell types within the hippocampus receive signals from L5a of MEC? While the compartmentalized arrangement of axons from L5a of MEC suggests selective targeting of CA1 layers, axonal topography does not necessarily reflect functional connectivity. Therefore, we targeted viral vectors expressing a channelrhodopsin2\u2013mCherry conjugate to L5a neurons in MEC and tested for connectivity using whole-cell ex vivo patch-clamp recordings from CA1 neurons.n = 64 cells, 27 mice, We focused initially on responses of pyramidal cells. Brief light pulses evoked depolarizing subthreshold postsynaptic potentials (PSPs) in 64% of SP pyramidal neurons recorded at their resting membrane potential  that were relatively invariant from trial to trial (The majority of PSPs (20 out of 26) maintained their polarity when the membrane potential was adjusted from rest (Vm = \u221266.7 \u00b1 0.6 mV) to \u221250 mV, indicating that they were glutamatergic. In these neurons, EPSPs were maintained when GABAmatergic . The resto trial  and wereto trial . Consistto trial . TogetheA smaller population of pyramidal neurons showed PSPs with characteristics of indirect inhibitory connections (6 out of 26). These responses either reversed polarity when the cell\u2019s membrane potential was held above the chloride reversal potential  or had aFS and non-fast spiking: SPNFS; FS including PV+ interneurons, 46% of the SPNFS, 64% of SR, and lower proportions of recorded neurons in SL (31%), SM (23%), and SO (16%). Except for interneurons in SO, PSPs in all layers showed characteristics of monosynaptic connectivity KL100Gsat/Mmucd). The Vgat-Venus mouse line was generated by Dr. Atsushi Miyawaki at RIKEN, Wako, Japan Hze/J; Jax 022730). Wild-type mice were obtained from Charles River Laboratories C57Bl6J stock. Double transgenics were generated by crossing the Rbp4-Cre line to the Vgat-Venus or the Pvalb-Flp lines. Rbp4-Cre and Pvalb-Flp lines were maintained as heterozygous and all mice were on C57Bl/6 background.All animal experiments were approved by the University of Edinburgh animal welfare committee and were performed under a UK Home Office project license. The o, Japan . The PvaX) between the transverse sinus and lambdoid suture . The injection pipette was at a 9\u00b0 angle towards the caudal end of the brain. 100 nl of virus was slowly released at four Z-depths 3.0, 2.8, 2.6, and 2.4 mm from the surface of the brain. Three minutes after delivering the virus the injection needle was retracted to the next injection depth until the most dorsal location where 10 min past before the needle was fully retracted. For anterograde tracing of L5a axons and terminals AAV-EF1a-DIO-mCherry , AAV-CAG-FLEX-GFP , and AAV-hSyn-FLEx-mGFP-2A-Synaptophysin-mRuby (Addgene:71760) were used.Eight- to fourteen-week-old male and female mice were used in all experiments. For targeting deep MEC a craniotomy was made 3.4\u20133.65 mm lateral to the bregma  or rAAV2retro-hSyn-mCherry  was injected at each depth.For injections in CA1 a craniotomy was made 3.5 mm lateral and 3.30 mm caudal to the bregma in C57BL6J mice. 100 nl virus was delivered at 3.0, 2.8, 2.6, 2.4, and 2.2 mm from the surface. For retrograde labelling of hippocampus-projecting EC neurons, 100 nl of a To assess whether telencephalic projection neurons in L5a of MEC co-express Etv1 , fluoroprAAVretro-EF1a-Cre-WPRE  was made using an AAV-retro helper plasmid (Addgene plasmid ID 81070) as described previously  viruses were injected either in NucAcb  or RSC  in wild-type mice. A Cre-inducible reporter virus was also injected in the MEC as described above. GFP expression driven by the CAG promoter at the target site was used for verification of injection location.To achieve Cre expression in target-specific subpopulation of L5a neurons in the MEC , a rAAVreviously . A cocktAAV2-EF1a-DIO-hChR2(H134R)-mCherry-WPRE-pA  in the MEC of the Rbp4-Cre or the Rbp4-Cre X Vgat-Venus or the Rbp4-Cre X Pvalb-FLP double transgenic mice. Additionally, in order to fluorescently label PV-expressing interneurons in CA1 a Flp recombinase-dependent AAV vector was constructed in house and injected in the hippocampus . To produce the virus, the viral construct pAM-FLEX-GFP (12 genome copies [GC]/ml).To achieve expression of channelrhodopsin-2 in L5a axons, we injected FLEX-GFP   or 2% bovine serum albumin  in 0.3% PBST (PBS with 0.3% Triton X-100) for 2 hr. Sections were then transferred to primary antibody solutions made with either 5% NGS or 2% BSA in 0.3% PBST and incubated overnight. After three washes, each 20 min, in 0.3% PBST, sections were transferred to secondary antibody solutions made with 0.3% PBST and, if required, NeuroTrace 640/660  and incubated overnight. After three washes, sections were incubated in DAPI  solution made in PBS for 20 min at room temperature, where required, and then mounted on microscope slides using Mowiol 4-88 (Aldrich: 81381). Slides were covered with glass coverslips and left to dry overnight at 4oC in the dark. The following primary antibodies were used: rat anti-mCherry , chicken anti-GFP , rabbit anti-Etv1  using either the Zeiss LSM 800  or Leica SP8 confocal microscope  at \u00d720 magnification. Regions of interest were drawn around L5a and cell counting was carried out either manually using Fiji software or using cell-counting tools in Vision4D .The boundaries for L5a were determined using either a DAPI or Neurotrace counterstain. The border between medial and lateral divisions of EC was determined in each section using layer 2 as a guide \u2013 in lateral EC, layer 2 is separated into two distinct layers, while this separation is not seen in medial EC .The distribution of axons of L5a neurons in the radial axis of the hippocampal CA1 was quantified in slide-scanner images  or confon = 5 mice). GFP and mCherry fluorescence signals were amplified with immunostaining as described previously. The borders of CA1 were drawn on brain section images using a customisable digital microscopy analysis platform (Visiopharm), and the proximo-distal border was defined as the border equidistant from the proximal and distal ends of CA1. The mean fluorescence density, defined as the total number of pixels above a set threshold in an area divided by the total number of pixels in the area, was measured in proximal and distal CA1. The threshold was determined manually by ensuring that only pixels representing axons were detected as signal. A median unsharp background correction was used to remove background noise from axons outside of the focus plane of the image. The mean fluorescence density values were then normalized within each brain by scaling the values such that the total fluorescence density value  in each brain is equal to 1.A Zeiss Axioscan slidescanner was used to image every second brain section at \u00d710 magnification. The projection strengths of MEC L5a neurons to proximal and distal halves of CA1 were quantified in horizontal sections located at depths between 2.56 and 4.12 mm from the surface of the brain, with injection sites located across medial, mid and lateral MEC  provided by the MAPseq facility (Cold Spring Harbor Laboratories). To cover the whole mediolateral and dorso-ventral extent of the MEC virus was injected in two locations: 3.4 and 3.6 mm lateral to bregma. 100 nl virus was delivered to 3.0, 2.8, 2.6, and 2.4 mm below the surface of the brain. After 44 hr, mice were sacrificed, brains were extracted and immediately immersed in oxygenated cold artificial cerebrospinal fluid (ACSF prepared in ultrapure water). All surgical tools and surfaces were treated with RNaseZAP (Invitrogen) prior to the start of the experiment and in-between samples. 400 \u03bcm fresh sagittal brain sections were cut using a vibratome . The isocortex division included the somatosensory, motor, visual, posterior parietal, anterior cingulate, and retrosplenial areas combined. The CNU division was restricted to striatum. The olfactory/cortical subplate division (Olf/Ctxsp) was a combination of olfactory areas and cortical subplate including amygdalar nuclei. The remaining two divisions were dorsal (dHip) and ventral hippocampus (vHip) including subiculum. Some brain areas were excluded from the study because of the difficulty in dissecting or identifying brain areas in the sagittal plane. All sections >3.7 mm lateral to bregma are not annotated in Allen Brain Reference Atlas and were excluded. Therefore, neocortical areas in the most lateral sections such as perirhinal cortex, ectorhinal cortex, and temporal association areas were not included in the study. The claustrum and adjacent neocortical areas  were excluded as it was not possible to separate these areas precisely to prevent contamination between the assigned divisions. Since borders between the brain divisions CNU and OLF/Ctxsp were not always clear, dissections avoided these areas hence the brain areas in these divisions are partially included. White matter between the hippocampus and the neocortex carrying axon tracts were also excluded. Brainstem and cervical spinal cord tissue were used as control. When L5a neurons in the MEC were labelled using strategies explained in n = 3).For the tissue dissections, identification of brain areas was done by using Allen Brain Reference Atlas (n = 2 mice) NeuN-labelled neurons were counted. Using the formula F = (1 \u2212 (1 /N))k\u22121 where N is the barcode diversity (2 \u00d7 106)   and k is neurons .Barcode counts were first normalized in each area by the relative number of spike-in RNAs for each sample. Orphan barcodes, barcodes which did not have counts in the injection site (dMEC or vMEC) were removed. We then calculated the 90th percentile of the barcode counts in our negative controls and based on this set all barcode counts of 1\u20130. A small number of barcodes had a higher count in any target area compared to the injection site which might be a result of incomplete dissection of the injection site or viral expression in areas that the virus spilled into as we retracted the pipette. These barcodes were removed. Since our goal was to find whether neurons projecting to the telencephalon also project to the hippocampus we removed the barcodes that had no counts in any of the telencephalic target areas. For the same reason, barcodes that were detected only in the hippocampus were also removed. Finally, we excluded all barcodes with counts of less than 400 in the injection site to minimize the possibility of incomplete transport of RNA barcodes to axons in target areas due to weak expression at the cell bodies or low counts due to PCR or polymerase errors. Barcodes with low counts in all target areas (<10) were also excluded to account for potential false positives.To quantify the proportion of barcodes that were present in both a target division (dHip = (countsdHip)/(countsdHip+vHip) \u00d7 100, and likewise for vHip. Barcodes were sorted by maximum projection site.For a visual display of the relative barcode counts in dHip and vHip of barcodes originating from \u2018dorsal MEC\u2019 or \u2018ventral MEC\u2019, we generated heat maps showing normalized projection strengths between dHip and vHip for all barcodes . The nor2PO4, 2.5 KCl, 25 NaHCO3, 25 glucose, 75 sucrose, 0.5 CaCl2, 7 MgCl2, bubbled with 95% O2/5% CO2. They were then sectioned horizontally (400 \u00b5m) using a vibratome (Leica VT1200) ACSF. Tissue was collected and maintained in extracellular solution of the following composition (mM): 124 NaCl, 1.2 NaH2PO4, 2.5 KCl, 25 NaHCO3, 20 glucose, 2 CaCl2, and 1 MgCl2, continuously supplied with 95% O2/5% CO2. Slices were allowed to rest for 15 min at 35\u00b0C followed by a minimum of 30 min recovery time at room temperature before the start of the experiment.Slice preparation and subsequent data acquisition were done as previously described . Three tWhole-cell patch-clamp recordings were made in pyramidal neurons and interneurons in all layers of hippocampal CA1. Typically, two to three slices from the intermediate hippocampus were used where morphologies of the pyramidal cells and interneurons were confirmed to be intact with post hoc staining and imaging of biocytin filled neurons. Data were collected using AxoGraph (v1.7.6) software.2, 0.1 EGTA, 2 Na2ATP, 0.3 Na2GTP, 10 NaPhosphocreatine, 5.4 Biocytin. The intracellular solution was adjusted to a pH of 7.2\u20137.4 with KOH and osmolarity 290\u2013300 mOsm. All recordings were made in current clamp mode with pipette capacitance neutralization and bridge balance applied. Subthreshold membrane properties were measured from the changes in membrane potential upon depolarizing and hyperpolarizing current injections . Rheobase was established from responses to a depolarizing current ramp .Pipettes with 4\u20136 M\u03a9 resistance were pulled from borosilicate glass (Sutter Instruments) and filled with an intracellular solution of following composition (mM): 130 K gluconate, 10 KCl, 10 HEPES, 2 MgClFor optogenetic stimulation of ChR2, an LED of wavelength 470 nm (ThorLabs) was attached to the epifluorescence port of the microscope. Where necessary, the irradiance of the LED (max 9 mW) was controlled by voltage commands. Pharmacological tests were done by bath application of the following reagents with the indicated final concentrations in standard extracellular solution: Gabazine , NBQX , D-AP5 , 4-AP , and TTX . To allow for morphological reconstructions cells were filled with Biocytin  during electrophysiological recordings.Vsteady state/Vmin from the largest hyperpolarizing current step. Rheobase and action potential (AP) threshold were determined from the first AP during injection of steadily increasing current in a ramp. From the same protocol, after-hyperpolarization (AHP) was calculated as the difference between AP threshold and the most negative peak of the hyperpolarization following the first spike. Half-width was measured as the width of the AP at half its maximum spike amplitude. Firing frequency was measured from a 1-s current injection of 200 pA. Maximum firing frequency was measured between the first two APs, and base frequency between the last two APs in a train.Electrophysiological properties were analysed using built-in and custom routines in IGORpro8 (WaveMetrics) and Matlab (MathWorks). All basic properties were established from the I\u2013V protocol described above. Input resistance was determined from the largest depolarizing current step injected. Sag ratio was calculated as t-test. The PSP amplitude and latency were measured using the Neuromatic toolbox in IGORPro8.For optogenetic stimulations, responsiveness was confirmed for each cell by a significant difference between the detected peak of change in membrane potential after light stimulation and the average baseline using a two-tailed, type one Student\u2019s Cell types were determined based on biophysical properties extracted from electrophysiology data. Pyramidal cells were identified from a max firing frequency of <50 Hz, AHP <10 mV, AP half-width \u22650.7 ms, and sag \u22640.9, with descending hierarchical order. Fast spiking interneurons were categorized from a max firing frequency \u2265100 Hz, AHP \u226512 mV, AP half-width <0.5 ms, and sag >0.9. Non-fast spiking interneurons were classified from a max firing frequency 50\u2013100 Hz, AHP \u226512 mV, AP half-width 0.5\u20130.9 ms, sag ratio >0.9. and max firing frequency 50\u2013100 Hz.Neurons with a resting membrane potential less negative than \u221250 mV or a bridge balance higher than 40 M\u03a9 were excluded from the analysis, as were neurons that did not fit within a class from above criteria.The proximo-distal position and sublayer identity of neurons filled with biotin during patch-clamp recordings were determined post hoc on immunostained slices after streptavidin staining. CA1 was divided into two halves by drawing a line equidistant to CA1/CA2 and CA1/Sub border. Only neurons that were clearly located in the proximal and distal halves were included in the analysis. Neurons from the first two cell layers of SP were assigned a superficial, neurons in deeper parts of SP towards SO were assigned a deep sublayer profile. Neurons in intermediate positions were excluded from the analysis since it was not possible to unequivocally determine whether they belong to deep or superficial SP.oC. Slices were washed with PBS three times, 20 min each, and incubated in Streptavidin-Alexa 488 (S11223) or Alexa 647  and DAPI  overnight at 4\u00b0C. Slices were washed in 0.3% PBST four times and mounted on glass slides with Mowiol 4-88. Mcherry fluorescence in the MEC was examined with confocal imaging and those animals where virus injection missed MEC L5 were excluded.For identification of cells following recordings, tissue was fixed in 4% paraformaldehyde overnight at 4For staining of the interneuron marker Parvalbumin (PV), slices were prepared as described above, then incubated in primary antibody solution containing mouse anti-PV  and 5% NGS in 0.3% PBST for 48 hr at 4\u00b0C. Slices were then washed and incubated in secondary antibody solution with Alexa-conjugated Streptavidin and DAPI prepared in 0.3% PBST and mounted on glass slides after overnight incubation.A Zeiss LSM800 microscope was used for image acquisition of the slices. Pinhole was set to 1 Airy Unit. Objectives used include \u00d710 (air), \u00d720 (air), and \u00d740 (oil) to image the hippocampal formation, morphology of biocytin filled cells, and immunolabelling of interneurons, respectively.Experimental design sketches were generated using Brainrender  or Affint-test, chi-squared test, Wilcoxon signed rank test) were performed using R (https://www.r-project.org) 3.6.0, 4.0.0, and 4.0.3. Normality was tested for using both the Shapiro\u2013Wilk test and Q\u2013Q plots. All data are presented in the format of mean \u00b1 standard error of the mean unless otherwise stated. A p value <0.05 was regarded as significant.Statistical tests .Thank you for submitting the paper \"Telencephalic outputs from the medial entorhinal cortex are copied directly to the hippocampus\" for consideration by Comments to the Authors:eLife. While the reviewers felt the paper presented robust analyses of novel anatomical pathways, without functional evidence for how these pathways interact or support memory processing, the interest may be limited to specialists and not broad enough for the readership of eLife. However, if additional experiments were to lead to insight regarding the function of these pathways, eLife would consider a new submission of the manuscript. Please note in this case, the manuscript would be treated as anew submission.We are sorry to say that, after consultation with the reviewers, we have decided that this work will not be considered further for publication by Reviewer #1:This manuscript characterizes where deep layers of the medial entorhinal cortex (layer 5a and layer 5b) project to in the brain. Using a variety of circuit mapping techniques , the authors find that the same neurons in the layer 5a of the medial entorhinal cortex send projections to both the telencephalon and the hippocampus. They also find that the projections target hippocampal pyramidal cells and interneurons and has a unique topography. While these findings are interesting and suggest that deep layers of the entorhinal cortex may coordinate hippocampal-cortical interactions in memory processing, but this is just speculation based on the anatomical connections.eLife.The manuscript does a great job of characterizing the anatomical projections with a variety of approaches and the results are interesting. However, without functional evidence of how these projections interact with other brain regions to support cognition or memory processing, I think the current version of the manuscript does not quite meet the impact level for publication at Reviewer #2:In this study, the authors have characterized a novel projection from layer 5a of entorhinal cortex to CA1 of hippocampus. Overall the data are convincing that 5a cells project to CA1. The optogenetic experiments provide strong evidence that the projection to CA1 is glutamatergic and targets pyramidal cells and several classes of interneurons. The authors present some evidence that the same 5a cells send axon collatorals to retrosplenial cortex + CA1 and nucleus accumbens + CA1 - however these data could be strengthened by making it more clear whether only 5a cells were targeted within the MEC. This reviewer does not have the expertise to comment on the MAPseq experiments.The data characterize the projection well - but lack any kind of functional insight. Without this, it is difficult to place these findings in context. For example, are the projections to different interneuron subtypes different? Are there conditions when input to CA1 would be strong or weak - in other words - what are the dynamics of the layer 5MEC to CA1 pathway? Does silencing this projection effect behavior?In summary - these data nicely characterize a novel projection from layer 5a of MEC to CA1, but leave open questions as to the function of this projection.Regarding the axon collateral experiments: In Figure 3- supplement 1, it was unclear to me whether only layer 5a is targeted. Could you label layers on the figure to ensure this? Did you consider doing retrograde injections in CA1 and in RSC/NucAcb to look for double labeled 5a cells?Regarding the optogenetic aided circuit mapping experiments: These comprise a large set of recordings which are technically challenging. I was hoping, however, that you might go beyond the characterization of connections to give some functional context to this pathway. In the abstract you state \u2013 'Our results suggest that rather than serving as a relay, deep EC may coordinate hippocampal-neocortical interactions in spatial cognition and memory'. I think this statement requires experiments aimed at understanding the dynamics and magnitude of the connection at the very least.Reviewer #3:Layer 5 neurons of the entorhinal cortex are thought to play a key role in memory consolidation because they receive inputs from the hippocampus and send axons to large parts of the neocortex . Six years ago, it was shown that Layer 5 neurons of the entorhinal cortex can in fact be divided into 2 populations (Layer 5a and 5b) with very different axonal profiles . Neurons in Layer 5a, but not those in layer 5b, project to the neocortex. Based on these findings, Layer 5a neurons appear perfectly suited to influence neocortical areas and contribute to memory consolidation.The current manuscript by Tsoi et al. reveals an important new twist to our understanding of this Layer 5 output pathway: a large proportion of layer 5a neurons that project to the telencephalon (including the neocortex) also provide excitatory inputs to the hippocampus. The idea that neurons in the deeper layers of the entorhinal cortex send axons to the hippocampus is not new , but, until now, it was not clear if the population of neurons projecting to the neocortex was also sending axons to the hippocampus.Tsoi and colleagues make use of two modern tracing strategies to test whether Layer 5a neurons projecting to the telencephalon also project to the hippocampus. First, they inject a retrograde AAV expressing Cre-recombinase in extra-hippocampal areas targetted by Layer 5a neurons. A second AAV expressing a reporter protein in a Cre-dependent manner was also injected in the deep layer of the medial entorhinal cortex. The Cre-dependent reporter protein was observed in the CA1 areas. The second approach used is MAPseq. A MAPseq barcode RNA virus library was injected into the deep layers of the medial entorhinal cortex. With this technique, the majority of infected neurons are expected to express a unique RNA sequence, which will be present both in the cell body the axon terminals. The entorhinal cortex, hippocampus, and other telencephalic structures were then processed to identify the bar codes present in the different target areas of Layer 5a neurons. The majority of barcodes found in the telencephalic structures were also found in the hippocampus, suggesting that a very large proportion of Layer 5a neurons projecting to the hippocampus also project to the hippocampus.The manuscript has a clear message and the conclusions are generally well supported by the data presented. Two complementary methods are used to show that MEC deep layer neurons projecting to the telencephalon also project to the hippocampus. The reported proportion of Layer 5a neurons having projections towards the telencephalon and the hippocampus is very high, suggesting that this is an important feature defining this cell population.One limitation of this work is that the functional role of the axon collaterals in the hippocampus is not explored in detail. The main target cells in the CA1 areas have however been identified. In addition, the authors describe a mouse line in which Cre-recombinase in the entorhinal cortex is limited to Layer 5a neurons. These mice will surely prove useful in future studies investigating the role of Layer 5a neurons.It is not clear whether there are differences in connectivity between Layer 5a and CA1 superficial or deep pyramidal cells.The MAPSeq technique is relatively new and the percentage of shared barcodes between the hippocampus and the telencephalic areas is very high (approaching 99%). Given these high values, more control data would be beneficial. What is the overlap of barcodes across animals or between telencephalic brain areas? It would be interesting to know whether the layer 5a neurons projecting to the Isocortex also project to the CNU? The results on the control brain area shown in the Methods section (line 433) could be moved to the Results section.Line 209: Does light stimulation lead to suprathreshold PSP in CA1 pyramidal cells? If so, how confident can we be that the responses observed in interneurons are not due to feedback connectivity between spiking CA1 pyramidal cells and inhibitory neurons? Is the latency sufficiently low to rule out indirect connectivity? It would be beneficial to show the response latency for these responses.Line 159: The MAPSeq technique is relatively new compared to viral tracing and many readers might not know how it works. I would suggest adding a few sentences explaining this method. [Editors\u2019 note: The authors appealed the original decision. What follows is the authors\u2019 response to the first round of review.]Reviewer #1:The manuscript does a great job of characterizing the anatomical projections with a variety of approaches and the results are interesting. However, without functional evidence of how these projections interact with other brain regions to support cognition or memory processing, I think the current version of the manuscript does not quite meet the impact level for publication at eLife.eLife in its current state.We are pleased that all reviewers found our results robust, clear and interesting. The reviewer naturally questions the functional role of this pathway. We believe the characteristics of the pathway that we revealed in this manuscript and the tools we introduce will be the foundation of many experiments targeted to reveal its function. Our findings will also influence new circuit models of learning and memory, a popular area of theoretical neuroscience. Therefore, we firmly believe it is impactful and appropriate for the readership of Reviewer #2:Regarding the axon collateral experiments: In Figure 3- supplement 1, it was unclear to me whether only layer 5a is targeted. Could you label layers on the figure to ensure this?. We also added extra text in the figure legend to clarify this point and refer the reader to a previous publication from our group that shows back-labeled neurons from neocortical target structures in sagittal sections by using the same injection strategy.We thank the reviewer for this point. We understand that readers might not be familiar with the appearance of layers of the EC in coronal sections. The curved geometry of the medial entorhinal cortex makes layer organization in caudal sections in the coronal plane unintuitive: L5 cells look like a clump as opposed to a thin layer of neurons that most readers would be familiar with from horizontal or sagittal sections as in Figure 1A-D. Therefore, it is difficult to demarcate layer borders without misleading the reader. In the revised manuscript, we now provide labels on Figure 3- supplement 1 for guidance but without demarcating layer bordersDid you consider doing retrograde injections in CA1 and in RSC/NucAcb to look for double labeled 5a cells?We did consider doing these experiments. However, because incomplete back-labeling causes under-representation of overlap between the labeled populations we opted for MAPseq and combinatorial viral labeling instead.Regarding the optogenetic aided circuit mapping experiments: These comprise a large set of recordings which are technically challenging. I was hoping, however, that you might go beyond the characterization of connections to give some functional context to this pathway. In the abstract you state \u2013 'Our results suggest that rather than serving as a relay, deep EC may coordinate hippocampal-neocortical interactions in spatial cognition and memory'. I think this statement requires experiments aimed at understanding the dynamics and magnitude of the connection at the very least.We are encouraged by the reviewer\u2019s comment as it demonstrates the impact of our study and that our discovery will beget more research on this front to test emerging predictions from our study. To make sure our speculations are not mistaken as conclusions we revised the text in the abstract and result sections to emphasize that our findings are limited to the anatomical properties of deep EC projections. We now discuss our predictions on the functional role of this pathway in the \u201cIdeas and speculation\u201d section.Reviewer #3:It is not clear whether there are differences in connectivity between Layer 5a and CA1 superficial or deep pyramidal cells.We thank the reviewer for the suggestion. We now added this analysis to the manuscript and Figure 5 D. We also added a section in the methods that describe our criteria for assigning positional identities to the neurons used in this analysis.The MAPSeq technique is relatively new and the percentage of shared barcodes between the hippocampus and the telencephalic areas is very high (approaching 99%). Given these high values, more control data would be beneficial.As we understand it, the reviewer is concerned about false positives. There are three main possible reasons for false positives in these experiments. First is RNA contamination between samples during the dissection. Second is PCR errors. Third is infection of more than one neuron with virus carrying the same RNA barcode sequence. We explain each of these and our control measures in detail in the methods section. Given these control measures, we are confident that the shared barcode values we present here are accurate.What is the overlap of barcodes across animals or between telencephalic brain areas?We are unable to understand the value of investigating barcodes that overlap across animals since we do not pool the barcodes across animals. Theoretically there should be minimal overlap since a random selection of barcodes will be expressed in each experiment. We predict that the reviewer might be under the impression that the barcodes were pooled because we present a total number of barcodes across three animals in the legend of Figure 4B. We now present barcode numbers from each of the 3 mice separately as opposed to a total number to prevent misdirecting the readers.It would be interesting to know whether the layer 5a neurons projecting to the Isocortex also project to the CNU?This is indeed a very interesting point and the organizational logic of EC layer 5a projections to its telencephalic targets are currently being investigated by our group. However, we believe it is not within the scope of our manuscript as we prefer to keep the focus on establishing the principles of hippocampal projections.The results on the control brain area shown in the Methods section (line 433) could be moved to the Results section.We agree with the reviewer that this is an important point that we may not have emphasized enough in the main text. We now make clear in the main text the usage of control tissue and refer the reader to the methods section where control measures are discussed in detail. We also modified the sketch in figure 3 to emphasize the location of where the control tissue was extracted from.Line 209: Does light stimulation lead to suprathreshold PSP in CA1 pyramidal cells? If so, how confident can we be that the responses observed in interneurons are not due to feedback connectivity between spiking CA1 pyramidal cells and inhibitory neurons? Is the latency sufficiently low to rule out indirect connectivity? It would be beneficial to show the response latency for these responses.We did not observe suprathreshold responses from pyramidal neurons under our stimulation conditions but some neurons did show large EPSPs . Nevertheless, we did consider the possibility of indirect activation of the interneurons and addressed this by measuring latencies and pharmacology .Line 159: The MAPSeq technique is relatively new compared to viral tracing and many readers might not know how it works. I would suggest adding a few sentences explaining this method.We appreciate the suggestion from the reviewer. We added more explanation on the technique to the relevant section."}
+{"text": "Mycobacterium tuberculosis (Mtb) resistant to rifampicin (RIF) has mutations in the rpoB gene, while most Mtb resistant to isoniazid (INH) has mutations in the katG gene or inhA promoter. We used gene chip technology to detect mutations in these genes to determine the resistance of Mtb to RIF and INH. A total of 4148 clinical specimens with sputum smear positivity for acid-fast bacilli (AFB) were detected. Then, taking the results of the drug sensitivity test (DST) as the reference standard, the detection efficiency of sputum samples from different grades of positive smears was compared in detail. We found that the sensitivity of the gene chip method for detecting sputum samples with a grade\u2009\u2265\u2009AFB 2\u2009+\u2009was higher than that of sputum samples with a grade\u2009\u2264\u2009AFB 1\u2009+\u2009(P\u2009<\u20090.05). When the grade of the sample was\u2009\u2264\u2009AFB 1\u2009+, the sensitivity of the gene chip method was 72.6% for RIF, 67.3% for INH, and 60.0% for MDR-TB. When the grade of the sample was\u2009\u2265\u2009AFB 2\u2009+, the sensitivity of the gene chip method was 84.5% for RIF, 78.2% for INH, and 73.9% for MDR-TB. The results show that gene chip technology can be directly used to diagnose drug-resistant tuberculosis in clinical specimens, and the diagnostic efficiency for the detection of sputum specimens with a grade\u2009\u2265\u2009AFB 2\u2009+\u2009is better than that of other sputum specimens.Most  Mtb. It is an infectious disease that greatly endangers human health. Since rifampicin was first introduced as an anti-TB drug in 1972, standardized treatment regimens have been used to treat TB for nearly half a century; however, Mtb still threatens the health of nearly one-third of the world's population, many of whom will suffer from the disease during their lives1. Globally, there were 10 million new TB cases in 2019, and 1.4 million people died of TB2. China is one of the countries that is the most threatened by TB3. Governments need to invest a large amount of money and manpower to prevent and treat TB every year. Although new and highly effective bacillus Calmette-Gu\u00e9rin (BCG) vaccines have been used in newborns and young children as vaccinations to prevent TB, they cannot effectively prevent adults from being infected with Mtb4. Drug treatment is still very important for the prevention and treatment of TB. More seriously, the occurrence and prevalence of drug-resistant TB, especially multidrug-resistant TB (MDR-TB), is a serious threat to TB prevention and treatment in China. According to the World Health Organization Tuberculosis Report (2020), in 2019 alone, approximately 546,000 patients in China were infected with MDR-TB. And China is one of the three countries with the largest burden worldwide.Tuberculosis (TB) is a serious global public health problem caused by Mtb, while secondary resistance is caused by the acquisition of drug resistance abilities after the infection of people with drug-resistant strains due to factors such as drug treatment5. MDR-TB is a disease caused by Mtb that is resistant to at least the two of the most commonly used first-line anti-TB drugs, RIF, and INH6. Many mechanisms cause drug resistance in Mtb, but most of the drug resistance in clinical Mtb strains is due to chromosomal mutations7. Studies have shown that there are many types of gene mutations in drug-resistant TB. Among them, rpoB gene mutations account for 95\u201399% of RIF-resistant strains; among the strains resistant to INH, katG gene mutations account for 60\u201395%, and inhA promoter mutations account for 8\u201343%8.Drug-resistant TB can be classified as primary resistance or secondary resistance. Primary resistance is caused by the direct infection of patients with drug-resistant Mtb, although it is time-consuming and labour-intensive9. Traditional Mtb drug susceptibility tests can take 4\u20138\u00a0weeks or even longer to obtain TB drug susceptibility test results, which obviously does not allow for the early treatment of drug-resistant TB11. The diagnosis time is too long, leading to inappropriate medication, which not only increases the treatment time and the patient's financial burden but also may lead to more serious drug resistance, resulting in the aggravation of the disease and even death among patients14. With the advancement of science and technology, a new solution to this problem has been developed. In recent years, a variety of molecular biology techniques have been applied for the detection of drug resistance in Mtb17. Examples include loop-mediated isothermal amplification (LAMP), simultaneous amplification testing (SAT), Xpert MTB/RIF , MTBDRplus, TB-Biochip and TB-Biochip-2 technologies 18. CapitalBio  developed a DNA microarray chip method based on a variety of molecular analyses with PCR and reverse hybridization to detect drug resistance in TB bacteria19.Generally, the culture-based conventional drug sensitivity test has long been considered the gold standard for diagnosing drug-resistant Mtb isolates from clinical TB patients. It can detect the resistance of samples to RIF and INH within 6\u00a0h in full and provide the corresponding gene mutations at the same time. The detection indicators include 3 genes related to resistance to RIF and INH: wild-type and different mutant types of the rpoB gene, katG gene, and inhA gene promoter. Among them, the rpoB gene is related to RIF resistance, and a total of 13 mutations are detected at 6 codons, including codon 531 TCG\u2009\u2192\u2009TGG (Ser531Trp) and TCG\u2009\u2192\u2009TTG (Ser531Leu); codon 526 CAC\u2009\u2192\u2009GAC (His526Asp), CAC\u2009\u2192\u2009TAC (His526Tyr), CAC\u2009\u2192\u2009CTC (His526Leu), and CAC\u2009\u2192\u2009CGC (His526Arg); codon 511 CTG\u2009\u2192\u2009CCG (Leu511Pro); codon 513 CAA\u2009\u2192\u2009CCA (Gln513Leu) and CAA\u2009\u2192\u2009AAA (Gln513Lys); codon 516 GAC\u2009\u2192\u2009GTC , GAC\u2009\u2192\u2009TAC (Asp516Tyr), and GAC\u2009\u2192\u2009GGC (Asp516Gly); and codon 533 CTG\u2009\u2192\u2009CCG (Leu533Pro). For the INH resistance-related genes, the katG gene and inhA gene promoter, one gene codon was examined for each: two mutations in codon 315 of the katG gene, AGC\u2009\u2192\u2009ACC (Ser315Thr) and AGC\u2009\u2192\u2009AAC(Ser315Asn), and the inhA gene promoter codon -15 C\u2009\u2192\u2009T mutant  and high population mobility. According to a report issued by the local Center for Disease Control and Prevention (CDC), the prevalence of TB in Lianyungang City from 2008 to 2010 was 51.49, 53.26, 55.83 per 100,000 people, and the incidence of drug-resistant TB has also been increasing annually. Therefore, the early diagnosis of drug-resistant TB is very important; thus, with the help of the Jiangsu Provincial Government, the Fourth People\u2019s Hospital of Lianyungang City introduced the CapitalBio DNA microarray chip method in 2010.Lianyungang is a city located in eastern China and northern Jiangsu Province. It has a high population density , positive predictive value (PPV), negative predictive value (NPV), and k values of the microarray method for RIF resistance detection were 81.4%, 97.6%, 96.5%, 71.3%, 98.6%, and 0.74, respectively. The values for INH resistance detection were 74.0%, 97.1%, 94.7%, 75.0%, 96.9%, and 0.72. The values for MDR-TB are 69.8%, 99.0%, 97.6%, 78.3%, 98.5%, and 0.73. In addition, we compared the diagnostic efficacy of each drug sensitivity test according to the different AFB grades of the sputum smear results  accounted for 70.5%, Ser315Asn (katG315 AGC\u2009\u2192\u2009AAC) accounted for 4.2%, inhA-15 (C\u2009\u2192\u2009T) accounted for 22.7%, and katG plus inhA mutations accounted for 2.6% . Previous studies have indicated that if the grade of the sample is\u2009\u2265\u2009AFB 2\u2009+, the MTBDRplus test will perform best28. Therefore, we grouped the patients again according to a grade of\u2009\u2264\u2009AFB 1\u2009+\u2009and\u2009\u2265\u2009AFB 2\u2009+\u2009; the sensitivity to detect INH resistance was 67.3% and 78.2%, respectively ; and the sensitivity to detect MDR-TB was 60.0% and 73.9%, respectively . The P values were both less than 0.05, and the difference was statistically significant. At the same time, there was no significant difference in specificity after testing (P\u2009>\u20090.05). Moreover, when the sputum smear grade was\u2009\u2265\u2009AFB 2\u2009+\u2009, the consistency of the gold standard for RIF resistance and MDR-TB detection showed \"very good agreement\" (k\u2009>\u20090.75) with the gene chip method, so we believe that when the grade was\u2009\u2265\u2009AFB 2\u2009+\u2009, the DNA microarray method was more sensitive and accurate. This is consistent with the experimental results obtained by the MTBDRplus method. Gauthier et al. proposed a new algorithm for the diagnosis of drug-resistant TB from the perspective of the economic burden. Their research showed that when\u2009\u2265\u2009AFB 2\u2009+, MTBDRplus is faster and cheaper than liquid-based tests and is the preferred method for the rapid detection of MDR-TB22. Our research suggests that this algorithm may also be used for the CapitalBio DNA microarray method, but further research is needed to confirm this hypothesis. However, in this study, 36.26% (1504/4148) of samples were sputum smear-positive grade\u2009\u2264\u2009AFB 1\u2009+. How to improve the accuracy of the rapid detection of TB drug resistance in this population is a question that cannot be ignored.In our study, the DNA microarray method was used to detect large numbers of clinical samples over a long period of time, and the phenotypic drug resistance results of culture and the DST were used as reference standards. Through comparative analysis, we found that the two methods for detecting TB drug resistance have good consistency . In all specimens, the sensitivity, specificity, AR, PPV, NPV, and k values for detecting RIF-resistant 30. However, the emergence and prevalence of drug-resistant RIF strains have posed a dilemma for TB control. The drug resistance of Mtb is mainly caused by mutations rather than gene transfer from other bacteria via mobile genetic elements31. According to reports, mutations in the rpoB gene are the main cause of resistance to RIF in Mtb32. In this study, we found that the most common mutant codons were 531 (45.3%), 526 (20.3%), 516 (10.0%) and 511 (9.4%), and the most common types of mutations included Ser531Leu (42.5%), Leu511Pro (9.4%), His526Tyr (9.1%) and Asp516Va (7.2%) . It is particularly effective in killing semi-dormant or dormant bacillikatG, which encodes an INH activator, and the second most common mechanism of INH resistance is a mutation in the inhA-15 (C\u2009\u2192\u2009T) promoter region, which leads to inhA overexpression and titration of the drug37. Therefore, the CapitalBio DNA microarray method also detects the mutation of these two genes to determine the resistance to INH. In our study, Ser315Thr (AGC\u2009\u2192\u2009ACC) was the most common mutation type, accounting for 70.5% of the total detections. The inhA\u2009\u2212\u200915 (C\u2009\u2192\u2009T) promoter mutation accounted for 22.7%, which was the second most common type of mutation. This is similar to the results of others' research40. It is worth noting that among 432 INH-resistant strains, we found 20 cases of Ser315Asn (AGC\u2009\u2192\u2009AAC) mutations (including two katG 315\u2009+\u2009inhA mutations), which has rarely been reported in previous studies26. This may be attributed to regional differences but also reflects the diversity of drug-resistant bacteria in the region.On the other hand, INH is one of the common anti-TB drugs used to treat and prevent TB. The leading mechanism of INH resistance is a mutation in rpoB gene mutations41. In addition to the katG gene and inhA promoter, the mutant genes that cause INH resistance also include at least 23 genes, such as ahpC, kasA, ndh, iniABC, fadE, furA, Rv1592c and Rv177243. Therefore, the CapitalBio DNA microarray method also needs to increase the detection range to increase the detection rate of drug-resistant TB. Moreover, compared with traditional culture and drug sensitivity experiments, this method requires sophisticated equipment and highly specialized technical personnel, which also results in only a few areas where this method can be carried out.There are also limitations to this work. First, because of the difficulty of obtaining samples from patients with extrapulmonary TB, this study focused on sputum specimens only. Second, all the samples included in this study were sputum smear-positive for acid-fast bacilli; we did not collect sputum smear-negative patients with a clinical diagnosis of active TB. According to this study, we found that the CapitalBio DNA microarray chip method has some shortcomings. The sensitivity of the DNA microarray method to detect the resistance to RIF and INH was 81.43% and 73.97%, respectively. This shows that if this method is used alone, there will be a certain degree of risk of missed detection. In addition to the errors that occur during operation, the DNA microarray method itself also has certain shortcomings. Soumitesh Chakravorty et al. used the Xpert MTB/RIF Ultra method to detect 25 types of Mtb. This study confirmed that this method can directly detect the target gene in clinical specimens with a complex composition. Compared with traditional culture and the DST, this method reduces the testing time required from 8\u00a0weeks to 6\u00a0h, so it can allow clinical adjustments to the medication plan in time. Since the DST uses live bacteria, it must be carried out in a BLS-3 laboratory. The DNA microarray method significantly reduces the risk of biohazards after the thermal lysis step, allowing it to be performed in a BLS-2 laboratory22.Nevertheless, the CapitalBio DNA microarray method is still a very suitable method for detecting the drug resistance of rpoB gene is the main cause of the resistance of Mtb to RIF, and the Ser315Thr mutation of the katG gene is the main reason for the resistance of Mtb to INH. Through comparisons with the results of the drug sensitivity test (DST), we confirmed that this method is an efficient, accurate, and rapid method for diagnosing the drug resistance of TB, which is very suitable for the direct detection of clinical specimens. The detection efficiency of clinical specimens with a sputum smear grade\u2009\u2265\u2009AFB 2\u2009+\u2009was very good. In summary, this study will help clinicians choose more reasonable testing methods and reduce the economic burden on the government and patients.We used the CapitalBio DNA microarray chip method to detect 4148 clinical specimens with sputum smear positivity for acid-fast bacilli from Lianyungang City. Among them, the Ser531Leu mutation of the The Ethics Committee of the Fourth People's Hospital of Lianyungang City approved the study , and informed consent was waived by the ethics committee due to the retrospective nature of the study . Except for the experimental results, the personal information of all participants was kept confidential. At the same time, we confirmed that all methods were implemented by the industry standards and regulations of China.nontuberculous mycobacteria (NTM), and 19 had no drug susceptibility test results. Meanwhile, there were 522 negative and 564 NTM in the samples tested by the gene chip method. As a comparison, we included 4148 samples with results from both methods in the study  digestion solution, neutralized with phosphate buffer solution, and centrifuged. Then, the phosphate buffer solution was removed by pouring, and the bacterial solution was resuspended in 2\u00a0ml. A total of 0.1\u00a0ml of sample digestion solution was inoculated in 2 Roche Neutral Solid Medium  and incubated in a 37\u00a0\u00b0C incubator, and the results were observed weekly. If the strain was growing, the strain was smeared, and a duplicate sample was stained with the acid-fast staining method. If it was positive, the strain was used for the drug sensitivity test; if it was negative, it was considered \u201ccontamination\u201d. If the culture result was negative after 8\u00a0weeks, it was considered \"culture-negative\".Mycobacterium Drug Sensitivity Roche Medium Kit, the culture-positive isolated strains were ground into a 1\u00a0mg/ml suspension by the ratio method, and then diluted to 10\u20132\u00a0mg/ml and 10\u20134\u00a0mg/ml, respectively. A 22 SWG standard inoculation loop was used to pick a full loop  of the suspension and inoculate it into the medium containing RIF and INH. The same method was used to inoculate the control medium without drug and the identification medium containing P-nitrobenzoic acid (PNB). After 4\u00a0weeks of continuous incubation at 36\u2009\u00b1\u20091\u00a0\u00b0C, the results were observed. If there was no growth on the control medium, it was judged as a \"DST failure\"; if there was colony growth on both the control medium and PNB medium, then the result was \"NTM\". If there was colony growth on the control group but no colony growth on the PNB medium, the result was \"Mtb\". The number of colonies on the medium was counted, and the resistance rate was calculated as follows: resistance rate (%)\u2009=\u2009(number of colonies grown on the drug-containing medium/number of colonies grown on the control medium)\u2009\u00d7\u2009100%. A resistance rate\u2009<\u20091% was considered sensitive, and a resistance rate\u2009>\u20091% was considered resistant.Drug susceptibility test: Using a BASO One millilitre of sputum sample was added to 1\u20132 times 4% sodium hydroxide solution, vortexed, and shaken for 1\u00a0min to perform sputum digestion and mixing. A total of 1.0\u00a0ml of the digestion solution treated with 4% sodium hydroxide was added to a 1.5\u00a0ml centrifuge tube and centrifuged at 12,000 r/min for 5\u00a0min. The supernatant was discarded, 1\u00a0ml of pH 6.8 phosphate buffer was added, and the sample was shaken and mixed. After homogenization, it was centrifuged at 12,000 r/min for 5\u00a0min, and the supernatant was discarded. A total of 80\u00a0\u03bcl nucleic acid extraction solution was added, and the sample was mixed thoroughly, transferred to a nucleic acid extraction tube, vortexed and shaken to mix well, and placed in an ultrasonic oscillator for 5\u00a0min. It was incubated in a dry bath at 95\u00a0\u00b0C for 15\u00a0min, centrifuged at 12,000 r/min for 1\u00a0min, and set aside. After following the kit instructions for PCR amplification, chip washing, drying, and chip hybridization, a LuxScan 10K-B microarray chip scanner was used to scan and automatically interpret the results , and negative predictive value (NPV) of the CapitalBio DNA microarray. A chi-squared test or two-tailed Fisher\u2019s exact test was used for statistical analysis, and the difference was considered significant when P\u2009<\u20090.05. The degree of agreement between the DST and the GeneChip assay was also assessed using Cohen\u2019s kappa (k) coefficient. k values\u2009>\u20090.75 indicate that the two methods show very good agreement, and k values of 0.40\u20130.75 show that the two methods show fair to good agreement. k values of\u2009<\u20090.40 indicate that the two methods show poor agreement. All statistical analyses were performed with SPSS 24.0 .Supplementary Information 1.Supplementary Information 2.Supplementary Information 3."}
+{"text": "Mycobacterium tuberculosis (MTB) is a causative agent of tuberculosis (TB) that causes death worldwide. MTB was subjected to phenotypic drug-susceptibility tests (DST), and drug-resistant genes were sequenced. katG (S315T), rpoB (S450L), rpsL (K43R), and embB (M306V). Previously treated patients were more likely to have positive smear results and exhibit drug resistance. New patients were more likely to be mono SM-resistant and less likely to be INH- and RIF-resistant. The most common mutations were The proportion of mono-SM-resistant TB among new patients was higher.  Mycobacterium tuberculosis (MTB), remains a major threat to the public health worldwide. Although the directly observed treatment, short-course (DOTS) strategy has significantly reduced the incidence of TB in recent years, the emergence of drug-resistant TB has severely hampered TB prevention and control, especially regarding multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB)Tuberculosis (TB), usually caused by katG gene are a major cause of INH resistancerpoB gene, especially in the 81-bp RIF resistance determining region (RRDR)rpsL and rrs, which encode the S12 ribosomal protein and 16S rRNA, respectively, are associated with intermediate or high levels of SM resistanceembB codon 306, which occur in 30%-69% of clinical EMB-resistant strains, are associated with resistance to EMBkatG, inhA, rpoB, rpsL, rrs, and embB, is thought to represent a rapid screening method for the detection of first-line drug resistance in clinical isolates.TB epidemics are unevenly distributed in China, and there is a high prevalence in rural areas, especially those that are underdeveloped in the northwest and southwest of ChinaThe current analysis presents data on the drug resistance profiles of drug-resistant TB as well as gene mutations from a larger MTB DNA sample of the most recent prevalent drug-resistant isolates in rural areas of China. Based on positive sputum culture specimens, MTB isolates were identified, phenotypic drug sensitivity tests (DST) were conducted, and drug-resistant genes were sequenced. Patient information was registered and verified at the Community Health Care Center. Specimens were collected from specialist TB hospitals in Hangzhou, China, from 2017 to 2019. Sputum samples were collected from patients suspected to have TB. Samples were subjected to acid-fast staining and microscopy tests and were cultured on Lowenstein-Jensen medium according to the national guidelines2O) and a standard strain of H37Rv (American Type Culture Collection 27294) were used as negative and positive controls, respectively, in all experiments. Drug-resistant MTB genomic DNA was extracted and stored at 20 \u00b0C for further use. Genetic fragments associated with drug resistance were amplified using previously reported primersSamples were submitted to the tuberculosis reference laboratory of the Hangzhou Center for Disease Control and Prevention. Phenotypic DSTs for each drug were determined using the proportional method on Lowenstein-Jensen medium. The concentrations of INH (0.2 mg/L), RIF (40 mg/L), SM (4.0 mg/L), and EMB  software was used for the statistical analyses.Categorical data were summarized as counts or percentages (%), and the groups were compared using chi-square test or Fisher's exact test. Statistical significance was set at 2 = 23.861, p < 0.001). There were more patients between 76 and 91 years old in the new group (47.80%) than in the previously treated group . The group with no TB treatment history comprised more of clerks , retirees , and residents . Previously treated patients were more likely than new patients to have positive smear results  and drug resistance .As shown in Of these, 163 were resistant only to INH, 26 were resistant only to RIF, 168 were resistant only to SM, and 23 were resistant only to EMB . Among tkatG, inhA, rpoB, rpsL, rrs1, and embB genes showed that 68.68% (193/281) carried a single mutation in the katG gene at codon 315  had mutations in both the katG and inhA genes. The most frequent mutation was S315T . For RIF-resistant isolates, 90.60% (106/117) carried mutations in the RRDR of rpoB. Seven mutation sites were identified, and the most frequent mutation was at codon 450 . The second most frequent mutation was found at codon 445 . In addition, 11.97% (14/117) had mutations at codon 435, while three isolates had synonymous mutations at codons 427, 428 and 430, respectively. Furthermore, 87.55% (204/233) of the SM-resistant isolates harbored mutations in rpsL or rrs1. The most frequent mutation in the rpsL gene was K43R . A total of 70.91% (39/55) of the EMB-resistant isolates had mutations in the embB gene.Sequencing results of odon 315 , 9.25% (P < 0.05). A total of 20.25% (522/2 578) isolates were resistant to any of the four first-line anti-TB drugs, and the proportion of MDR-TB was 3.45%, much lower than that reported by Lv et al. This study analyzed the epidemiology of drug-resistant TB in seven rural areas in Hangzhou . Of all included patients, 76.96% (1 984/2578) were Hangzhou residents, and the proportion of previously treated patients was 25.02% (645/2 578), which was significantly lower than the data from other TB hospitals in ChinakatG and inhA genes for INH resistance, rpoB for RIF resistance, rpsL and rrs1 for SM resistance, and embB for EMB resistance. Therefore, we analyzed these genes in the current study. The predominance of the S315T substitution in the katG gene in INH resistance has been demonstrated globally, and estimates of this mutation range from < 25% to > 90%, and inhA also confers low-level INH resistance (> 10%)katG (81.14%) and inhA (25.62%) genes was higher than that found by Zhao et al., who reported findings  from the neighboring province of FujianrpoB RRDR remain important RIF resistance markers. In our study, all mutations conferring RIF resistance had mutations within the RRDR, with the most prevalent being S450L , followed by H445L . Mutations at codon 445 were the most diverse (H\u2192R/L/D/Y/C/P/N). In agreement with the studies reported previouslyrpoB gene, and although they were at different locations, most of them were located at three rpoB codons: 450, 445, and 435. Drug-resistant TB is often caused by mutations in genes, especially in nhA and katG genes were the most clinically relevant and determined resistance in most clinical isolates, and this was the main reason we included only inhA and katG in our study. We found that 5.69% of INH-resistant isolates were not associated with any genotypic mutations in inhA or katG, which was a much lower result than that reported by Hazbon et al. previouslyIrpsL nor rrs1, and 29.09% of EMB-resistant isolates had no mutations in the embB gene, so other related genes may be involvedGidB mutations have been found in both resistant and susceptible clinical drug-resistant MTB isolatesgidB gene in the analysis we used to detect SM resistance. We sequenced the associated gene fragments of 30 all-drug susceptible MTB isolates simultaneously; however, we found none of the mutations mentioned above.Regarding the two other first-line anti-TB drugs, 87.55% (204/233) of SM-resistant isolates and 70.91% (39/55) of EMB-resistant clinical isolates were identified using molecular techniques. The findings were similar to those reported by Zhao et al. katG , rpoB , rpsL , and embB , respectively. Additionally, we identified a rare substitution mutation of S450P in the RRDR of the rpoB gene. Furthermore, we found that new TB patients were more likely to be resistant only to SM and less likely to be resistant to both INH and RIF than previously treated patients. Our findings could be helpful in the development of rapid molecular diagnostic methods and may improve our understanding of drug resistance in Hangzhou, aiding the development of precision medicine for TB and the disturbance of drug-resistant TB transmission.Our study showed that the overall prevalence of the first-line drug-resistant TB in the rural areas of Hangzhou, China was low. However, the proportion of INH and SM resistance were higher. The most prevalent genetic mutations associated with INH, RIF, SM, and EMB resistance were"}
+{"text": "The association of short-term particulate matter concentration with cardiovascular disease (CVD) among cancer survivors is yet unclear. Using the National Health Insurance Service database from South Korea, the study population consisted of 22,864 5-year cancer survivors with CVD events during the period 2015\u20132018. Using a time-stratified case-crossover design, each case date (date of incident CVD) was matched with three or four referent dates, resulting in a total of 101,576 case and referent dates. The daily average particulate matter 10 (PM10), 2.5 (PM2.5), and 2.5\u201310 (PM2.5\u201310) on the day of case or referent date (lag0), 1\u20133 days before the case or referent date , and the mean value 0\u20133 days before the case or referent date (lag0\u20133) were determined. Conditional logistic regression was conducted to calculate the adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for CVD according to quartiles of PM10, PM2.5, and PM2.5\u201310. Compared to the 1st (lowest) quartile of lag0\u20133 PM10, the 4th (highest) quartile of lag0\u20133 PM10 was associated with higher odds for CVD . The 4th quartiles of lag1 , lag2 , lag3 , and lag0\u20133  PM2.5 were associated with higher odds for CVD compared to the respective 1st quartiles. Similarly, the 4th quartile of lag0\u20133 PM2.5\u201310 was associated with higher CVD events  compared to the 1st quartile. Short-term exposure to high levels of PM may be associated with increased CVD risk among cancer survivors. Particulate matter (PM), minute solid particles or liquid droplets suspended in the air, has been shown to be associated with a number of health-related outcomes such as cancer, cardiovascular disease (CVD), and mortality . For exaWith recent advances in cancer management, the prevalence of cancer survivors, those who successfully received treatment for and survived cancer, is increasing globally. However, cancer survivors are at higher risk for developing subsequent serious illnesses, such as CVD. Recently, it has been shown that cancer survivors are at higher risk for developing CVD compared to those without cancer . As cancTherefore, in this time-stratified case-crossover study using a nationwide administrative data, we aimed to determine the association of short-term PM exposure with CVD events.The National Health Insurance Service (NHIS), which provides health insurance for all citizens in South Korea, covers nearly all forms of health services. Due to this, the NHIS has health administrative records of nearly the entire population of South Korea in the form of health claims data . A part 2, with the mean  area being 55.1 (79.9) km2 [Daily PM levels categorized according to size  were derived from the Air Korea database, which was constructed from the National Ambient Air Monitoring System (NAMIS). The NAMIS collects daily air pollution levels and other weather-related information from more than 280 atmospheric monitoring sites located nationwide, with at least one monitoring site located in approximately 280 administrative districts in South Korea. The area of the administrative districts, which are a unit of municipality in South Korea, ranged between 2.8 and 775.0 km9.9) km2 . Based o9.9) km2 .We extracted newly diagnosed cancer patients during the period 2006\u20132013. Cancer patients who survived for at least 5 years were defined as 5-year cancer survivors during the period 2011\u20132018. Among them, 23,044 cancer survivors with subsequent CVD events after the 5-year mark of cancer survival were determined. Those residing in areas without PM values (n = 180) were excluded, resulting in 22,864 5-year cancer survivors with subsequent CVD events during the period 2015\u20132018. Based on the CVD event (case) dates, referent periods were defined as dates in the same day of the week within the same month . Therefore, three or four referent dates were matched for each case (CVD) date, resulting in a total of 78,712 referent periods for 22,864 case periods. Based on the combined 101,576 case and referent periods, a time-stratified case-crossover design was used to determine the association of daily PM levels with short-term CVD events. Detailed descriptions of the case-crossover design are described elsewhere ,15. The PM10, PM2.5, and PM2.5\u201310 levels were determined based on the case and reference dates. The mean daily PM level on the date of CVD event or its matched referent dates were defined as lag0. PM levels 1\u20133 days prior to the case or reference dates were defined as lag1, lag2, and lag3, respectively. The mean PM level over the 4-day period between the date of and 3 days prior to the case or referent date was defined as lag0\u20133. After determining lag0, lag1, lag2, lag3, and lag0-3 values, the PM levels were grouped according to quartiles of increasing PM levels, with the 1st , 2nd (26\u201350 percentile), 3rd (51\u201375 percentile), and 4th  quartiles having increasing levels of PM for each lag period.The operational definition for CVD, which was defined as being hospitalized for coronary heart disease (CHD) or stroke for 2 or more days, was adapted from a previous study that also used the NHIS database for identifying CVD events . The diap for interaction > 0.05). Based upon this, a one-pollutant model was used to determine the association of short-term PM with CVD incidence. Upon regression analysis, mean daily temperature , determined from the NAMIS Air Korea database, was adjusted for. Mean daily temperature was adjusted for via quartiles of the mean daily temperature value on the lag day. CVD odds were determined with the 1st (lowest) quartile group being the reference. p for trend was determined by assessing CVD odds per 1 quartile  increase in PM. Stratified analysis on the association of PM with acute CVD events among 5-year cancer survivors according to subgroups of age, sex, and household income was conducted. Household income was derived from the health insurance premium. Finally, the association of PM with short-term CHD and stroke events among 5-year cancer survivors was determined.Conditional logistic regression analysis was conducted to determine the adjusted odds ratios (aORs) and 95% confidence intervals (CIs) for short-term CVD events according to PM10, PM2.5, and PM2.5\u201310 levels among 5-year cancer survivors. Interactions of PM with other measures of PM and mean daily temperature were assessed in the association of PM with CVD incidence. No significant interactions were observed between PM2.5 with PM10, PM2.5 with PM2.5\u201310, PM10 with PM2.5\u201310, as well as between PM with mean daily temperature . Upon statistical analysis, the \u201cproc logistic\u201d command with the \u201cstratified\u201d option was used to fit the conditional logistic regression model.Statistical significance was defined upon a 3, 25.1 (14.2) \u03bcg/m3, and 19.8 (17.1) \u03bcg/m3, respectively. The mean (SD) daily temperature was 12.9 (10.3) \u00b0C. The mean (SD) age for the study population was 71.7 (10.8) years. The number of participants (%) who were men or women were 12,750 (55.8) or 10,114 (44.2), respectively. Finally, 41.8% of the participants were within the highest quartile of household income.p for trend < 0.05).The association of short-term PM10 exposure among 5-year cancer survivors with subsequent CVD events is shown in p for trend <0.05). There was a tendency towards higher CHD and stroke odds upon increasing levels of short-term PM2.5 exposure, as shown in The short-term CVD odds according to PM2.5 are depicted in p for trend <0.05).In summary, short-term exposure to higher levels of PM10, PM2.5, and PM2.5\u201310 was associated with increased CVD events among 5-year cancer survivors. This association of higher CVD risk with increasing levels of short-term PM did not appear to be modified significantly according to age, sex, and household income. To our knowledge, this was the first study to demonstrate that higher short-term PM exposure was associated with higher CVD risk among 5-year cancer survivors.Although we could not find any previous studies on the association of short-term PM exposure with CVD among cancer survivors, long-term and short-term PM exposure has previously been associated with the development of both CVD and cancer risk among the general population. Recently, a cohort study using the US National Health Interview Survey data showed that higher levels of PM2.5 were associated with increased risk for heart disease mortality . Similar3 increase) [3 increase) [While the harmful effects of PM on cardiovascular health among the general population is generally well-accepted, there has been an emphasis on identifying susceptible populations on the detrimental effects of PM . Determincrease) , which wncrease) . This prMultiple possible mechanisms may contribute to the development of CVD upon exposure to high levels of PM . First, Multiple limitations must be considered upon the interpretation of our results. First, while short-term PM exposure was measured on an individual level, PM exposure was nonetheless based on the daily PM levels according to the area of residence for each subject. Therefore, the short-term PM exposure defined in this study may not completely reflect each participant\u2019s actual PM exposure. While multiple large-scale epidemiological studies using the NHIS database have previously used area of residence PM exposure, there is a need for future studies with a more accurate measure of short-term PM exposure ,13. SecoDespite these limitations, this study has a number of strengths. First, there is a relatively low possibility of bias from individual factors, such as age and sex, due to the time-stratified case-crossover design in which comparisons are made within each subject among different (case and referent periods) time frames. This is of particular importance in cancer survivorship studies, as the database lacked detailed clinical information such as cancer management regimens and the pathological stage. While such factors could lead to bias when comparing between subjects, such as in cohort studies, case-crossover studies are relatively less susceptible to confounding effects of such characteristics . NonetheBeing exposed to high levels of short-term PM10 and PM2.5 may lead to acute CVD events among cancer survivors. Therefore, cancer survivors may be susceptible to the acute CVD-risk increasing effects of daily PM. Cancer survivors who reduce short-term exposure to high levels of PM10 and PM2.5 may benefit from reduced risk of acute cardiovascular events. Future studies must investigate whether intervention methods aimed at reducing short-term PM exposure actually lead to lower CVD events in cancer survivors."}
+{"text": "Physical frailty is a common characteristic of older people with the ageing process and has been viewed as a major public health issue. The longitudinal association between different social engagement and physical frailty among older people has not been explored adequately in China. Marital status forms a critical context for the link between social engagement and frailty among older people, which might constitute a moderating process. The purpose of the present study is to investigate the longitudinal association between social engagement and the changes in physical frailty among Chinese older adults, and to examine whether the association between social engagement and frailty differs by marital status.The data use in this study were from the data from the China Health and Retirement Longitudinal Study aged 60+ years from 2011 to 2015. A total of 6575 respondents who participated in at least one follow-up wave were included in the analysis. The relationship between social engagement and changes in frailty over time, and the moderating role of marital status were estimated using individual fixed-effects models. Sensitive analyses were conducted to test the robustness of the results.P\u2009<\u20090.001), engaging in hobby groups , engaging in sports groups , and volunteering  with a frequency of almost daily had a significantly lower frailty risk than participants who never engaging in those activities. The association between frequent engaging in hobby groups and physical frailty was strongest for unmarried than married older adults .After adjusting the confounders, participants who interact with friends (Coef: -1.309, Frequent social engagement might help to decrease the risk of frailty in the Chinese older population. This finding has important implications for public health policy and encourages the incorporation of a broad range of social engagement into the daily lives of older individuals. Specially, encouraging unmarried older adults to engage in intellectual activities, such as playing chess or Mahjong with others, may be an effective way to reduce physical frailty.The online version contains supplementary material available at 10.1186/s12877-021-02194-x. It is defined as a multifactorial syndrome that reduces one\u2019s physiologic reserve [The population is ageing worldwide. Population ageing presents tremendous challenges and has far-reaching implications for individuals, families, and communities. One of the most profound challenges is the increasing multiple adverse health conditions as people age. The term  reserve  and lead reserve \u20134. A rec reserve . Physica reserve , 7. Frai reserve . Therefo reserve .Active Ageing\u201d proposed by the World Health Organization [Physical frailty is a reversible process when effective interventions are employed . Most innization , is definization . Social nization , and is nization . One stunization . NeverthMarital status forms a critical context for the link between social engagement and frailty among older people, which would constitute a moderating process. Many studies have shown that married people are healthier than unmarried people in later life. For example, married people tend to have lower risk of mortality  and suff, which is a nationwide survey aiming to provide sufficient micro-level longitudinal data for aging studies. Its design is similar to that of the Health and Retirement Study (HRS) in the United States, the English Longitudinal Study of Aging (ELSA), and the Survey of Health, Aging and Retirement in Europe (SHARE). We hypothesize that: (a) Four types of social engagement, i.e., interaction with friends, engaging in hobby groups, sports groups, and volunteering, are all associated with the reduced frailty among Chinese older people; (b) The association between social engagement and frailty is moderated by marital status.The purpose of the present study is 2-fold. First, while social engagement has been found to be associated with positive physical and mental health among older people, whether this association would extend to physical frailty is still unclear, especially lacking the longitudinal evidence in mainland China. Thus, the first aim is to investigate the longitudinal association between social engagement and the changes in physical frailty among Chinese older adults. Second, marital status forms a critical context for the link between social engagement and frailty among older people, and married people are healthier than unmarried people in later life, thus the second aim is to examine whether the association between social engagement and frailty differes by marital status. To achieve the two aims, we use data from the China Health and Retirement Longitudinal Study (CHARLS)We used panel data from the three waves of the China Health and Retirement Longitudinal Study (CHARLS) in 2011, 2013, and 2015. The CHARLS collected high-quality data via in-person interviews with a structured questionnaire, and adopted a four-stage, stratified, cluster sampling method to enroll community-dwelling residents from 450 villages and 150 counties in 28 provinces in China. Details of its sampling technique have been reported elsewhere . With thIn this study, physical frailty was defined as the accumulation of health deficits during an individual\u2019s later life, and we used the Frailty Index (FI) to estimate the frailty of each respondent. FI can provide a more comprehensive picture of older adults\u2019 overall health, and is particularly useful for predicting adverse health outcomes , 35. AltSocial engagement was defined as involvement in any type of social activity during the study period. Respondents were asked how often they took part in the following four types of activities in the last month: (1) interaction with friends; (2) hobby groups ; (3) sports groups ; (4) voluntary work . For each activity, an additional question was asked about the frequency of engagement, which was categorized as: often , some of the time , not regularly (about once a month), and never.Marital status was divided into two categories, i.e., married and unmarried status . Cohabiters were included in the unmarried groups, because they might not receive the same levels of socio-psychological and economic benefits as married individuals due to a lack of institutional legitimacy and less commitment based on previous studies .Potential confounding variables in this study included age, financial situation (household income per capita), currently working (yes or no), smoking status, alcohol consumption, multi-morbidity (co-existing of two or more chronic diseases), self-perceived health status, depression, physical activity, and wave. We did not control for time-independent factors, such as sex, education, baseline age, etc., because fixed effects regression models only use within-person variation, and time-invariant factors will be absorbed in the fixed effects. We measured depression using the 10-item Center for Epidemiologic Studies Depression Scale (CES-D), and participants with CESD-10 scores above 10 were classified as having depressive symptoms . PhysicaWe first examined the descriptive characteristics of the study sample separated by wave. Then, longitudinal linear fixed-effects (FE) models were used to examine the association between changes in social engagement and changes in FI within each older adult. Applying a fixed effects model on panel data reduces potential bias due to unobserved heterogeneity . In modeFIit denotes the frailty index for individual i at wave t. Zi refers to the fixed effects, which includes all observed and unobserved time-invariant covariates. Xit and Mit indicate four types of social engagement and marital status for individual i at wave t, respectively. Ut denotes wave effects, and \u0190it is the error term.To test the feasibility and robustness of the FE model, we conducted several sensitivity analyses. First, FE models may yield biased and inconsistent results when \u201cstrict exogeneity\u201d cannot be assumed. To minimize the potential impact of reverse causality, we conducted a test for the endogenous selection  by using the following equation :2\\documFIt-1) net of the proxy for the fixed effect We tested whether social engagement (t) can be predicted by the previous wave\u2019s frailty  and FE model. We then used Hausman-test between the FE model and the random-effects (RE) model. Next, we investigated whether it was necessary to control for time-fixed effects in the models. Finally, to test the robustness of the results, we used multiple imputation techniques to reduce the potential biases resulting from missing data in the frailty index and control variables. Multiple imputation involves replacing missing values with predictions based on other observed variables by chained equation method . All anaTable P\u2009<\u20090.001), engaging in hobby groups , engaging in sports groups , and volunteering  almost daily were all significantly associated with reduced frailty. We also identified a similar association in not regular and some of the time groups, but the coefficients declines, such as interaction with friends  and hobby groups . Regarding marital status, we found that being married was significantly associated with reduction in frailty compared with unmarried older adults .The results of the FE regression are shown in Table P\u2009=\u20090.037). The interactions between marital status and the other three types of social engagement were not significantly at 5% level.Table\u00a0P-value was statistically significant (P\u2009<\u20090.001), which indicated that the former might be biased. Then we used Hausman-test to assess whether random effect models were preferred over the FE models, and the test yielded P-value less than 0.001, indicating a preference over the FE models. Next, we investigated whether it was necessary to control for time-fixed effects in our models. After estimating the FE models with the wave dummy variables, we tested the null hypothesis that the coefficients of those variables were all equal to zero. Results (P\u2009<\u20090.001) indicated the necessity to control for time-fixed effects in the models. Finally, the results of multiple imputation did not differ much from the main results (see Supplementary Table First, Supplementary Table The current study systematically examines the associations between four types of social engagement and physical frailty among a sample of individuals aged 60 and above from a longitudinal study in China, as well as whether these associations differs by marital status. Overall, after eliminating the time-invariant unobserved heterogeneity and any individual-level idiosyncratic disturbances over time, our study have offered longitudinal evidence that frequent interaction with friends, engaging in hobby groups, sports groups, and voluntary work are all associated with physical frailty reduction among older people, even after fully adjusting for potential confounding factors.Few studies have examined the association between social engagement and frailty among older adults. One notable exception is Kwan et al. , which sChinese older adults usually participate in hobby groups after retirement, such as playing Mahjong, chess, or card. Unlike engagement in voluntary work and sports groups, not only frequent engagement, but also not regular or some of the time engaging in hobby groups is associated with frailty reduction. Earlier studies  found thTheoretically, social capital theory  providesThis study also shows that being married is significantly associated with the frailty reduction in older adults, which is consistent with previous studies , 28\u201330. The results of interaction effect shows that marital status plays a moderating role in the relation between engaging in hobby groups and physical frailty reduction. Specifically, the association between frequent engaging in hobby groups and frailty reduction is particularly pronounced for participants who are not married. According to the model of marital resource , marriedThe present research contributes to existing literature by examining the longitudinal effects of social engagement on physical frailty among older people using longitudinal panel data from three waves of the CHARLS. The results suggests the concept of social engagement would have clinical relevance and provides new insights for physical frailty prevention. Methodologically, the associations may reflect confounding by unobserved characteristics or reverse causality from frailty to social engagement. The present study has two strengths in its analytical strategies. First, we use individual fixed-effects models to adjust for unobserved time-invariant confounders. Fixed-effects model exploit longitudinal nature of the data by assessing the association between changes in social engagement and changes in physical frailty within individuals, and thus controlling for unmeasured individual heterogeneity that may be correlated with both social engagement and frailty, such as socioeconomic status, personality traits, and intellectual ability . This meThis paper has several limitations. First, while we have found that frequent engaging in volunteer activity is associated with reductions in frailty, it might be possible to assess the exact threshold or dose-response relationship if more fine-grained data on volunteering hours are available. Unfortunately, our data do not allow for this, thus we leave this issue for future research. Second, although the FE models can eliminate the time-invariant unobserved heterogeneity and we have controlled for some time-variant confounding, there may still be residual time-varying confounding not controlled in our study. For example, older adults\u2019 social support may be an important resource that can increase their health and thus protect against frailty reduction. We do not control for social support due to data limitation, which might have led to an over-estimation of our estimates. Finally, engaging in community-related organizations has not been included in our study because there is a limited number of older adults who engage in community-related organizations. In this case, the relationships between engaging in community-related organizations and frailty await future studies with a larger sample size. Our study may serve as a basis for future research evaluating the value of social engagement in designing targeted interventions of frailty. Prospective studies are needed to identify the causal relationship for social engagement and frailty, and further research is required to identify the specific mechanisms that explain the association between social engagement and frailty among older people.In conclusion, this large, longitudinal study suggests that frequent social engagement, including interaction with friends, engaging in hobby groups, sports groups, and volunteering, potentially results in lower physical frailty among older Chinese population. Furthermore, the association between engaging in hobby groups and frailty reduction is particularly pronounced in unmarried groups. These findings have important implications for public health policy and encourage the incorporation of a broad range of social engagement into the daily lives of older individuals to protect against frailty status among Chinese older people. Specially, encouraging unmarried older adults to engage in hobby groups, such as playing chess or Mahjong with others, may be an effective way to reduce frailty.Additional file 1."}
+{"text": "Depression is a common mental disorder characterized by disturbances in mood, thoughts, or behaviors. Serious games, which are games that have a purpose other than entertainment, have been used as a nonpharmacological therapeutic intervention for depression. Previous systematic reviews have summarized evidence of effectiveness of serious games in reducing depression symptoms; however, they are limited by design and methodological shortcomings.This study aimed to assess the effectiveness of serious games in alleviating depression by summarizing and pooling the results of previous studies.A systematic review of randomized controlled trials (RCTs) was conducted in accordance with the PRISMA  statement. The search sources included 6 bibliographic databases , the search engine \u201cGoogle Scholar,\u201d and backward and forward reference list checking of the included studies and relevant reviews. Two reviewers independently carried out the study selection, data extraction, risk of bias assessment, and quality of evidence appraisal. Results of the included studies were synthesized narratively and statistically, as appropriate, according to the type of serious games .P=.12). Very low-quality evidence from 5 RCTs showed a statistically and clinically significant difference in the severity of depressive symptoms (P=.004) between exergame and control groups, favoring exergames over no intervention. Very low-quality evidence from 7 RCTs showed a statistically and clinically significant effect of computerized CBT games on the severity of depressive symptoms in comparison with no intervention (P=.003).From an initial 966 citations retrieved, 27 studies met the eligibility criteria, and 16 studies were eventually included in meta-analyses. Very low-quality evidence from 7 RCTs showed no statistically significant effect of exergames on the severity of depressive symptoms as compared with conventional exercises (Serious games have the potential to alleviate depression as other active interventions do. However, we could not draw definitive conclusions regarding the effectiveness of serious games due to the high risk of bias in the individual studies examined and the low quality of meta-analyzed evidence. Therefore, we recommend that health care providers consider offering serious games as an adjunct to existing interventions until further, more robust evidence is available. Future studies should assess the effectiveness of serious games that are designed specifically to alleviate depression and deliver other therapeutic modalities, recruit participants with depression, and avoid biases by following recommended guidelines for conducting and reporting RCTs.PROSPERO International Prospective Register of Systematic Reviews CRD42021232969; https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=232969 An individual's mental health is fundamental to living a healthy and enjoyable lifestyle. Studies estimate that 1 in 3 people may suffer from a mental illness during their lifetime . DepressDepressive disorders are a family of mental disorders ranging in severity from mild temporary episodes of sadness to more severe and persistent depression . DepressThe use of serious games, defined as games that have a purpose other than entertainment, has seen a rise in recent years . SeriousGaming as a therapeutic tool in mental health can potentially offer several specific advantages that may be missing from traditional forms of delivery. The gaming industry is, as ever, popular globally  and arguMany studies have assessed the effectiveness of serious games to alleviate depression. Aggregating the evidence from these studies is very important to draw more definitive conclusions about the effectiveness of serious games as viable therapeutic interventions in depressive disorders. Several published reviews have summarized the evidence about the effectiveness of serious games for depression ,19,35-37We conducted a systematic review in accordance with the PRISMA  statement  38]..38].We utilized the following bibliographic databases to retrieve relevant studies: MEDLINE (via Ovid), PsycInfo (via EBSCO), CINAHL (via EBSCO), IEEE Xplore, ACM Digital Library, and Scopus. These databases were searched on March 30, 2021 by the first author. When applicable, we set auto alerts to conduct an automatic search weekly for 12 weeks . We also searched the search engine \u201cGoogle Scholar\u201d to identify grey literature. We checked only the first 10 pages  because Google Scholar retrieved a vast number of studies and it ordered them based on their relevancy. To identify further studies of relevance to the review, we conducted backward reference list checking  and forward reference list checking .The search query in this review was developed by consulting 2 experts in digital mental health and by checking systematic reviews of relevance to the review. These terms were chosen based on the target intervention , target outcome , and target study design . This review included only RCTs that assessed the effectiveness of serious games for alleviating the severity of depressive symptoms. To be more precise, the intervention of interest in this review was serious games that were delivered on any digital platform such as computers, consoles , mobile phones, tablets, handheld devices, or any other computerized devices. The intervention had to utilize elements of gaming as an integral and primary method for therapeutic or prevention purposes. We did not consider nondigital games and those used for other purposes such as monitoring, screening, and diagnosis. We included RCTs whether they were parallel RCTs, cluster RCTs, crossover RCTs, or factorial RCTs but we excluded quasi-experiments, observational studies, and reviews. We focused on studies in which one of the measured outcomes was depression or depressive symptoms regardless of the outcome measures. Only trials in the English language were eligible for inclusion in this review. RCTs published as journal articles, conference proceedings, and dissertations were included, whereas we excluded conference abstracts and posters, commentaries, preprints, proposals, and editorials. We did not apply restrictions related to the population, year of publication, country of publication, comparator, and study settings.We followed 3 steps to identify the relevant studies. In the first step, we exported the retrieved studies to EndNote to identify and remove duplicates. Then, 2 reviewers (EA and MA) independently screened the titles and abstracts of all retrieved studies. In the last step, the 2 reviewers independently screened the full texts of studies included from the second step. A third reviewer (AA) resolved any disagreements between the 2 reviewers in the second and third steps. Cohen \u03ba in this review indicated a very good level of interrater agreement in the first (0.85) and second (0.90) steps .Two reviewers (EA and MA) independently extracted data from the included reviews using Microsoft Excel . Two reviewers (EA and MA) independently assessed the risk of bias in the included studies using the Risk-of-Bias 2 (RoB 2) tool, which is recommended by the Cochrane Collaboration . This toWe utilized narrative and statistical approaches to synthesize the extracted data. Specifically, in narrative synthesis, texts and tables were used to describe the characteristics of the included studies, population, intervention, comparator, and outcome measures. Then, we grouped and summarized the findings of the included studies according to the type of serious games . A meta-analysis was conducted when at least 2 studies of the same type of serious game reported enough data for the analysis . When this information was not reported in any included study, we contacted the first and corresponding authors to get the missing information.d) because the outcome data (severity of depressive symptoms) were continuous and tools used to measure the outcome were different between the included studies. The random effects model was used in the analysis given the clinical heterogeneity between the meta-analyzed studies in terms of serious game characteristics , population characteristics , and outcome measures .Review Manager (RevMan 5.4) was used to conduct the meta-analysis. We measured the effect of each trial and the overall effect using the standardized mean difference  is defined as the smallest change in a measured outcome that a patient would consider as worthy and significant and which mandates a change in a patient\u2019s treatment. The MCID boundaries for an outcome were calculated as \u00b10.5 times the SMD of the meta-analyzed studies.P value and I2, which measures the statistical significance of heterogeneity and the degree of heterogeneity, respectively. A Chi-square P value \u2264.05 indicates heterogeneous meta-analyzed studies [2 was 0%-40%, 30%-60%, 50%-90%, or 75%-100%, respectively [We checked the characteristics of participants, interventions, comparator, and outcomes in studies included in the meta-analysis to assess their clinical heterogeneity. We also examined the statistical heterogeneity of the meta-analyzed studies by calculating a Chi-square  studies . The degectively .We assessed the overall quality of evidence from the meta-analyses using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, which assesses the quality of evidence based on 5 domains: risk of bias, inconsistency , indirectness, imprecision, and publication bias . Two revAs shown in The included studies were published between 2012 and 2021 . The yeaThe sample size in the included studies varied from 32 to 540, with an average of 104. The mean age of participants in the included studies ranged between 9.41 years and 84.5 years, with an average of 43.9 years. The percentage of the sample who were men reported in 26 studies ranged from 0% to 100%, with an average of 46.1%. Participants\u2019 health conditions were varied between studies, and depression and stroke were the most common (n=4 each). Participants in most studies were recruited from clinical settings (n=20).The intervention in the included studies was only serious games in 19 studies, serious games plus occupational therapy in 2 studies, and serious games plus psychotherapy in 1 study . The mosAs shown in The random allocation sequence for the randomization process was appropriate in 23 included studies. However, only 10 studies concealed the allocation sequence until participants were enrolled and assigned to interventions, and groups were not comparable in 4 studies. Accordingly, the risk of bias due to the randomization process was rated as low for only 8 studies .Participants and individuals delivering the interventions were aware of assigned interventions during the experiment in 22 and 20 studies, respectively. Deviation from the intended intervention occurred in 2 studies due to the experimental contexts. Only 14 studies used an appropriate analysis  to estimate the effect of assignment to intervention. Therefore, the risk of bias due to the deviations from the intended interventions was judged as low in only 8 studies .Outcome data were not available for all or nearly all participants in 21 studies, and there was evidence that the findings were not biased by missing outcome data in only 5 studies. The reasons for missing outcome data could not be related to the true value of the outcome in 18 studies. As a result, 17 studies were judged as having a low risk of bias in the \u201cmissing outcome data\u201d domain.All included studies assessed the outcome of interest  using appropriate measures and used measurement methods comparable across intervention groups. However, the assessor of the outcome was blinded in only 9 studies. For this reason, only these studies were rated as low risk of bias in the \u201cmeasuring the outcome\u201d domain .In 17 studies, a prespecified analysis plan  was not published. Only 4 studies reported outcome measurements different from those specified in the analysis plan. There is no evidence that all included studies selected their results from many results produced from multiple eligible analyses of the data. Accordingly, the risk of bias due to the selection of the reported results was considered low in 4 studies .In the last domain \u201coverall bias,\u201d the risk of bias was considered high in 20 studies as they were judged as having a high risk of bias in at least one domain; 6 studies were judged to have some concerns in the domain of overall bias as they had some concerns in at least one of the domains and were not at high risk for any domain. The remaining study was judged to be at low risk of bias for the domain of overall bias given that it was rated to be at low risk of bias for all domains. Reviewers\u2019 judgments about each \u201crisk of bias\u201d domain for each included study are presented in As mentioned earlier, we identified 5 types of serious games based on the therapeutic modality that they deliver in the included studies. The first type is exergames, which refer to video games that require physical exercises  in order to be played. The second type is computerized CBT games, which are video games that provide CBT for the users. The third type is exposure therapy games, which are video games that apply exposure principles to reduce anxiety in users with phobias. The fourth type is brain training games, which are video games that are based on cognitive interference tasks to reconsolidate traumatic memories. The last type is REBT- and REBE-based games, which are video games that enable users to replace irrational beliefs  with rational beliefs . Results of the included studies were grouped into 3 categories based on the types of serious games.Exergames were the intervention in 16 studies -58. ExerP<.05) in depressive symptoms between the groups, favoring exergames over ergometer training. Another study assessed the effect of exergames (Wii Fit Plus) on the severity of depressive symptoms (measured using the HADS-D) among patients with unilateral peripheral vestibular loss [P=.49) in the severity of depressive symptoms between the exergame group and conventional exercise group [P=.07) between both groups in the severity of depressive symptoms. A study by Ozdogar et al [P>.05) in the severity of depressive symptoms between the 2 groups. A study examining the effect of exergames (Kinect-RehabPlay) on the severity of depressive symptoms (measured using the BDI) among patients with coronary artery disease in comparison with conventional exercises found no significant difference in the severity of depressive symptoms between the 2 groups [P=.27) in the severity of depressive symptoms between the 2 groups. A study assessed the effects of exergames (Wii-Sports) and conventional exercises on the severity of depressive symptoms (measured by HADS) among patients with a high risk of cardiovascular diseases [In 9 studies, the effect of exergames was compared with that of conventional exercises on the severity of depressive symptoms -51. Althlar loss . The stuse group . Schumacse group  assessed2 groups , no sign2 groups  compareddiseases . No statdiseases . In the diseases .P=.12) in the severity of depressive symptoms was found between the exergame group and conventional exercise group . There was substantial heterogeneity in the evidence . The quality of the evidence was very low, as it was downgraded by 5 levels due to a high risk of bias, heterogeneity, and imprecision , PAID (P=.007), and ADS-L (P=.002) [P<.05) between the groups, favoring no intervention over exergames [P<.001) in the severity of depressive symptoms between the 2 groups, favoring exergames over no intervention. Jahouh et al [P=.43) and GADS (P=.21) was detected between the exergame group and the control group [P>.05) in the severity of depressive symptoms between the 2 groups [P=.09) was found in the severity of depressive symptoms between the 2 groups [In 7 studies, the effect of exergames was compared with that of no intervention on the severity of depressive symptoms -55. Alth(P=.002) . A study(P=.002)  examinedxergames . In anotxergames  compareduh et al  assessedol group . Another2 groups . The eff2 groups , and no 2 groups .P=.004) between exergame and control groups, favoring exergames over no intervention . The quality of the evidence was very low, as it was downgraded by 6 levels due to a high risk of bias, heterogeneity, and imprecision  in the severity of depressive symptoms between the exergame group and physical education group [P=.56) in the severity of depressive symptoms between the exergame group and occupational therapy group [In 3 studies, the effect of exergames was compared with that of active interventions on the severity of depressive symptoms, and no statistically significant difference was found between the groups -58. To bon group . The secpy group . The thiComputerized CBT games were the intervention in 8 studies -66. CompP=.001) but not the Reynolds Adolescent Depression Scale  [P<.001, and at a 6-month follow-up, P=0.01) between the groups, favoring SPARX-R over LIFESTYLE [P=.26) and RADS-2 (P=.16) [P=.96) in the severity of depressive symptoms between the SPARX group and the control group [P=.25) in the severity of depressive symptoms was detected between the groups [P=.76) between the groups. In the last study in this comparison, the effects of a computerized CBT game (ZeroPhobia) and no intervention on the severity of depressive symptoms (measured using the PHQ-9) among patients with acrophobia were investigated [P=.12) in the severity of depressive symptoms was found between the 2 groups [In 7 studies, the effect of computerized CBT games was compared with that of no intervention on the severity of depressive symptoms -65, and ; P=.08) . In the ; P=.08) , the effIFESTYLE . In cont (P=.16) . Similarol group . Anotherol group . No state groups . V\u00e4lim\u00e4kstigated . No stat2 groups .P=.003) indicating that computerized CBT games are more effective than no intervention in alleviating depressive symptoms: . This difference was also clinically important as the overall effect was outside the MCID boundaries (\u20130.10 to 0.10) and its CI did not cross the \u201cno effect\u201d line (zero effect) and both MCID boundaries. For this outcome, MCID boundaries were calculated as \u00b10.5 times the SMD value (\u2013.20). The heterogeneity of the evidence was not a concern . The quality of the evidence was very low, as it was downgraded by 3 levels due to the high risk of bias and impression  between the groups. Another study compared the effect of a computerized CBT game (MindLight) with that of an entertainment video game (Triple Town) on the severity of depressive symptoms (Child Depression Inventory 2) among patients with autism spectrum disorder and anxiety [P>.05) in the severity of depressive symptoms was detected between the groups [P=.58) in the severity of depressive symptoms between the groups.V\u00e4lim\u00e4ki et al  compared anxiety . No state groups . A studye groups  assessedP=.95) in the severity of depressive symptoms was detected between the groups [P=.95) in the severity of depressive symptoms between the 2 groups was detected [P=.03) and no intervention (P<.001).One study compared the effect of an exposure therapy game (Spider App) with that of an entertainment video game (Bubble Shooter) on the severity of depressive symptoms (measured using the BDI-II) among patients with arachnophobia . No state groups . Butler e groups  examineddetected . In anotdetected . The stuThis review assessed the effectiveness of serious games on the severity of depressive symptoms as reported by RCTs. Although 27 RCTs were included in the current review, 16 studies were included in the meta-analysis. Very low-quality evidence from 7 RCTs showed no statistically significant effect of exergames on the severity of depressive symptoms as compared with conventional exercises. Furthermore, 3 studies that compared the effect of exergames with that of other active interventions  on the severity of depressive symptoms and were not included in the meta-analyses found no statistically significant difference between the groups. These findings indicate that exergames are as effective as active interventions, which are usually delivered and supervised by health care providers .Very low-quality evidence from 5 RCTs showed a statistically and clinically significant effect of exergames on the severity of depressive symptoms when compared with no intervention.P<.001) of exergames on depression. Additionally, another recent meta-analysis of 8 RCTs conducted by Li et al [Findings in this review are comparable to other reviews. Specifically, a recently published meta-analysis of 5 RCTs conducted by Yen and Chiu  showed aLi et al  showed aLi et al ,37 compaLi et al .Very low-quality evidence from 6 RCTs showed a statistically and clinically significant effect of computerized CBT games on the severity of depressive symptoms when compared with no intervention. In contrast, 3 studies that compared the effect of computerized CBT games with those of active interventions  on depressive symptoms and were not included in the meta-analyses found no statistically significant difference between the groups. This insignificant effect can be attributed to the fact that conventional CBT is comparable to the active interventions, thereby comparing the effect of 2 comparable interventions usually produces no significant difference, which indicates that computerized CBT games are at least as effective as these active interventions. None of the previous reviews ,19,35-37This review bridged the gaps of previous reviews by focusing on all types of serious games, including only RCTs, targeting all age groups, searching technical databases, assessing the quality of evidence, and synthesizing the data statistically. Therefore, it is more comprehensive than previous reviews ,19,35-37The risk of publication bias in this review is minimal, as we searched the most popular databases in information technology and health fields; conducted backward and forward reference list checking; used a comprehensive search query; searched grey literature databases; and did not restrict our search to a certain country, year, setting, population, and comparator.The risk of selection bias in this review is minimal because 2 reviewers independently performed the study selection, data extraction, risk of bias assessment, and quality of evidence evaluation with a very good interrater agreement for all processes. The quality of the evidence was appraised to enable the reader to draw more accurate conclusions. When possible, we synthesized data statistically, and this improved the power of studies and increased the estimates of the likely size of the effect of serious games on depression.The intervention of interest in this review was restricted to serious games delivered on any digital platform and used as a therapeutic intervention. Thus, this review cannot comment on the effectiveness of nondigital serious games and those used for other purposes such as monitoring, screening, or diagnosis. The outcome of interest in this review was depression; therefore, we cannot comment on the effectiveness of serious games on other mental health outcomes.The review was restricted to RCTs written in the English language; therefore, many studies were excluded because they were quasi-experiments or written in other languages. This restriction was necessary because RCTs have higher internal validity than any other study design  and owinMost included studies recruited patients without depression; thereby, the effect of serious games on the severity of depression symptoms was not significant. Further, the overall risk of bias was high in most included studies, and the quality of evidence for the meta-analyses was very low. Accordingly, findings in this review must be interpreted with caution.Although the severity of depression was one of the measured outcomes in all included studies, only 5 studies recruited patients with depression. This might lead to underestimating the effect of serious games. Therefore, future studies need to recruit participants with depression to assess the effectiveness of serious games on depression.The therapeutic modalities provided by serious games in most included studies were either exercises or CBT. Further, serious games were not designed specifically to alleviate depression in about half of the studies. Thus, there is a pressing need to assess the effectiveness of serious games that are designed specifically to alleviate depression and deliver other therapeutic modalities such as art therapy, psychotherapy, relaxation-based exercises, psychoeducation, rational emotive behavioral therapy, and exposure therapy, and the list goes on.Most included studies were carried out in high-income countries; thereby, our findings may not be generalizable to low-income countries. Researchers should conduct more studies to assess the effectiveness of serious games in low-income countries. We excluded many studies that assessed the effectiveness of serious games on other mental disorders such as anxiety and dementia. Further systematic reviews need to be carried out to investigate the effectiveness of serious in alleviating other mental disorders.The overall risk of bias was high in most included studies mainly due to issues in the randomization process, deviations from the intended outcomes, and selection of the reported result. Further, several studies were not included in the meta-analysis due to missing outcome data. For this reason, we encourage researchers to follow recommended guidelines or tools  when coThis review hopefully augurs the possible potential of serious games in mental health disorders, but it also underlines that this field, albeit full of potential, is still in its infancy. More studies are needed to prove the significant role of serious games in alleviating depression.Overall, this study showed that serious games can be effective in alleviating depression in comparison with no intervention, and they can be comparable to other traditional therapeutic interventions for alleviating depressive symptoms. However, findings in this review must be interpreted with caution because the overall risk of bias was high in most included studies, the quality of evidence in the meta-analyses was very low, few studies recruited patients with depression, and serious games in half of the studies were purpose-shifted. Therefore, we can only recommend health care providers consider offering serious games as an adjunct to existing interventions until further, more robust evidence is available.As mentioned before, serious games in more than half of the studies were not designed to specifically alleviate depression and did not deliver other therapeutic modalities such as art therapy, REBT, and psychoeducation. This may be attributed to the lack of such serious games in real life. Accordingly, there is a need to develop more serious games that are designed to specifically alleviate depression and deliver other therapeutic modalities.The most common platforms used for playing the games were computers and video game consoles and their accessories, which are relatively more expensive and less accessible than smartphones that were the platform for serious games in only 1 study. The number of smartphone users in the world exceeded 6.4 billion in 2021 , which fMost studies were carried out in high-income countries, and this may indicate the lack of serious games in low-income countries. People in low-income countries may be more in need of serious games than those in high-income countries because low-income countries have a greater shortage of mental health professionals than high-income countries  ,74. SeriGaming and mental health have traditionally been two distinctly separate fields and come with their own unique pedagogy and praxis. The potential of utilizing the advantages inherent to gaming, as described earlier, from its reach to its transformative potential in mental health holds a lot of promise in theory. However, to achieve this potential, experts from the two disciplines need to work together in order to understand the unique strengths and limitations of each field when designing serious games.Overall, serious games can be better than no intervention in alleviating depression and as effective in alleviating depression as other active interventions . However, definitive conclusions regarding the effectiveness of serious games could not be drawn in this review because the overall risk of bias was high in most included studies, the quality of the meta-analyzed evidence was very low, and few studies recruited patients with depression. Therefore, we can only recommend health care providers consider offering serious games as an adjunct to existing interventions until further, more robust evidence is available. To have sufficient evidence, future studies should assess the effectiveness of serious games that are designed specifically to alleviate depression and deliver other therapeutic modalities, recruit participants with depression, and avoid biases by following recommended guidelines for conducting and reporting RCTs ."}
+{"text": "Cognitive impairment is a mental disorder that commonly affects elderly people. Serious games, which are games that have a purpose other than entertainment, have been used as a nonpharmacological intervention for improving cognitive abilities. The effectiveness and safety of serious games for improving cognitive abilities have been investigated by several systematic reviews; however, they are limited by design and methodological weaknesses.This study aims to assess the effectiveness and safety of serious games for improving cognitive abilities among elderly people with cognitive impairment.A systematic review of randomized controlled trials (RCTs) was conducted. The following 8 electronic databases were searched: MEDLINE, Embase, CINAHL, PsycINFO, ACM Digital Library, IEEE Xplore, Scopus, and Google Scholar. We also screened reference lists of the included studies and relevant reviews, as well as checked studies citing our included studies. Two reviewers independently carried out the study selection, data extraction, risk of bias assessment, and quality of evidence appraisal. We used a narrative and statistical approach, as appropriate, to synthesize the results of the included studies.P=.04) and conventional exercises (P=.002) for improving global cognition among elderly people with cognitive impairment. Further, a subgroup analysis showed that cognitive training games were more effective than no intervention (P=.05) and conventional exercises (P<.001) for improving global cognition among elderly people with cognitive impairment. Another subgroup analysis demonstrated that exergames  are as effective as no intervention and conventional exercises (P=.38) for improving global cognition among elderly people with cognitive impairment. Although some studies found adverse events from using serious games, the number of adverse events  was comparable between the serious game and control groups.Fifteen studies met the eligibility criteria among 466 citations retrieved. Of those, 14 RCTs were eventually included in the meta-analysis. We found that, regardless of their type, serious games were more effective than no intervention (Serious games and specifically cognitive training games have the potential to improve global cognition among elderly people with cognitive impairment. However, our findings remain inconclusive because the quality of evidence in all meta-analyses was very low, mainly due to the risk of bias raised in the majority of the included studies, high heterogeneity of the evidence, and imprecision of total effect sizes. Therefore, psychologists, psychiatrists, and patients should consider offering serious games as a complement and not a substitute to existing interventions until further more robust evidence is available. Further studies are needed to assess the effect of exergames, the safety of serious games, and their long-term effects. Societies globally are rapidly aging at unprecedented rates. The United Nations projects that by 2050, 1 in 6 people in the world will be over the age of 65 years .Unfortunately, declining mental and cognitive abilities not only affect people and their relatives but also burden health care systems. Among the top chronic diseases causing the progressive decline and deterioration of cognitive abilities are mild cognitive impairment (MCI), Alzheimer disease (AD), and dementia. Globally, it is estimated that the number of prevalent dementia cases more than doubled (117%) between 1990 and 2016 . MoreoveResearch suggests that cognitive symptoms experienced by people with declining or deteriorating cognitive abilities are often associated or even preceded by behavioral symptoms. Therefore, treatments for improving cognitive functions and abilities often cannot be separated from behavioral treatments , and theNumerous prior studies have investigated the effectiveness of serious games for improving cognitive abilities. Aggregating and summarizing the findings from these studies is crucial for drawing conclusions about the effectiveness of serious games for improving cognitive abilities. Several systematic reviews have aggregated evidence from these studies; however, they are undermined by certain shortcomings that limit the generalization of the findings. Specifically, these reviews (1) focused on older adults who did not have cognitive impairment ,17,18;  guidelines  21]. Th. Th21]. The following 8 bibliographic databases were searched in order to retrieve studies that were relevant to this review: MEDLINE (via Ovid), PsycInfo (via Ovid), Embase (via Ovid), CINAHL (via EBSCO), IEEE Xplore, ACM Digital Library, Scopus, and Google Scholar. The search was conducted on August 6, 2021, by the first author . In order to retrieve studies that were added to the databases after that date, an automatic alert was set up, and it ran its course for 12 weeks . Due to the large number of studies retrieved on Google Scholar, only the first 10 pages  were considered, as they are automatically ordered based on their relevance . We applFor developing the search query for this review, we consulted 2 experts in digital mental health and checked the search query used in other systematic reviews within this field. The chosen search terms related to the target population , target intervention , and target study design . Only RCTs that assessed the effectiveness of serious games for improving cognitive abilities among elderly people with cognitive impairment were included in this study. For the purpose of this review, the target intervention was serious games that are available on digital platforms, such as computers, consoles , mobile phones, tablets, handheld devices, Nintendo, and other computerized devices. Further, gaming had to be an integral and primary component of the intervention and used solely for the purpose of therapy. Studies combining serious games with other interventions were eligible if the control group received the same adjacent intervention. Nondigital games and those used for other purposes, such as monitoring, screening, diagnosis, and research, were excluded.The population of interest was elderly people (\u226560 years) with cognitive impairment/disorder . Their diagnosis had to be confirmed by examining the inclusion criteria or baseline scores against standardized diagnostic criteria . Studies about healthy elderly people without cognitive impairment, health care providers, and caregivers were excluded. No restrictions were applied regarding gender and ethnicity.The main outcome of interest in this review was global cognition regardless of the tool used for measuring the outcome. Global cognition is a general measure of all cognitive abilities such as memory, language, learning, and attention. Although behavioral outcomes relate to cognitive outcomes, behavioral outcomes are out of the scope of this review. The secondary outcome of interest was adverse events, which we used as an indicator of the safety of serious games. Studies were excluded if they assessed only cost effectiveness, acceptance, feasibility, and satisfaction. This review focused on outcome data that were measured immediately after the intervention rather than follow-up data.All types of RCTs  were included, but pilot or feasibility RCTs, quasiexperiments, observational studies, and reviews were excluded. For practical reasons, only those trials in the English language were eligible for inclusion. Studies published from 2010 onwards were included. Studies published as journal articles, conference proceedings, and dissertations were included. Otherwise, conference abstracts and posters, preprints, commentaries, proposals, and editorials were all excluded. No restrictions related to the country of publication, comparator, and study settings were applied.Relevant studies were identified in the following steps. To begin, the obtained studies were imported into EndNote to identify and delete duplicate items. Following the PRISMA guidelines, the titles and abstracts of all retrieved studies were evaluated in the second phase by 2 reviewers working independently. Two reviewers independently evaluated the entire text of the studies included in the previous step. Any disagreements were resolved via discussion. The 2 reviewers were the first 2 authors of this paper: A Abd-alrazaq and MA. The interrater agreement (Cohen \u03ba) in steps 2 and 3 were 0.84 and 0.89, respectively, indicating a near-perfect level of interrater agreement .Data from the included papers were extracted by 2 reviewers  independently using Microsoft Excel. Cochrane Collaboration recommends assessment of the risk of bias by 2 independent reviewers using the Risk-of-Bias 2 (RoB-2) tool , and as d) was used to assess the overall effect of each study, as the type of data for the outcome of interest  was continuous and the instruments used to evaluate the outcome were diverse among the included trials. Due to the high clinical heterogeneity between the meta-analyzed studies in terms of serious game characteristics , population characteristics , and outcome measures , the random effects model was used for the analysis.For the purpose of synthesizing the gathered data, a narrative and statistical approach was employed. Texts and tables were used to describe the features of the included studies  in our narrative synthesis. The results of the experiments were compiled and categorized based on the comparator as follows: control, conventional exercises, conventional cognitive training, and other serious games. A meta-analysis was conducted when at least two studies of the same comparator submitted enough data . Meta-analyses were conducted using Review Manager (RevMan 5.4). The standardized mean difference (SMD)  refers to the smallest change in a measured outcome that a patient would consider worthwhile and significant enough to merit a change in treatment. The MCID bounds were computed as \u00b10.5 times the meta-analyzed studies\u2019 SMD.2 and the chi-square P value, respectively. A chi-square P value of .05 or less suggests heterogeneous meta-analyzed studies [2 ranged from 0% to 40%, 30% to 60%, 50% to 90%, and 75% to 100%, the degree of heterogeneity was judged as insignificant, moderate, substantial, and considerable, respectively [In order to examine the degree and statistical significance of heterogeneity in the meta-analyzed studies, we calculated I studies . When I2ectively .In order to assess the overall quality of evidence resulting from the meta-analyses, we used the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach . The GRABy searching the 7 electronic databases, 466 records were retrieved . Of thesThe included studies were published between 2013 and 2021 . The yeaThe sample size in the included studies ranged from 20 to 114, with an average of 70.4. The mean age of participants reported in 14 studies ranged between 67 and 84.2 years, with an average of 75.2 years. The percentage of males reported in 14 studies ranged from 23.5% to 70%, with an average of 44.5%. The mean MMSE score for participants in the included studies varied between 10.2 and 27. Participants in the included studies had MCI (n=10), AD (n=2), dementia (n=1), MCI and dementia (n=1), and neurocognitive disorders (n=1). Participants were recruited from clinical settings in 13 studies and the community in 2 studies.Serious games alone were used as interventions in 13 of the included studies, whereas the remaining 2 studies used serious games combined with other interventions . The incThe comparison groups received passive interventions in 9 studies, whereas active interventions were received in 8 studies  . Two stuSeven included studies generated an appropriate random allocation sequence for the randomization process. In 4 studies, the allocation sequence was concealed until participants were assigned to interventions. There were no imbalances between groups at baseline in 14 studies. Consequently, the risk of bias due to the randomization process was rated as low in only 4 out of 15 studies .Participants and those who delivered the interventions were aware of the assigned interventions during the trial in 13 and 14 studies, respectively. In all included studies, there was no evidence that the experimental contexts led to a deviation from the intended intervention. All 15 studies used appropriate analysis methods  to estimate the effect of the intervention. According to these judgments, the risk of bias due to the deviations from the intended interventions was low in 13 out of 15 studies .Missing outcome data were less than 5% in 7 studies. In only 1 study, there was evidence that the findings were not biased by missing outcome data. The missing outcome data could be related to participants\u2019 health status in 3 studies. Consequently, 11 studies were judged as having a low risk of bias in the \u201cmissing outcome data\u201d domain .In all included studies, global cognition was assessed using appropriate measures, and measurement methods were comparable across intervention groups. The assessor of the outcome was blinded to the assigned interventions in 9 studies. Assessment of the outcome may have been affected by knowledge of the intervention received in 3 studies. Accordingly, the risk of bias in the \u201cmeasuring the outcome\u201d domain was rated as low in 13 studies .Five studies published their protocol in sufficient detail. In all studies, reported outcome measurements did not differ from those specified in the analysis plan, and there was no evidence that studies selected their results from the many results produced from multiple eligible analyses of the data. Based on these judgments, the risk of bias due to the selection of the reported results was considered low in 5 studies .In the last domain \u201coverall bias,\u201d the risk of bias was considered high in 2 studies as they were judged as having a high risk of bias in at least one domain. Twelve studies raised some concerns in the domain of overall bias as they had some issues in at least one of the domains and were not at high risk for any domain. The remaining study was judged to be at low risk of bias for the domain of overall bias given that it was rated to be at low risk of bias for all domains. Reviewers\u2019 judgments about each \u201crisk of bias\u201d domain for each included study are presented in In this review, serious games were compared with control (no/passive interventions), conventional exercises, conventional cognitive activities, and other serious games. Results of the included studies are shown in the following subsections based on these comparisons. Then, the results of the subgroup analysis are shown. Lastly, the results of the included studies regarding the safety of serious games have been reported.The effect of serious games was compared with a control (no/passive interventions) in 9 studies -35. PassP=.04) between the serious games and control groups, favoring serious games over no/passive intervention . This difference was also clinically important as the overall effect was outside MCID boundaries (\u22120.15 to 0.15) and its CI did not cross the \u201cno effect\u201d line (zero effect). For this outcome, MCID boundaries were calculated as \u00b10.5 times the SMD value (0.29). The statistical heterogeneity of the evidence was substantial . The quality of the evidence was very low as it was downgraded by 5 levels due to a high risk of bias, heterogeneity, and imprecision  between the groups, favoring serious games over conventional exercises . This difference was also clinically important as the overall effect was outside MCID boundaries (\u22120.31 to 0.31) and its CI did not cross the \u201cno effect\u201d line (zero effect). For this outcome, MCID boundaries were calculated as \u00b10.5 times the SMD value (0.61). The statistical heterogeneity of the evidence was substantial . Like the meta-analysis seen in the previous section, the quality of this evidence was very low as it was downgraded by 5 levels due to a high risk of bias, heterogeneity, and imprecision  and GDS (P=.07) [One study compared the effect of serious games to conventional cognitive activities in terms of global cognition among patients with MCI . The stu (P=.07) .Gooding et al  comparedP=.05) and conventional exercises (P<.001) (P<.001) compared with both control and conventional exercises . This difference was also clinically important as the overall effect was outside MCID boundaries (\u22120.27 to 0.27) and its CI did not cross the \u201cno effect\u201d line (zero effect). For this outcome, MCID boundaries were calculated as \u00b10.5 times the SMD value (0.54). The statistical heterogeneity of the evidence was substantial . The quality of this evidence was very low as it was downgraded by 5 levels due to a high risk of bias, heterogeneity, and imprecision  have a different effect on global cognition. A subgroup analysis of 11 studies (14 comparisons) showed that cognitive training games had a statistically significant effect on global cognition compared to control ((P<.001) . The overecision .P=.38) in global cognition between the exergame group and control or conventional exercise group  . The quality of the evidence was very low as it was downgraded by 4 levels due to a high risk of bias and imprecision  showed no statistically significant difference (to 0.32) . The starecision .Five studies assessed the safety of serious games by checking adverse events -38. Of tThis review investigated the effectiveness of serious games for improving global cognition as reported by RCTs. Very low\u2013quality evidence from 9 RCTs (11 comparisons) and 6 RCTs (7 comparisons) showed that the effect of serious games on global cognition was statistically significant in comparison with no/passive interventions and conventional exercises. None of the previous reviews examined the effect of all types of serious games. Due to evidence paucity, no statistical analysis was carried out to compare serious games to other types of interventions .Additionally, very low\u2013quality evidence from 11 RCTs (14 comparisons) showed that the effect of cognitive training games on global cognition was statistically significant in comparison with no/passive interventions, conventional exercises, and both. Interestingly, the effect of serious games in comparison with conventional exercises was higher than their effect in comparison with no/passive interventions. Studies in both meta-analyses were comparable in terms of population, intervention, and outcome measures. This paradoxical finding may be attributed to the fact that passive interventions used in the included studies  improved global cognition among participants, and thereby, the difference in global cognition between the serious game group and passive intervention group decreased. This is evident in Our findings are in line with the findings of a previous review that compared the effect of cognitive training games to passive interventions, active interventions, and both in terms of global cognition among old adults with MCI . SpecifiP<.001) in global cognition between the groups, favoring exergames over both active and passive interventions [Very low\u2013quality evidence from 3 RCTs (4 comparisons) showed an insignificant effect of exergames on global cognition in comparison with both control and conventional exercises. Our findings are inconsistent with findings from a previous review that compared the effect of exergames to both active and passive interventions in terms of global cognition among adults with and without health issues . Specifiventions . This inventions ; (2) theventions ; (3) theAccording to 5 of the included studies, there was no significant difference in the number of adverse events between groups, indicating that serious games are safe. This finding was also concluded by a previous review about the use of cognitive training games for improving cognitive abilities among elderly people without cognitive impairment .In comparison with previous reviews ,17-20, tThere is no concern about the risk of publication bias as the authors sought to identify as many relevant studies as possible by searching the most popular databases in the information technology and health fields, searching grey literature databases, conducting backward and forward reference list checking, and using a well-developed search query.Given that all processes  were carried out by 2 reviewers independently, the risk of selection bias is not a concern in this review. This review enables the reader to draw more accurate conclusions given that we appraised the quality of the evidence using the GRADE approach. When possible, we synthesized data statistically, and this improved the power of studies and increased the estimates of the likely size of the effect of serious games on global cognition.The effectiveness and safety of serious games delivered on nondigital platforms and those used for other purposes  cannot be commented on, because this review excluded studies discussing these types of serious games. This review focused on the effectiveness and safety of serious games for promoting global cognition among elderly people with cognitive impairment; thus, the effectiveness and safety of serious games for improving specific cognitive abilities  or behavioral outcomes  among other age groups without cognitive impairment cannot be commented on.We excluded numerous studies as they were quasiexperiments, pilot RCTs, published before 2010, or written in non-English languages. Therefore, it is likely that we missed some relevant studies. We excluded these studies as quasiexperiments and pilot RCTs have lower internal validity than RCTs . BecauseThis review focused on the short-term effect of serious games by meta-analyzing only postintervention data rather than follow-up data, because only 3 studies reported follow-up data and the follow-up period was not consistent between studies. Therefore, we cannot comment on the long-term effect of serious games on global cognition. The quality of the evidence in all meta-analyses was very low, and this may decrease the internal validity of our findings.This review used postintervention data for each group to assess the effect size for each meta-analyzed study rather than the pre-post intervention change for each group, and thereby, it is likely that the effect size was overestimated or underestimated in this review. We used postintervention outcome data because the majority of studies did not report the mean and standard deviation for the pre-post intervention change in global cognition for each group, and there was no statistically significant difference in global cognition at baseline between groups in all studies.Non-RCTs can be used to assess the safety of interventions, such as serious games. However, such studies were not included in this review. Thus, it is likely that we missed several studies about the safety of serious games.Given that this review focused on the effectiveness and safety of serious games for improving global cognition among elderly people with cognitive impairment, it is recommended that researchers conduct further reviews to assess the effectiveness and safety of serious games for improving specific cognitive abilities  among people from different age groups with or without cognitive impairment.A few studies were carried out in developing countries, and as such, the generalizability of this review\u2019s findings to such countries may be limited. More studies should be conducted in developing countries, especially given the varying nature of their cultures and socioeconomic conditions. A handful of studies assessed the safety of using serious games for improving cognitive abilities; thus, more studies will be helpful to draw more definitive conclusions about the safety of serious games.This review did not assess the long-term effect of serious games given the lack of studies that reported follow-up data. Researchers should follow-up with participants to assess the long-term effect of serious games on global cognition. The majority of the included studies did not report the mean and standard deviation for the pre-post intervention change in global cognition for each group. It is important that future studies report this information to calculate more accurate effect sizes.The overall risk of bias was low in only 1 study given that most studies had issues mainly in the randomization process and selection of the reported results. Accordingly, researchers should follow recommended guidelines or tools  when coThis review showed that serious games and specifically cognitive training games are more effective than no intervention and conventional exercises for improving global cognition, whereas exergames are as effective as no intervention and conventional exercises. However, these findings should be interpreted carefully because the quality of evidence in all meta-analyses was very low given that the majority of the included studies were judged to have some concerns in overall bias, the heterogeneity of the evidence was high in all meta-analyses except 1, and the total effect sizes were imprecise. Accordingly, psychologists and psychiatrists should consider offering serious games as a complement and not as a substitute to existing interventions until more robust evidence is available.Still, the emerging evidence from this study presents promising opportunities to leverage serious games to alleviate the burden on health care systems due to exponential growth in the number of elderly people worldwide in the years to come. Serious games can allow elderly people with cognitive impairments to improve their psychological, physiological, sensory/motor, and social functions, thereby enjoying a higher quality of living . BecauseMobile devices (smartphones and tablets) were used as the platform for serious games in only 2 studies. Mobile devices are particularly appealing as they are cheaper than computers and more pervasive than gaming consoles. Mobile devices are also more accessible than computers and gaming consoles. It is estimated that there were about 15 billion mobile devices and more than 7.1 billion mobile users worldwide in 2021 . App andWhen examining the few studies conducted in developing countries, it seems that there is more focus on implementing serious games in developed countries despite the greater shortage of mental health professionals in developing countries . TherefSerious games and specifically cognitive training games have the potential to improve global cognition among elderly people with cognitive impairment. However, definitive conclusions could not be drawn regarding the effectiveness and safety of serious games for improving global cognition among elderly people with cognitive impairment. This is because the quality of evidence in all meta-analyses was very low mainly due to concerns raised about the bias in the majority of the included studies, high heterogeneity of the evidence, and imprecision of total effect sizes. Therefore, psychologists, psychiatrists, and patients should consider serious games as a complement and not as a substitute to existing interventions until further more robust evidence is available. Further reviews are required to assess the effectiveness and safety of serious games for improving specific cognitive abilities  among people from different age groups with or without cognitive impairment. Additional studies are needed to assess the effect of exergames, the safety of serious games, and their long-term effects."}
+{"text": "To determine the transfer of the monoclonal antibody natalizumab into breastmilk and to evaluate drug and serum neurofilament light chain ((s)NfL) levels in natalizumab exposed pregnancies and lactation periods.Eleven women with relapsing remitting multiple sclerosis treated with natalizumab during pregnancy and lactation were included in this study. Breastmilk samples were collected up to 302 days after delivery and analyzed for natalizumab concentration and NfL. Additionally, maternal drug levels and sNfL were determined preconceptually, in each trimester, at delivery and postpartum. Clinical and radiological disease activity was systemically assessed across pregnancy and postpartum period.The mean average natalizumab concentration in breast milk was low at 0.06 \u00b5g/ml [standard deviation (SD) 0.05] in the eight patients who provided serial breastmilk samples with an estimated mean absolute infant dose of 0.007 mg/kg/d (SD 0.005). The relative infant dose (RID), a metric comparing the infant with maternal drug exposure was low as well with a mean of 0.04% (SD=0.03). Most patients had a maximum concentration in breast milk at one to eight days after infusion. Pregnancy was associated with a non-significant decline of the median natalizumab serum concentration. All patients exposed to natalizumab prior (n=10) and during pregnancy (n=11) kept free of disease activity during gestation. While pregnancy was associated with low sNfL levels in patients treated with natalizumab prior and during pregnancy, the postpartum period was linked to a transient sNfL increase in some patients without any evidence of clinical or radiological disease activity. NfL was detectable in the majority of breastmilk samples with a median concentration of 1.7 pg/ml (range 0.004-18.1).We determined transfer of natalizumab into breastmilk with an RID far below the threshold of concern of 10%. Studies including childhood development assessment are needed in order to gain safety data about natalizumab-exposed breastfeeding. SNfL assessment might be a useful adjunct to monitor silent disease activity and therapeutic response during pregnancy and postpartum period. However, further investigations regarding transient postpartum sNfL increases are required to determine its association to parturition per se or to a silent disease activity in people with multiple sclerosis. Multiple sclerosis (MS) is a chronic autoimmune demyelinating disease of the central nervous system, which predominantly affects women, and is most commonly diagnosed in the early adulthood , 2. Thus\u00ae, Biogen, Cambridge, MA), as one of the highly efficacious treatments, can be continued in selected patients throughout pregnancy after weighting benefits for the mother against the potential risks to the fetus  reflects disease activity during pregnancy in pwMS . Upon neThe evaluation of the safety profile regarding the use of high-effective DMT during pregnancy and lactation could support the decision of taking of these drugs, particularly in the case of high disease activity. In this study, we determined NAT concentrations in breast milk of eleven nursing women with relapsing remitting multiple sclerosis (RRMS). Additionally, we aimed to advance our knowledge about pregnancy and postpartum associated variations of NAT's PK and sNfL levels in association to disease activity parameters.We included eleven women diagnosed with RRMS according to the 2010 revisions of the McDonald criteria with a highly active disease course . Ten patThe immunological sub study was performed according to the Declaration of Helsinki and the study protocol was approved by the Ethics Committee of the Faculty of Medicine of the Dresden University of Technology. All participants provided written informed consent.Serum samples were collected within the six months prior pregnancy, during each trimester, immediately after delivery and during the first six months after delivery. Samples were obtained immediately before and generally 20 minutes after NAT infusions.Eight of the eleven women provided at least nine serial breastmilk samples after the first up to tenth maintenance infusions after delivery. Samples were collected immediately before NAT infusion, one day and several days after infusion. The other three patients provided one (n=1), three (n=1) or four (n=1) breastmilk samples.Serum and breastmilk samples were frozen at -20\u00b0C after collection until final preparation. For analysis of NAT concentrations in breastmilk and serum samples, our previously described HL60 cell based FACS assay  was used .Serum and breastmilk samples were stored at -20 until after preparation. NfL levels were determined using a Simoa HD-1 instrument  , 27. The0-Tmax) for each of these women. The average concentration of NAT in breastmilk (CAVG) was calculated by dividing the AUC0-Tmax by the number of days from the first breastmilk sample (T0) to the last breastmilk sample (Tmax). Additionally, the maximum concentration (CMAX) and the time to the maximum concentration was determined. The absolute average and maximum NAT dose to the infant in a 24-hour period and the relative infant dose (RID) was calculated using a method described by Bennett as well as the assumption that the infant will consume approximately 150ml/kg of breastmilk per day , followed by standard deviation (SD) or range. For the eight women who provided serial breastmilk samples the trapezoidal rule was used to calculate the area under the breastmilk concentration time curves  and GraphPad Prism .Patient characteristics are reported in No new and/or enlarging T2 lesions were detected in the first and second postpartum brain MRI scans performed after a median of 1.5 months (range 1 to 3) and a median of 8\u00a0months (range 4 to 10), respectively.A total of 178 breastmilk samples were provided by eleven women, with eight patients donating at least nine serial samples (range 9-51) while the other three patients provided one to four samples, respectively. NAT was detectable in varying concentrations in all breast milk samples with a median concentration of 0.049\u00b5g/ml (range 0.002\u20130.306\u00b5g/ml). Pharmacokinetic curves for patients with serial samples are shown in AVG, the mean absolute infant dose of NAT was 0.008mg/kg/d (SD 0.005), and the mean RID based on CAVG was 0.04% (SD 0.03). Corresponding serum samples were available for 7/8 patients with a median NAT concentration of 15.5 \u00b5g/ml (range 3.6 to 55.9). The calculated breastmilk to serum ratio from samples obtained immediately before the next NAT infusion, ranged between 1/228 to 1/2100 equivalent to less than 1%.Taken into account that we do not have breastmilk samples from the first hours after NAT infusion, the maximum NAT concentration was measured one to eight days (in one case after 22 days) after infusion with a mean maximum concentration of 0.12 (SD 0.09 \u00b5g/ml). Based on Cp > 0.999).A median NAT serum trough (preinfusion) concentration of 27.9 \u00b5g/ml (range 10.5 to 85.6) was observed prior pregnancy. Gestation was associated with a non-significant decrease of the median NAT concentration with a percentage difference of 53.1% between levels measured prior pregnancy and in the third trimester. NAT serum trough concentration at month four to six was comparable to NAT concentration prior pregnancy  was determined pre-pregnancy in nine patients who received at least seven infusions prior conception. During pregnancies, sNfL levels remained at low and stable values in these nine patients. For the patient who received only five infusions prior conception a sNfL concentration of 23.7 pg/ml followed by a constantly decrease during pregnancy was observed. For the early postpartum period, a significant transient peak in the first (n=3) or second (n=1) month following delivery with an up to 6-fold increase to individual SS value (range 22.4-38.3 pg/ml) in four of five patients with available serum samples was revealed. For none of these four patients, clinical nor radiological disease activity in follow up brain MRI scan was documented. During the third and up to the sixth month following delivery low sNfL concentration were observed and linked to a stable disease course (including the one with only five infusions prior conception and the two patients who only received three NAT infusions after delivery)  with a median concentration of 1.7 pg/ml (range 0.004 to 18.1). A correlation with NfL levels in serum was not observed.In this study of NAT-treated highly active RRMS patients, therapeutic drug monitoring revealed a low transfer of NAT into breastmilk and a small, non-significant decrease of NAT serum trough concentration across pregnancy. Although no evidence of clinical and radiological disease activity was detected in NAT exposed pregnancies and postpartum period up to six months, puerperium was linked to a transient sNfL peak in some patients.AVG was 0.04% and the median RID estimated by CMAX was 0.11%, which is in line with recently published data and well below the 10% that has generally been considered safe for breastfeeding an infant  are largely precluded from transfer into breastmilk due to large molecule size and limited transport mechanisms . For then infant , 31. Then infant . Howevern infant . NAT wasvia nFcR . In recevia nFcR , 13. Hervia nFcR . HoweverPregnancy is known as a state where PK changes are more pronounced and more rapid than during any other period of life with potential effects on any level of the disposition of drugs. Given the importance to maintaining an adequate treatment response during pregnancy in highly active MS, it is mandatory to understand whether pregnancy affects drug levels. For NAT, median serum trough concentration measured prior to pregnancy was similar to those levels reported in previous studies , 34\u201337. Disease reactivation following NAT cessation prior to or during pregnancy is well recognized and can lead to the accumulation of permanent disability , 39\u201341. Beside clinical and radiological disease activity parameters, blood derived biomarkers gain more and more attention. The implementation of a blood derived and easy to measure biomarker would be a valuable adjunct for monitoring disease activity during pregnancy in women with MS. Here, we describe pregnancy and postpartum associated changes in sNfL levels in women exposed to NAT. We observed low pre-pregnancy sNfL values in nine patients who received a least seven NAT infusions while a sNfL concentration of 23.7 pg/ml was detected in a patient who received only five infusions prior pregnancy. sNfL levels remained low and stable in the nine patients and decreased in the patient with only five infusions. We revealed a short lasting peak in sNfL with a significant increase up to 6-fold in puerperium in 4/5 patients with available serum samples from early postpartum period. This transient peak was not accompanied by clinical or radiological disease activity. For the further postpartum period from three to six months, low sNfL levels were detected, which were linked to a stable disease course in all patients.In this study, patients were only monitored through cerebral MRI as spinal cord MRI was not performed regularly. Other factors such as pre-eclampsia, general anesthesia during delivery, trauma, stroke, metabolic diseases, which could be associated with the postpartum sNfL increase, were not reported. For patients treated with alemtuzumab, similar findings about transient sNfL peaks postpartum were reported . A recenIn accordance with previous study results, we can confirm that NAT is transferred into human breastmilk in reassuringly low amounts. The observed alteration of drug levels during pregnancy are small and unlikely to be of clinically significance regarding efficacy. Data presented here suggest a therapeutic benefit for women with highly active RRMS from treatment with NAT during pregnancy and lactation and highlight sNfL as a promising add-on biomarker for monitoring disease activity and therapeutic response in pregnancy and postpartum period.While rheumatology and gastroenterology societies have already provided guidelines about the use of mAbs during lactation, such guidelines are lacking for neurologic conditions , 46. TheThe 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 Ethics Committee of the Faculty of Medicine of the Dresden University of Technology. The patients/participants provided their written informed consent to participate in this study.Study concept and design: UP, KA, and TZ. Acquisition of data: UP. Analysis and interpretation of data: UP. Drafting of the manuscript: UP, KA, and TZ. Critical revision of the manuscript for important intellectual content: RH and HI. Statistical analysis: UP and RH. Study supervision: KA and TZ. All authors contributed to the article and approved the submitted version.UP received speaker fee from Merck, Biogen and Bayer and personal compensation from Biogen and Roche for consulting service. RH has received travel compensation from Celgene and Sanofi. HI received speaker fee from Roche. KA received personal compensation from Roche, Sanofi, Alexion, Teva, Biogen and Celegene for consulting service. TZ reports consulting or serving at speaker bureaus for Biogen, Celgene, Roche, Novartis, Celgene, Merck and Sanofi as well as research support from Biogen, Novartis, Merck and Sanofi.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 Driver and Vehicle Licensing Agency (DVLA) in England, Scotland and Wales are legally responsible for deciding if a person is medically unfit to drive. This means they need to know if a person holding a driving licence has a condition or is undergoing treatment that may now, or in the future, affect their safety as a driver. The driver is legally responsible for telling the DVLA about any such condition or treatment. Doctors should therefore alert patients to conditions and treatments that might affect their ability to drive and remind them of their duty to tell the appropriate agency. Patients with acute schizophrenia or an acute psychotic disorder must not drive and must notify the DVLA. In alliance with the above, the GMC advises that clinicians have a responsibility to explain the above information to the patient and inform them that they have a legal duty to inform the DVLA. Doctors should also inform patients that relevant medical information may need disclosing about them to the DVLA if they continue to drive against advice, and any advice given should be documented. The main objective of this audit is to identify if notification of DVLA for forensic patients living in supported accommodation, is in accordance with the DVLA guidelines.A total of 12 residents living in community forensic supported accommodation who have a notifiable diagnosis were included. Data collection took place in September 2021, looking through all previous records relating to the search words \u201cDVLA\u201d, \u201cdrive\u201d, \u201cdriving\u201d and \u201clicense\u201d. Data audited were from the trust's electronic patient records.Diagnoses included paranoid schizophrenia, delusional disorder and personality disorder. Antipsychotic medications included Olanzapine , Clozapine and Zuclopenthixol +/- antidepressants. Legal status included community treatment orders (civil section), voluntary community patients and those on a conditionally discharged restriction under secretary of State supervision. Two patients held full driving licences and a further two held provisional licences, with DVLA documented discussions and notification compliance at 100%. The remaining eight patients had no documentation regarding driving nor DVLA discussions or notification.This audit found that DVLA discussions are not currently well documented, with only four patient records that have this recorded. Although it is the clinical team's responsibility to advise the patient to notify the DVLA, it is ultimately the patient's responsibility to notify the DVLA themselves. DVLA discussions need to be had regardless of driving status and documentation should reflect this."}
+{"text": "The global population of older adults (aged >60 years) is expected to triple to 2 billion by 2050. Proportionate rises in older adults affected by loneliness and social isolation  are expected. Rapid deployability and social changes have increased the availability of technological devices, creating new opportunities for older adults.This study aimed to identify, synthesize, and critically appraise the effectiveness of technology interventions improving social connectedness in older adults by assessing the quality of reviews, common observations, and derivable themes.Following the guidelines of PRISMA , 4 databases  were searched between February 2020 and March 2022. We identified reviews with adults aged \u226550 years in community and residential settings, reporting outcomes related to the impact of technologies on social disconnectedness with inclusion criteria based on the population, intervention, context, outcomes, and study schema\u2014review-type articles \u2014and with digital interventions included. Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) was used to measure the strength of outcome recommendations including the risk of bias. The reviews covered 326 primary studies with 79,538 participants. Findings were extracted, synthesized, and organized according to emerging themes.Overall, 972 publications met the initial search criteria, and 24 met our inclusion criteria. Revised Assessment of Multiple Systematic Reviews was used to assess the quality of the analysis. Eligible reviews  were excluded because of their low Revised Assessment of Multiple Systematic Reviews scores (<22). The included reviews were dedicated to information and communications technology , videoconferencing , computer or internet training , telecare , social networking sites , and robotics . Although technology was found to improve social connectedness, its effectiveness depended on study design and is improved by shorter durations, longer training times, and the facilitation of existing relationships. ICT and videoconferencing showed the best results, followed by computer training. Social networking sites achieved mixed results. Robotics and augmented reality showed promising results but lacked sufficient data for informed conclusions. The overall quality of the studies based on GRADE was medium low to very low.Technology interventions can improve social connectedness in older adults. The specific effectiveness rates favor ICT and videoconferencing, but with limited evidence, as indicated by low GRADE ratings. Future intervention and study design guidelines should carefully assess the methodological quality of studies and the overall certainty of specific outcome measures. The lack of randomized controlled trials in underlying primary studies (<28%) and suboptimal methodologies limited our findings. Robotics and augmented or virtual reality warrant further research. Low GRADE scores highlight the need for high-quality research in these areas.PROSPERO CRD42022363475; https://tinyurl.com/mdd6zds The use of technology to support older adults against feelings of loneliness and social isolation provides novel opportunities that have grown in the field of aging, as technology demonstrates that information and communications technology (ICT) use and training  and roboSocial isolation and loneliness in older adults have been extensively researched. Many studies showed that the prevalence of these problems increases with age. For example, the prevalence of loneliness among young adults, early to middle\u2013aged adults, and late to middle\u2013aged older adults are 39.7%, 43.3%, and 48.2%, respectively . LonelinSocially disconnected older adults are also vulnerable to a range of health disorders, including infection , high blRapidly deployable technologies, along with socioeconomic changes that have reduced the cost of technology, have increased the accessibility of technological devices, creating new opportunities for older adults . InterneSeveral reviews have summarized works on technology interventions for older adults experiencing loneliness ,24, but For a standardized systematic report on these reviews, we must assess the quality of the reviews and find common observations and derivable themes. An umbrella review method can provide a focus for areas where there are competing interventions and amalgamate evidence from multiple quantitative and qualitative reviews . To our To bridge this gap in the literature, we aimed to explore the findings and limits of current knowledge on the impact of technology interventions on social disconnectedness in older adults. We also emphasize areas requiring further research. In a comprehensive umbrella review, we synthesized the various categories and types of the used technology interventions, discussed their effectiveness and limitations, and finally explored their potential and need for further research. Finally, we amalgamated all the evidence from the umbrella review and used Grading of Recommendations, Assessment, Development, and Evaluations (GRADE) to make recommendations for interventions targeting social connectedness. This review attempts to answer the following questions:What technology interventions are used to influence social connectedness in older adults?How effective are these technology interventions in improving social connectedness in older adults, and what aspects make them effective?This umbrella review followed the standardized procedures ,26,27 ofThe search strategy involved controlled vocabulary searching; phrase searching; and applying Boolean logic, limits, and filters. A comprehensive systematic search of 4 databases  was conducted between February 2020 and March 2022. The reference lists were also examined for additional reviews. The following search terms were used: \u201cageing,\u201d \u201caging,\u201d \u201colder adults,\u201d \u201creviews,\u201d \u201c2000-22,\u201d and synonyms for \u201csocial isolation and loneliness,\u201d \u201csocial connectedness,\u201d and \u201ctechnology interventions.\u201d As an example, Search terms usedSU (technology or computer or Internet) and TI  and SU (older OR aging OR aging OR aged OR elderly OR senior) and Search strategy appliedLimiters\u2014Published Date: 20\u00a0000\u00a0101-20\u00a0211\u00a0231; Language: English; Publication Type: Academic Journal; English Language; Language: English; Year of Publication: 2000-22; Publication Year: 2000-22; Publication Type: Peer Reviewed Journal; English; Language: English; Exclude Dissertations Search modes\u2014Boolean or Phrase Sort by best MatchThe inclusion criteria were formulated using the population, intervention, comparison or context, outcomes, and study schema ,30. TablThe abstracts and titles of all potentially relevant articles were screened. Full texts were then evaluated, and duplicates were removed. Uncertainties were discussed among the research team members to reach a consensus. Relevant data of the included articles were summarized in tables and checked for accuracy by a second investigator (CH).The data analysis was based on a thematic synthesis with an inductive, iterative process consisting of 3 main stages: (1) free line-by-line review of the results, synthesis tables, and discussion sections of the included papers; (2) organization of themes into related areas; and (3) the identification, development, and refinement of detailed descriptions of factors that impacted the effectiveness of technology interventions . All meaThe methodological qualities of the reviews were assessed using the Revised Assessment of Multiple Systematic Reviews (R-AMSTAR)  quality The overall certainty of the evidence was evaluated using the GRADE method, which analyzes the risk of bias  and assesses the quality of the included evidence, which we used to make recommendations . InitialThe article elimination process is summarized as a flowchart in Among the 24 selected articles, 3 (12%) articles with R-AMSTAR scores <22 were excluded because they failed a priori systematic review processes . The 21 remaining reviews were of moderate quality, with none meeting all of the R-AMSTAR criteria.Data from the 21 reviews were extracted using a piloted, standardized data extraction form that captures and summarizes findings. As both technology interventions and extracted outcome data were heterogeneous, they were deemed inappropriate for a quantitative synthesis using meta-analytic techniques. Instead, a narrative synthesis summarizing the effectiveness of interventions was implemented. Under the methodological considerations of umbrella reviews, the results were reported descriptively in tabular form (The 21 selected reviews included 16 (76%) systematic reviews , 2 (10%) integrative reviews , 2 (10%) scoping reviews , and 2 (10%) meta-analyses . Most of the reviews covered the beneficial impact of technologies on loneliness, whereas others focused on social isolation, connectedness, and quality of life. General ICT was the most commonly applied intervention technology. The publication period was from 2005 to 2022, but 19 of the selected reviews were published within the last 7 years. Of the 21 reviews, 1 (5%) review focused on assistive technology for communication. Overall, 19% (4/21) of reviews focused on general interventions for social connectedness but examined technologies such as general ICT and videoconferencing, and 10% (2/21) of reviews focused on communication technologies for social connectedness in older adults. In all, 38% (8/21) of reviews investigated the impact of general internet and computer technologies on social isolation and loneliness. Of 21 reviews, 1 (5%) review examined the impact of smart technologies on social connectedness, and another  study reported the impact of health promotion technologies on social isolation and loneliness. In all, 10% (2/21) of reviews explored the ability of general ICT to improve the quality of life. Of 21 reviews, 1 (5%) review examined interventions to reduce social isolation and loneliness during the COVID-19 pandemic; 2 (10%) reviews focused on the impact of SNS on loneliness, another  examined interventions for preventing loneliness in nursing homes, and another  evaluated the benefits of telehealth in alleviating loneliness and increasing medication compliance. Here, telehealth was implemented through video health care professional visits to older adults. The 21 reviews covered a total of 326 underlying primary studies on technology interventions. It is worth pointing out that we were not able to confirm the presence of gray literature or studies that looked at technology interventions in the reviews.The interventions discussed in the reviews were general ICT , videoconferencing , computer and internet training , telecare , SNS , and robotics . The reviews reported mixed results. Positive effects of ICT on loneliness were the most commonly reported, followed by the positive impacts of ICT on social isolation or connectedness. Reviewing data from the underlying primary studies in the reviews, the most effective intervention mode for social connectedness was identified as general ICT, followed by videoconferencing and robotics .Z=2.085; P=.04). However, Bornemann [Z=.44; P=.37)\u2014that is, the same 5 studies yielded different pooled meta-analysis results in the 2 reviews. This divergence indicates potential biases in the analytic approaches; for instance, Bornemann [Among the 21 selected reviews, only Choi et al  and Bornornemann  concludeornemann   dealt with general ICT , 4 (19%) with videoconferencing, 3 (14%) with computer and internet training, 2 (10%) with telecare, 2 (10%) with robotics, 2 (10%) with SNS, 3 (14%) with gaming, and 1 (5%) with 3D augmented reality (AR). Among the primary studies, general ICTs were the most commonly adopted interventions (with 119 studies), followed by computer training, SNS, telecare, and robotics . AlthougAll the reviews reported large numbers and diverse outcome measures of primary studies. Besides constructs of social disconnectedness , many studies assessed factors such as quality of life, self-esteem, stress, and depression. Although not directly related to social disconnectedness, these factors may affect or be affected by social disconnectedness and may be useful to include outcome measures alongside social connectedness. A minority of the reviews also reported outcome measures of empowerment.When analyzing these quantitative primary studies, the reviews commonly applied validated tools, such as the University of California Los Angeles (UCLA) Loneliness Scale (or a modified version) and the De Jong Gierveld Scale . The UCLThe definitions and uses of outcome measures differed across the reviews. A total of 62 outcome indicators of social connectedness were used in the primary studies. Most reviews did not report on the lack of intervention effects ; moreover, the primary studies adopted a mixture of validated and nonvalidated outcome measures, making such reporting difficult. Consequently, they could not conclude whether the primary studies had validatable statistically significant outcomes.The social concepts used for determining outcomes varied in range and diversity. In many reviews, the source papers did not define social participation or social isolation but instead evaluated these factors as general or neighboring concepts ,35-37. LA few of the reviews highlighted that inconsistency and lack of specific definitions hindered the grouping and evaluation of their chosen papers ,37,39. MCattan et al  also notIbarra et al  correctlsocial facilitation for creating mechanisms through which older adults can interact with peers. From an alternative perspective, they measured the facilitation of social connections. The article by Williams et al [Gardiner et al  and Willms et al  was espeIn conclusion, different definitions and measurements of loneliness, social isolation, and social connectedness have led to diverse findings and wide variations across and within disciplines, defying a coherent picture of the research. Although some of the more recent studies and reviews have addressed this heterogeneity, reliable and succinct findings will remain elusive without further investigations.Many interventions implemented in the individual papers of the reviews were broadly divisible into group and one-to-one interventions. In general, group interventions were more frequently implemented than one-to-one interventions, although both types were effective ,37,40,41The imbalance between the group and one-to-one interventions impairs comparisons between the 2 types and conclusions regarding their comparative successes. Nevertheless, some of the reviews pointed out the possible advantages and limitations of these intervention approaches. Poscia et al  noted thOverall, group interventions appear to improve social disconnectedness, but the insufficient number of one-to-one interventions prevents an objective comparison and firm conclusions of the best interaction type. However, the GRADE assessment of the quality of evidence suggested a very low advantage of group interventions over one-to-one interventions .Technology interventions that enhance social connectedness include general ICT, video games, robotics, and the Personal Reminder Information Social Management system . Less conclusive evidence exists for the beneficial effects of SNS ,37,41,42Overall, technologies appear to positively affect loneliness, social isolation, and other psychosocial aspects of older adults\u2019 lives. Khosravi et al  examinedWhen technologies were intended to strengthen existing connections, their positive impacts on loneliness and social isolation were more consistent ,40,41. IP=.07 and P<.001, respectively). However, there were no statistically significant differences in loneliness among the members of the intervention groups before and after the intervention (P>.05).Choi and Lee  presenteIndividual reviews reported less conclusive outcomes of the overall technology use. The results of Morris et al  ranged fSpecifically, the following technology interventions appear to reduce social isolation but lack rigorous statistical support for a positive effect: robotics, telecare, and SNS ,36,42,45Overall, 86% (18/21) of reviews examined the impact of technology intervention on loneliness. The reviews covered 324 primary studies involving 66,565 participants. Of the 18 reviews, 15 (83%) reported a positive effect of technology on loneliness; the remaining 3 (16%) studies found a 0 or negative effect. From the reviews, it can be concluded that technology interventions exert an overall positive influence on social isolation and loneliness , but their effectiveness depends on the design of the study. Longer training times, shorter study durations, and facilitation of existing relationships tended to increase the effectiveness of the intervention. The quality of evidence supporting the effectiveness of technology interventions on social connectedness (GRADE assessment) was moderate to low.This section explores the findings of general ICT interventions reported in the reviews. General ICT is an umbrella term for generic technology devices, services, applications, and internet platforms . ICT incMany of the reviewed studies found that ICT interventions not only significantly reduce loneliness but also exert a positive impact on other aspects of social isolation, providing social support and connectedness, communication with family and friends, and ICT-accessible information sources ,35,42,43Damant et al  alone reOnly 2 reviews provided a homogenous meta-analysis. Both reviews reported positive impacts of general ICTs on social disconnectedness. In total, these reviews included 119 primary studies: 86 reporting a positive impact on social isolation or loneliness and 33 reporting unclear results or no impact. The studies agreed that increasing the frequency of general ICT use enhances social connectedness, improving the ease with which older adults can interact and maintain contact with others, thus reinforcing social connections with friends and family. The evidence that frequent ICT use facilitates the creation of new relationships or contacts is much weaker, further supporting, in part, the conclusions of Damant et al .Together, these results suggest that general ICT can facilitate established connections and might supplement or replace older communication methods. Its role in establishing new connections is uncertain. Our results suggest that when considering ICT interventions (at least for older adults), it is important to distinguish between their ability to maintain relationships, potential ability to deepen relationships, and inability to help create new relationships. The GRADE strength of the ICT category, although only moderate, was the highest among the categories because a large number of primary studies, including RCTs, were reviewed in this category, and there was consensus and clarity on the outcome measures.Although SNS is a subcategory of ICT, it warrants its own heading because 33% (7/21) of reviews discussed separate finding on SNS. The reviews gave mixed results. Whereas some studies supported the use of SNS in reducing loneliness, a sizable number showed no impact or even an increase in loneliness after SNS use ,42,46. BThese findings may partly depend on the type of SNS, as different types of SNS support different features. For example, Facebook may promote socialization more effectively than YouTube, whereas YouTube may better facilitate explicit knowledge acquisition and information transfer than Facebook. Ibarra et al  discoverOn the downside, SNS use raises several concerns: privacy, lack of perceived usefulness, and possibly demographic factors ,47. NewmOverall, 61 primary studies examining SNS were found in the reviews: 31 reporting positive impacts of SNS on social isolation and loneliness and 30 reporting unclear or no impacts of SNS. Therefore, the effectiveness of SNS is inconclusive. The results suggest that older users can obtain support, acquire knowledge, and maintain their existing relationships through SNS. In terms of combating social disconnectedness and establishing new relationships, SNSs are less effective and can be detrimental at times. However, the effectiveness of SNS in developing new relationships, fostering and maintaining existing ones, and acquiring knowledge and support has not been explored in depth, and the idiosyncrasies of SNSs must be unraveled in further research. The strength of evidence (GRADE assessment) of the reviews in this category is low because of indirectness, missing information, and publication bias.Overall, videoconferencing appeared to exert a positive impact on loneliness and social connectedness. The visual aspect of this intervention seemed especially appealing to older adults ,40,44,49Gardiner et al  and IbarWhen used in health support, videoconferencing yields mixed results. The intervention often decreases the loneliness and social isolation of residents in care and nursing homes, but a few studies have found no difference from the baseline ,35,43. MOverall, 14 primary studies in this subcategory were found in the reviews. Of these studies, 11 reported a positive impact on social isolation or loneliness. Owing to reviews such as by Schuster and Hunter , with clAmong the studied reviews, only Ibarra et al  alone deIn total, 13 reviews evaluated the impact of computer and internet training on various guises. All reviews found a positive impact of these interventions on social connectedness and loneliness ,41,43,45Mixed results were also obtained for this category. Baker et al  reviewedUnusually, among the reviews, Williams et al  found thAs some reviews did not differentiate between the impacts of training and subsequent use, any assumptions would be dubious. Morris et al  noted a Telecare was among the less frequent interventions in the review studies, but when included, it appeared to reduce social isolation and loneliness ,48,49. HAlthough none of the authors described the key features of successful telecare interventions, an emergent theme from successful primary studies was a high frequency of contacts. Interventions designed for regular and frequent contact were apparently more successful than interventions delivered on demand . Overall, 34 primary studies in the analyzed reviews covered this category. The impact of telecare on social connectedness was inconclusive, and uncertainty was further increased by the poor reporting of the results. Consequently, the GRADE strength of evidence in this area was very low.Robotics is a cutting-edge field and was mentioned in only 6 reviews. Some studies found that a pet robot provides the same level of benefit as animal-assisted therapy, which is known to reduce loneliness and social isolation ,36,42. IKhosravi et al  and AntuKhosravi and Ghapanchi  concludeAlthough these reviews indicate that social connectedness can be increased through robotics, this category is still new, and further studies on AI conversational agents and other robotic interventions are required. Therefore, the GRADE strength of evidence in this category is moderate to low.According to Khosravi et al , Video gSimilar to robotics, 3D environments have been newly introduced as a loneliness-reduction intervention technique and are rarely reported. Khosravi et al  reportedThere were few reviews that examined the usability of technology and its impact on the effectiveness of interventions. Some reviews identified a link between usability and acceptance of technology; more accessible devices were distinctly more likely to be embraced by users than less accessible devices ,40,48,58However, systematic reviews typically neglect the human-computer interaction components of intervention technology. Moreover, standardized measures of usability  for intervention studies have not been defined ,40. The Overall, the reported studies showed that whether technology can reduce loneliness depends on its usability. An intervention perceived as difficult to use by older adults cannot be effective. This aspect must be further investigated to improve the success of technology interventions.Owing to a lack of evidence, the GRADE confidence in the effect of usability on the success of intervention technologies is very low.On the basis of the results, We have also summarized the key recommendations for study design targeting social isolation, connectedness, and loneliness in This umbrella review, as highlighted in the analyzed reviews, found that different studies adopted a vast diversity of outcome measures and nonstandard definitions of loneliness and isolation ,35,39,42An umbrella review following the JBI methodology ,26 was wThe designs and qualities of the reviewed primary studies varied widely. Several reviews included RCTs and pilot, qualitative, and quantitative studies. In addition, the studies reviewed by Khosravi et al  were conThe findings of many underlying primary studies in the reviews were compromised by poor study designs, leading to conflicting information. For example, when reviewing the effects of computer and internet training on loneliness, Chen and Schulz  reached The reviewers generally agreed on the effectiveness of group-based interventions. Reviews examining the designs of the reviewed studies noted group-based interventions yielded positive effects on social disconnectedness ,37,40,41The reviews varied in scope, from assessments of the effectiveness of interventions, such as videoconferencing, to overviews of studies published in the field. The inclusion criteria and quality assessments of the primary studies also differed among the reviews, diminishing confidence in their findings. Our study confirmed a low quality of evidence in this field, whereas improved technology interventions for older adults are increasingly demanded by both policymakers and health professionals. Although the existing guidelines can encourage standardization of systematic reviews, these guidelines were largely ignored by researchers; accordingly, the strength of the reviews is diminished, which in turn led to the quality of evidence GRADE scores also being generally low.The scope of the reviews varied from a specific focus on the effectiveness of a targeted intervention (such as computer training) to an overview of the published studies in the field. The inclusion criteria for the primary studies and their quality assessment depended on tools used for rating rigor and bias. Such variations cast doubt on the conclusions of these reviews. This review confirms the lack of high-quality evidence in the field and highlights the failure to adhere to the existing guidelines. Standardization of systematic review reporting is expected to strengthen confidence in the review conclusions.Unlike their younger counterparts, older adults often lack the skills, functional capacity, and accessibility to adopt digital technology , which hTo improve the quality of results, interventions should be tailored to match the specific needs of older adults, and sufficient training should be provided for using the interventions. This tailoring requires the involvement or participation of participants in training in a variety of formats ,41. As uOur umbrella review is one of the few works that have looked at technology interventions for social connectedness for loneliness, following a well-established systematic approach such as the JBI umbrella review method. In examining other works, we came across reviews that focused on interventions generally -67 as opMost of the reviews demonstrated a need for stronger evidence on the effectiveness of technology interventions that reduce loneliness. Weak methodologies have limited the ability of reviews to establish conclusive remarks on their effectiveness ,42. ManyThe present review may also have been biased by accepting only English-language publications. However, many of the shortcomings and limitations of this umbrella review stem from the underlying problems of the primary papers included in the reviews. Among the common shortcomings were small-scale implementations with small sample sizes, low levels of evidence, and short periods of assessment.social connectedness and social disconnectedness to describe combinations of singular aspects such as social isolation and loneliness. Nevertheless, the methodology was the greatest limitation. Finally, the absence of gray literature in the reviews may have increased publication bias and led to the lack of inclusion of evidence for interventions that are not typically indexed in bibliographic databases. Future systematic reviews should consider including gray literature in the included studies.Another recurring limitation was the inconsistent definitions of social concepts. Social concepts such as loneliness, social isolation, and social connectedness were formally defined, but the authors did not use these definitions consistently; instead, they were often used interchangeably, inherently confounding measurements of these outcomes. The reviews were generally heterogeneous in focus  and discussed various interventions and syntheses of outcomes . Accordingly, the present review interchanges the terms The methodological limitations of the reviewed studies impaired the internal validity and usefulness of the reviews for technical and policy decision-making, as highlighted by the reviewers ,24. The The reviewed quantitative studies collected their data with questionnaires using scales developed for the study purpose. The reliability and validity of these nonstandardized scales are difficult to evaluate. Most reviews pointed out the suboptimal methodological quality of studies in this field, particularly the scarcity of RCTs (<28% of studies) and the dominance of quasi-experimental studies, which challenge the delivery of robust conclusions.Therefore, the results of this review should be interpreted with caution.Various technology interventions in different formats offer many ways to engage older adults. However, usability was rarely discussed in the reviews and was not assessed as an outcome measure. Although the existing guidelines encourage the standardization of systematic reviews, they have not been followed with the required rigor. Equally, the underlying primary studies of the reviews failed to address causation in a rigorous study design, and their heterogeneity limited their generalizability. It appears that there is a need for more studies on the multidimensional impact of technology on social connectedness, along with the assessment of other measures that may be interacting with technology use . Robotics is a relatively new technology that has emerged to be promising, but there are very few studies in this domain. Research on mobile technology interventions for social isolation is also encouraged as mobile phone technology provides opportunities for increasing the uptake of technology interventions targeting loneliness in older adults. Our results on the grading of evidence revealed that the strength of evidence was generally low to very low, indicating that the efficacy of the interventions is unclear and that more rigorous research is needed.Our review provides insights into strategies to reduce loneliness and isolation for older adults using technology interventions, with implications for future research, policy, and practice. Attention to social connections needs to be incorporated into existing preventative efforts for chronic diseases in older adults. Chronic illnesses develop slowly over decades. Since social connectedness is known to impact multiple mechanistic pathways in both the development and progression of disease, it warrants attention in primary, secondary, and tertiary prevention efforts. Given the lower economic costs of technology interventions for individuals, families, employers, and the broader health care system, we urge health care and health policy professionals to prioritize the investigation of technology interventions for social connections in prevention efforts.This umbrella review consolidates the state-of-the-art knowledge on the types of technology interventions that influence social connectedness in older adults and their effectiveness. The data were collected from the last 2 decades. Technology purportedly enables long-distance interactions, allowing older adults to become socially connected, obtain support, expand their social networks, and strengthen their existing ties. Some important themes that would improve the effectiveness of technical interventions for older adults emerged from the literature, namely group interventions, short-duration training and study programs, the use of general ICT, and videoconferencing. These implementations are more effective for maintaining existing connections than for building new ones. Certain technologies, such as robotics , AI-based conversational agents, and MIMs, show promising potential but have been underexplored.We attempted to determine which technology interventions can effectively improve social connectedness. The following conclusions emerged from our study. Reports on the effectiveness of computer and internet training on loneliness and social isolation provided mixed and inconclusive results. General ICT and internet-mediated communications were shown to reduce loneliness and social isolation in most studies, although the results apparently depend on the frequency of use and the time frame of the study, with shorter studies being more successful than longer ones. ICT interventions help socially isolated older adults through a range of mechanisms, including gaining social support, providing connections to the outside world, introducing new friends, and boosting self-confidence. All of these mechanisms must be studied hand in hand to gain a complete understanding of these processes. Finally, in our GRADE evaluation, most of the evidence was rated as moderate low to very low, reflecting methodological issues, the small number of RCTs, diverse outcome measures and definitions, and mixed results. Such low scores highlight the need for high-quality research in this area."}
+{"text": "KIT. A 58-year-old male patient presented with gastric subepithelial lesions accompanied by cutaneous hyperpigmentation, which were subsequently diagnosed as multinodular GISTs. Endoscopic surgery was initially conducted to remove the larger lesions, and pathological examinations were then conducted for the diagnosis of GISTs. Family history revealed that some other family members had similar cutaneous pigmentations. Whole-exome sequencing was used to search for potential driver mutations, and Sanger sequencing was used for mutation validation. A novel primary driver mutation of KIT  was detected in these hereditary GISTs, which has been reported in some targeted chemotherapy-resistant GISTs. Cell models were subsequently established for the rapid screening of candidate drugs and exploring potential mechanisms. This mutation could lead to cell proliferation and imatinib resistance by ligand-independent activation of KIT; however, ripretinib administration was identified as an applicable targeted therapy for this mutation. The mutation activated the JAK/STAT3 and MAPK/ERK pathways, which could be inhibited by ripretinib administration. To the best of our knowledge, this is the first report of the KIT-A829P mutation in familial GISTs, complementing the pathogenesis of familial GISTs and providing valuable information for the precision treatment of this disease.Familial gastrointestinal stromal tumor (GIST) is a rare autosomal dominant genetic disorder with only a few affected families reported to date. Here, we report a case of familial GISTs harboring a novel germline mutation within exon 18 of  KIT mutations (present in approximately 75\u201380% of cases) and PDGFRA mutations (in approximately 10% of cases). Molecular sub-classification of GISTs also incorporates succinate dehydrogenase complex (SDH) deficiency, BRAF mutation, and genetic alterations in the RAS-mitogen-activated protein kinase (MAPK) pathway [Gastrointestinal stromal tumors (GISTs) originating from Cajal cells are common mesenchymal tumors in the digestive system and are mainly located in the stomach and small bowel . GISTs a pathway . The cli pathway . The gol pathway . GeneticKIT and PDGFRA are the most common mutated genes in familial GIST [KIT (c. 2485G > C) that is associated with hyperpigmentation and lentigines was reported, but this mutation has not been detected in familial GISTs to date [Familial GIST is extremely rare and is mainly driven by germline mutations of related genes. Similar to GIST caused by somatic mutations, ial GIST . The cliial GIST ,7. Multiial GIST ,8,9,10.  to date ,12,13.KIT was confirmed. This case highlights that the determination of GIST molecular subtypes is crucial for the accurate evaluation and treatment of patients.Here, we report a Chinese pedigree with progressive multiple GISTs and cutaneous hyperpigmentation, in which the same missense mutation of In May 2016, a 58-year-old male patient was referred to Shengjing Hospital of China Medical University due to multiple gastric subepithelial lesions. An endoscopic ultrasound (EUS) showed that all lesions, some of which were exophytic, originated from the muscularis propria. The three largest lesions were located on the greater curvature side of the antrum\u2013gastric body junction (one lesion) and the lesser curvature side of the gastric antrum (two lesions). The section sizes were 23 mm \u00d7 20 mm a, 30 mm According to the patient\u2019s statement, two other family members also suffered from cutaneous hyperpigmentation. Based on these clinical presentations, we conjectured that this may be a rare familial genetic disorder. Therefore, the family was followed up in an intensive study. The proband\u2019s brother (P4) was also found to have multiple gastric subepithelial lesions, at the age of 54 years in 2016. The proband\u2019s niece d,f; P2 wDuring the follow-up period, the proband was regularly evaluated by CT. Comparing the CT images from 2019, 2021, and 2022 a, the prKIT  at codon 2485, which has not been reported in familial GISTs to date. This novel mutation was also detected, using targeted Sanger sequencing, in the formalin-fixed paraffin-embedded samples and EUS-fine needle aspiration tissues of the proband and normal tissues  from all members of the family with cutaneous hyperpigmentation  , including the wild type and three mutational types  were constructed. The plasmids were then transfected into the HEK 293T cell line for functional validation. Immunocytochemistry showed that the KIT protein was successfully expressed in the HEK 293T cells a. Sanger of KIT) a. Cell vferation b and ledferation b. Drug-sferation b,c. Riprferation b,c. AccoKIT oncogene have been reported in several tumors, including multiple GISTs [KIT are in-frame deletions involving codons 557 and 558 of exon 11. Other primary somatic KIT genetic aberrations refer to exons 9, 13, and 17. The activation-loop domain is encoded by exon 17, where mutations can also lead to ligand-independent activation of this protein. In addition, several other germline mutations associated with GIST have been reported in recent years, although familial GIST remains rare among gastrointestinal tumors [KIT or NF-1 gene mutations [KIT exon 18. Apart from mutations in mastocytosis and GISTs, mutations of KIT in melanomas have been found throughout the coding regions, with increased frequency in the juxta-membrane domain and tyrosine kinase domains. Germline KIT mutations are reported to be associated with other pigmented disorders, often accompanied by GISTs or mastocytosis [KIT is a transmembrane complex containing five domains that are encoded by a total of 21 exons ,17. The le GISTs ,18. The l tumors . Multiplutations ,20. The ocytosis . A recenc-KIT mutant GISTs typically involve activation of the PI3K-AKT-mTOR, RAS-RAF-ERK, and JAK/STAT signaling pathways, which influence the proliferation, survival, and anti-apoptosis activity of tumor cells [According to the characteristics of this family, stromal tumors caused by KIT-A829P have the features of early onset and rapid progression. Moreover, this mutation could lead to whole stomach involvement in the later stage of the disease. Therefore, the rapid identification of sensitive therapeutic targets is the key to the subsequent accurate management of this family. Owing to its rarity, there is limited research on the TKI sensitivities of A829P variant-associated GISTs. The molecular mechanisms of or cells ,22. AccoKIT/PDGFRA oncogenic signaling as a therapeutic vulnerability. Importantly, the KIT and PDGFRA genotypes predict imatinib activity, thereby providing very valuable clinical information [KIT exon 11 predict deeper and prolonged responses. GISTs harboring mutations in KIT exon 13 and 17 have been predicted to show less sensitivity or resistance to imatinib. The TKIs sunitinib and regorafenib are the standard second and third lines of treatment, respectively [Drug development in GIST has been oriented toward exploiting the high reliance on ormation ,23. The ectively ,25. In tectively . TherefoKIT and PDGFRA mutations and, therefore, has emerged as an innovative therapeutic approach against the heterogeneity of mechanisms of resistance [PDGFRA exon 18 mutations. Avapritinib also potently inhibits KIT N822K mutant autophosphorylation  = 40 nM), downstream signaling, and cellular proliferation (IC50 = 75 nM) [Ripretinib is an orally available type II switch-control TKI designed to inhibit the full spectrum of sistance ,28. Clinsistance ,30,31. I= 75 nM) . HoweverThere are some limitations of this study. The mechanistic experiments are not sufficiently strong to prove that KIT-A829P drives tumorigenesis and influences the prognosis of this hereditary disease. Further exploration, such as constructing a transgenic mouse model, is ongoing to gain an in-depth understanding of the genotype\u2013phenotype interaction.Our study characterized a rare mutation of KIT (A829P) in familial GISTs. This novel mutation led to cell progression and TKI resistance due to the constitutive activation of KIT. Drug-sensitivity experiments identified ripretinib administration as potentially the most applicable targeted therapy for this family, demonstrating GISTs to be notable candidates for precision medicine.Conventional endoscopy and endoscopic ultrasound were used in the imaging diagnosis of stromal tumors. Further pathological examinations were conducted according to the Chinese Society of Clinical Oncology (CSCO) criteria and modified NIH criteria. For the proband, endoscopic full-thickness resection (EFTR) was conducted for his partial gastric GISTs in 2016, and radical gastrectomy was conducted in 2022. Another patient of this family (the proband\u2019s niece) was treated by endoscopic submucosal dissection (ESD) in 2022. Other patients are still under follow-up.Whole-exome sequencing (WES) was conducted for finding potential genetic drivers. Genomic DNA was extracted from blood and sequenced according to standard protocols for next-generation sequencing . Sanger sequencing was used to verify the suspected somatic variants identified by WES. Genomic DNA was extracted from FFPE samples of GISTs resected by EFTR and normal oral epithelial cells. PCR amplification was conducted, and the PCR products were sent for automatic DNA sequencing (Takara).2 at 37 \u00b0C. The cell line was confirmed to be Mycoplasma-free (cat# CUL001B). KIT mutants were generated by QuikChange II Site-Directed Mutagenesis Kit purchased from Agilent Inc. . Wild-type and mutant KIT were cloned into the pcDNA3.1(+) vector using NheI and EcoRI restriction sites, respectively. The plasmids were transfected using Lipo3000 assay . For cell-viability assays, 3000 cells were plated in 96-well plates and cultured overnight. Serum starvation was conducted for another 24 h. Compounds  or stem cell factor  were then added in serial dilutions. Cellular ATP levels were determined after 48 h by the Cell Titer-Glo\u00ae Luminescent Cell Viability Assay . The absorbance of the plates was measured on a THERMO max microplate reader.The HEK 293T cell line was cultured in Dulbecco\u2019s Modified Eagle Medium containing 10% fetal bovine serum  and 2 mM glutamine under a humidified atmosphere in 5% COImmunoblotting was carried out using standard techniques. In brief, the cells were lysed in ice-cold 1X RIPA lysis buffer, and protein concentrations were determined. Aliquots (50 \u03bcg) of protein were denatured in Laemmli loading buffer and separated on precast 4\u201310% NuPAGE Novex 4\u201312% Bis-Tris Protein Gels . Proteins were transferred to polyvinylidene difluoride membranes, which were blocked and probed with primary antibodies and then detected using appropriate horseradish peroxidase-labeled secondary antibodies. Primary antibodies used in the study were as follows: AKT , Phospho-Akt (Thr308) , Phospho-Akt (Ser473) , p70 S6K , Phospho-p70 S6K (Thr421/Ser424) , Stat3 , Phospho-Stat3 , c-Kit , Phospho-c-Kit (Tyr703) , Phospho-c-Kit (Tyr719) , Erk1/2 , and Phospho-Erk (Thr202/Tyr204) . Proteins were visualized using enhanced chemiluminescence  on Hyperfilm .p-value < 0.05 was considered statistically significant in all cases and is indicated by one asterisk. A p-value < 0.01 and <0.001 is represented by two and three asterisks, respectively. Error bars shown in the figures represent standard deviations.Statistical analyses were carried out using Microsoft Excel software and GraphPad Prism . Student\u2019s t-test and one-way analysis of variance were used to analyze the differences between two groups and among multiple groups, respectively. Experiments were repeated in triplicate. A"}
+{"text": "However, in veterinary medicine, the significance of these mutations in canine gastrointestinal stromal tumors has not been sufficiently explored yet. The aim of this study is to investigate the mutational status of c-KIT and PDGFRA, by PCR and sequencing, in 17 canine gastrointestinal stromal tumors. Mutations of c-KIT were detected in 47% of cases; in one case, PDGFRA mutation was also identified. Although follow-up data were not available for all specimens, based on the information collected, we observed at the time of diagnosis the presence of metastases in cases with c-KIT mutation. In conclusion, this study provides evidence for the presence of c-KIT and PDGFRA mutations in canine gastrointestinal stromal tumors and suggests a potential association of c-KIT mutation with the more aggressive biological behavior of the tumor.Gastrointestinal stromal tumors represent the most common mesenchymal tumor of the canine gastrointestinal tract. Activating mutations in c-KIT and PDGFRA drive GIST oncogenesis and are used to predict the response to RTK-inhibitors in human oncology. Currently, the frequency and significance of these mutations in canine GIST have not been adequately explored. Therefore, we investigated the mutational status of c-KIT  and PDGFRA (exons 12 and 18) genes by PCR followed by fragment analysis for c-KIT deletions and PCR followed by screening with DHPLC and direct sequencing confirmation for single nucleotide variations in 17 formalin-fixed paraffin-embedded canine GISTs confirmed by KIT immunopositivity. c-KIT mutations were detected in 47% of cases, with a mutation detection rate significantly higher  and always involving exon 11. A PDGFRA gene mutation (exon 18) was identified in one case. Even if follow-up data were not available for all cases, four cases with documented abdominal metastases displayed c-KIT mutations. These data confirm that c-KIT exon 11 mutations occur frequently in canine GISTs, and identify the presence of a PDGFRA mutation similar to human GISTs. This study also suggests a potential association of c-KIT mutation with more aggressive biological behavior.Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the canine gastrointestinal tract and are diagnosed by the immunohistochemical expression of the receptor tyrosine kinase (RTK) KIT. Activating mutations of the proto-oncogenes  In fact, retrospective studies regarding dogs and humans have shown that most of the gastrointestinal mesenchymal neoplasms, previously classified as leiomyomas or leiomyosarcomas, express CD117 and are then to be diagnosed as GISTs [Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms affecting the gastrointestinal (GI) tract wall in humans ,2 as welas GISTs ,14,15,16c-KIT in exon 11, 9, 13 and 17 of the c-KIT gene have been observed in human GISTs. The mutation with the highest frequency (of more than 65%) is located in exon 11, in the juxtamembrane domain [Mutations in the proto-oncogene e domain ,19. The e domain ,12,15.PDGFRA encodes a transmembrane type-II tyrosine kinase receptor that is normally activated by platelet-derived growth factors (PDGFs), mitogens for cells of mesenchymal origin [PDGFRA mutations were identified in 35% of human GISTs with wild-type c-KIT [PDGFRA.l origin . PDGFRA pe c-KIT  and mostpe c-KIT  found thc-KIT and PDGFR [c-KIT [PDGFR mutations [c-KIT mutations, suggesting the possibility of the effectiveness of the treatment [c-KIT and PDGFR mutations in canine GISTs so that appropriate therapeutic options can be identified.Two Tyrosine kinase inhibitors (TKIs), toceranib and masitinib, have recently entered into veterinary clinical practice ,22 and and PDGFR , is the R [c-KIT  and PDGFutations ,25. A fareatment ,15. Strireatment  showed creatment . Therefoc-KIT exon 8, 9 and 11 and PDGFRA exon 12 and 18 mutations, and the potential relationship between mutational status and histotype and biological behavior, were investigated.In this study, the type and frequency of The study was conducted at Department of Veterinary Medical Sciences (DIMEVET) University of Bologna  and was carried out on formalin-fixed paraffin-embedded histological samples of canine GISTs received at the Pathological Service for histological diagnosis over a period of 16 years (2000\u20132016). All specimens previously diagnosed as GISTs by histomorphology and CD117 immunopositivity were included in this study. From records, anamnestic information about the caseload, including breed, gender and age of the patient, tumors localization, and the presence of metastasis, when detectable, were extracted.The classification was based on histopathological observations according to the criteria established by the WHO specific for the tumors of gastrointestinal tract , and on All the GISTs were subjected to immunohistochemical (IHC) evaluations with CD117, PDGFR\u03b1, SMA and Desmin.c-KIT , desmin , actin smooth muscle , and PDGFR\u03b1 . After incubation with 0.3% hydrogen peroxide in methanol for 20 min (to block the activity of endogenous peroxidases), and treatment in a microwave oven (750 W) for the unmasking of the antigen in citrate buffer at pH 6.0 , the sections were then incubated overnight at 4 \u00b0C in a humid chamber with the primary antibody diluted, according to the appropriate dilutions, in PBS . The sections, subsequently washed in PBS, were first incubated with the secondary antibody (anti-rabbit IgG conjugated with biotin) for 30 min at room temperature, then incubated with the streptavidin\u2013peroxidase complex for 25 min at room temperature. After a 12-minute passage in the DAB chromogen solution , the sections were immediately rinsed in PBS, then in running water, stained with a counterstain , dehydrated and mounted with DPX .The immunohistochemical staining was performed following the streptavidin-biotin-peroxidase technique . The antibodies and their dilutions are as follows: CD117-protein The results were graded as follows: \u2212, negative reaction; +, foci of positivity (<50% of neoplastic cells show a positive reaction); ++, widespread cellular positivity (>50% and <75% of cells immunoreactive); +++, most (>75%) of neoplastic cells are positive. Appropriate positive controls were used in order to evaluate the specificity of the reactions and ascertain the appropriate cross-reactivity in the dog tissue. Normal skeletal muscle and normal dog intestine were used as positive controls for mesenchymal markers . Purkinje cells from cerebellar tissue and mast cell tumors were used as a positive control for CD117 ,30. As ac-KIT and PDGFRA mutations status in genomic DNA.Tissue sample from each case was analyzed to determine the \u00ae FFPE DNA kit  according to the manufacturer\u2019s instructions. Different primers for c-KIT and PDGFRA  showed high cellularity, with a storiform pattern. Upon histological observation, they appear highly cellular, consisting of densely packed sheets of cells, with a markedly spindle morphology, arranged in palisades or intertwined and/or intercalated bundles or structured in a \u2018storiform\u2019 arrangement, with shaped nuclei oval and eosinophilic cytoplasm. The cell limits appear indistinct a. In thrIn 7 cases out of 17 (41%), the neoplasm shows a fasciculate pattern with less marked cellularity than the previous ones, and on histological observation it appears to consist of cells with fusiform morphology, which are less densely packed and arranged in loosely intertwined palisades and of smaller extension b.In 5 out of 17 samples (29%), the neoplastic cells showed an epithelioid-like appearance, and appear arranged in trabecular-like structures or, more frequently, in a solid-compact carpet. The cells, with a polygonal to rounded shape, have an often-vacuolated cytoplasm and high pleomorphism c.In one case (#12), it is possible to observe a myxoid pattern, characterized by poorly structured cells, spindle-shaped and separated by an abundant myxoid matrix. The nuclei are very elongated with clearly evident nucleoli. Interspersed with these areas it is possible to observe more or less extensive areas with a fascicular or storiform pattern d.At the evaluation of the histological grade, 10/17 cases (59%) showed a Grade I; 6/17 cases (35%) have Grade II, and only one case (n\u00b0 11) is Grade III . EvaluatIn all 17 GISTs, the cells show a strong and widespread cytoplasmic immunopositivity of the granular type for the KIT protein (CD117) with a percentage of immunopositive cells greater than about 90%. In only one case (#4) immunoreactivity occurs mainly in the cell membrane a and in Ten samples were tested with SMA antibody; only one of these (#6) was found to be diffusely immunopositive c, while Eight samples were tested for Desmin antibody. Of these, seven were negative, while only one sample (#1) was focally positive at the cytoplasmic level, in about 30% of the cells.Of all samples tested for PDGFR\u03b1 antibody, three cases were completely negative, eight samples were weakly positive and six samples moderately positive. The immunopositivity pattern for this marker is localized at the cytoplasmic level with variable intensity, from weak to moderate, with a percentage of positive cells greater than 70% d.c-KIT mutations were detected in 8/17 cases (47%) and always involved exon 11 (deletion of 3\u201346 bp), while exons 8 and 9 were wild-type in all cases. PDGFRA gene mutation was identified only in one case in the exon 18  (utation) . The mutc-KIT exon-11.Although follow-up information was not available for all cases, abdominal metastases were documented in four cases, and all showed a mutation in p = 0.2605), histological grade and histotype (p = 1.0000), histological grade and presence of mutations (p = 0.5840). The presence of mutations, on the other hand, appears to be significantly (p < 0.05) correlated with the presence of metastases (p = 0.0004).No significant correlations were found between the presence of metastases and histological pattern  represent a distinctive diagnostic neoplastic entity that, although quite uncommon and not yet fully characterized in dogs, has acquired over the years a well-defined clinical and histopathological identity and generates considerable interest for several reasons. Indeed, canine GISTs bear a very close resemblance to their human counterparts, representing an excellent spontaneous comparative model. This similarity paves the way for significant therapeutic perspectives in dogs, since features and the mutational aspect are almost completely superimposable ,33,34,35In our caseload, collected over a long period 16 years), GISTs are scarcely represented. However, these data follow epidemiological data from the literature  years, G,13,14,16CD117 positivity in over 70% of neoplastic cells is the prerequisite for diagnosing these neoplasms as GISTs . In all c-KIT.Some attempts have been made to correlate the CD117 immunohistochemical pattern to the presence of mutations in canine GISTs, similar to what has already been extensively analyzed for mast cell tumors in dogs , but witc-KIT gene in the juxtamembrane domain. On the other hand, no mutation was recognized in the other exons investigated . These results confirm what has already been reported in canine cases [c-KIT, which is found in humans, but not in our series.Eight distinct mutations of the same type (deletions) were identified in this study, all involving exon 11 of the ne cases ,7,12 in p = 0.0004).Almost half of the cases analyzed have this KIT mutation 47%; 8/17), in a percentage that falls within the range of the few studies that have evaluated the frequency of this mutation in canine GISTs  in correspondence to a human equivalent mutational hotspot. Until now, no significant activating mutation affecting PDGFRA had ever been reported in the dog, unlike what had been found for some time in humans, in which activating mutations of this gene represent a share ranging from 10 to 35% [KIT Exon 11. This finding, both mutations in the same tumor, has never been observed in human GISTs.One case showed the presence of an activating point mutation at the level of exon 18 of the 0 to 35% ,19,34. IPDGFRA-mutated GISTs are frequently found in the stomach, presented an epithelioid histotype and are often associated with a PDGFR\u03b1 strong immunopositivity [In humans, sitivity . Of notec-KIT mutations, imatinib mesylate (Gleevec) has been used for its ability to inhibit protein kinases and results in a marked tumor response and an increase in survival time [c-KIT and PDGRFA mutations in canine GISTs must be extensively studied to understand their potential therapeutic efficacy.There are several \u2018inhibitory\u2019 molecules that target tyrosine kinase receptors (TKIs), including imatinib mesylate, which has long been successfully used in the treatment of human GISTs with KIT mutations. To date, due to its effective therapeutic response, this TKI has recently been considered the treatment of choice for patients with advanced stage GISTs. In a study conducted on two dogs with documented exon-11 val time ,15. The val time . Therefoc-KIT mutations were detected in half of the cases included in this study, and their presence appears to be associated with malignancy. An activating mutation of PDGFRA, which in humans is represented in a significant fraction of GISTs, had not yet been reported in canine GISTs. Unfortunately, follow-up data were not available and, therefore, no meaningful prognostic information can be provided from this study.c-KIT play an important role in canine GISTs, are related to a malignant biological behavior and, therefore, have strong therapeutic implications. Further studies are needed involving a larger number of cases with follow-up data in order to demonstrate the prognostic and predictive role of KIT or PDGFRA mutations in canine GISTs.In conclusion, the study confirms that activating mutations of Our results indicate, confirming what has already been stated in the literature, to what extent KIT mutations can have a prognostic and therapeutic significance in canine GISTs."}
+{"text": "KIT and PDGFRA receptor genes led to a deep revolution in the knowledge of this tumor. This paved the way to the introduction of imatinib and other tyrosine-kinase inhibitors (TKIs), which terrifically revolutionized the prognosis of GIST patients. Currently, it is well established that tumor mutational status is the main player in clinical outcome; however, with the research advances, it has been slowly understood that GIST landscape is more complex than expected and the TKIs available are not effective for all the GIST subtypes. For this reason, in the era of tailored/personalized medicine, each GIST patient should be considered individually and genetic consult should be the first step to take in consideration in the therapeutic decision making process.Gastrointestinal stromal tumors (GISTs) are rare entities, which, however, represent the most common mesenchymal tumor of the gastrointestinal tract. The discovery of gain of function mutations on  Before the groundbreaking identification of activating mutations in the KIT tyrosine kinase receptor (TKR) gene in 1998, GISTs were considered as a devastating disease due to scarce response to chemotherapy and radiotherapy. Luckily, the discovery of gain-of-function mutations on KIT and PDGFRA receptor genes led to a deep revolution in the knowledge of this tumor and from a poor characterized entity, GIST became a paradigm of target therapy. Indeed, at the beginning of 2000, the FDA approved the tyrosine kinase inhibitor (TKI), imatinib, for the management of metastatic and inoperable GISTs. Imatinib - the magic bullet, originally developed for chronic myeloid leukemia - is the first example of molecular-targeted drug with a known mechanism of efficacy; it represents the worldwide paradigm of targeted therapy specifically tuned vs. specific molecules - peculiar of the cancer cells.Gastrointestinal stromal tumors (GISTs) are rare entities, which, however, represent the most common mesenchymal tumor of the gastrointestinal systemKIT/PDGFRA mutant (about 85%-90%), or KIT/PDGFRA wild-type (WT) GISTs or, often called, WT GISTs. WT GIST are a small group harboring a plethora of alterations on different genes, including succinate dehydrogenase (SDH), NF1, BRAF, KRAS,6. However, with the fast advances in sequencing technologies, different studies have showed novel genetic mutations in the WT GISTs subgroup. It became clear that GISTs are a heterogeneous family of tumors, fragmented in different subtypes with specific and peculiar features,8, which influence prognosis as well as clinical outcome. In 2014, a report from NIH suggested to classify GISTs in SDH competent, with characteristics in common with the classic KIT/PDGFRA mutant GISTs, and SDH-deficient.After the identification of driver genetic events in 1998 and until a few years ago, GISTs were classically dichotomized in KIT or PDGFRA genes. Specifically, approximately 80% of GISTs carry pathogenic activating mutations on KIT, whereas 5%-10% harbor PDGFRA mutations,11. These mutually exclusive mutations are gain of function mutations, leading to a constitutively and ligand independent activation. This means that the receptors promote the activation of downstream pathways involved in many key biological processes of carcinogenesis, including RAS/RAF/MEK and PI3K/AKT/mTOR, and MAPK cascades,13. Genetic alterations in KIT or PDGFRA genes - that can be simple aminoacid substitutions, in frame deletions or insertions - involve two main regions of the receptors, the regulatory domains and the enzymatic domains. Considering KIT receptor, the vast part of mutations (~65%) involve the exon 11, followed by 10% of cases who present a mutation on exon 9. In rare cases (~2%), primary KIT mutations can also hit exon 13 and exon 17. With regard to PDGFRA, the most common mutation (~5%) affects the exon 18 at codon 842, and promotes a substitution of an aspartic acid (D) with a valine (V) (D842V), while mutations on exon 12 and 14 are less frequent. The main difference between KIT and PDGFRA mutation is the location within the receptor. Indeed, the majority of KIT mutations in GISTs arise in the juxtamembrane domain (exon 11), but only ~10% of PDGFRA mutations are in this region (exon 12). On the contrary, alterations within the activation loop of KIT (exon 17) are rare events (< 1%), but are prevailing in PDGFRA-mutant GISTs (exon 18).As previously mentioned, the majority of GISTs harbor a mutation in BRAF/RAS mutant and NF1 mutant GISTs. GISTs with mutations in BRAF/RAS or NF1 might be referred to as RAS-pathway mutant GISTs. It is estimated that among patients with no mutations on KIT or PDGFRA (KIT/PDGFRA WT), 5%-13% may have a genetic alteration on BRAF; in particular, > 90% of BRAF mutations occur in exon 15 on codon 600, usually V600E-18. The V600E mutation promotes a BRAF activity due to creation of a salt bridge with the residue K507. V600E-K507 interaction mimics the conformational changes occurring during dimerization, so that BRAF V600E does not depend on dimerization for increased kinase activity. BRAF is an intracellular protein kinase, playing a critical role in the RAS-RAF-MEK-ERK signaling pathway. Mutations on KRAS are events even more rare than BRAF ones; KRAS mutations in GISTs have a low frequency, spanning from ~1% to 11% of KIT/PDGFRA WT GIST. Besides the mutations on BRAF/RAS, it has been reported that the autosomal-dominant inherited disease, neurofibromatosis Type 1 (NF1), promotes an increased incidence of GIST. In general, about 7% of cases with NF1 mutations will develop a GIST during their lifetime. This type of neurofibromatosis is characterized by genetic alterations on NF1 gene, which had more than 60 exons, and encodes neurofibromin, a tumor suppressor that downregulates the RAS/RAF/MEK/ERK pathway.Among the SDH competent GISTs are included KIT or PDGFRA but present alterations on one of the SDH genes. SDH is a mitochondrial enzyme composed by four different subunits, encoded by four different genes SDHA, SDHB, SDHC, and SDHD. The SDH loss of expression is often due to germ-line and/or somatic loss-of-function mutations in any of the SDH subunits. In addition to the canonical DNA mutations, recently, different papers have showed that SDH inactivation may be due to epigenetic mechanisms as hypermethylation of SDHC promoter. The SDH-complex is involved in the Krebs cycle and is responsible for the conversion of succinate to fumarate. Consequently, SDH deficiency leads to accumulation of succinate, which in turn promotes HIF1a overexpression and expression of hypoxia-associated tumorigenic responses and angiogenesis. Considering the clinical features, SDH-deficient GISTs show a number of unique characteristics, such as young age at onset, female gender predilection, gastric localization, frequent lymph node metastatic involvement, and an indolent behavior,23.The SDH deficient GISTs\u2019 group includes GIST patients who lost the SDH complex functionality. Indeed, 10%-15% of adult GISTs do not harbor genetic alterations on -26. Currently, the only first-line approved treatment for metastatic and inoperable GIST is imatinib. Imatinib, introduced in GIST management at the beginning of 2000, deeply changed the prognosis of these patients, who were considered irresponsive to the majority of available chemotherapic treatments. Imatinib is a selective TKI, which targets diverse tyrosine kinase receptors, including ABL, BCR-ABL, KIT, PDGFRA, PDGFRB and CSF1R. The majority of GISTs respond well to the imatinib standard dose of 400 mg/day, but, commonly, after 24-36 months, a large proportion of patients develop secondary mutations and the tumor progresses. To face the progressive acquisition of resistance, within the last 20 years, a second and a third line, sunitinib and regorafenib, respectively - have been introduced in GIST management. Sunitinib and regorafenib are TKIs,30 with a wider range of kinase inhibition - compared to imatinib, including KIT, PDGFR, VEGFR, FLT3, TIE2, RET, FGFR1, RAF,31. However, despite the efficacy of the currently available three-lines of therapy, patients usually progress even under sunitinib and regorafenib; unfortunately, there are no other therapeutic options and rechallenge of imatinib or sunitinib may represent a reasonable option in advanced GIST patients after failure of previous treatments. In the last years, the research progress and the advance in deep sequencing techniques promoted the identification of novel potential targets and different trials are ongoing.GIST management for immunohistologically confirmed GISTs plans: (1) surgical resection for resectable GISTs without metastasis, or (2) administration of TKIs for unresectable, metastatic, or recurrent GISTsIt is well established that tumor genotype play a critical role in GIST clinical outcome. Indeed, among GIST patients treated with TKIs, it has been observed a wide inter-individual variability and mutational analysis appear to be critical to make a clinical decision about adjuvant therapy.. It has been reported that about 10%-15% of GISTs treated with imatinib show primary resistance. Specifically, a meta-analysis of four studies involving 215 GIST patients evaluated resistance according to KIT and PDGFRA genetic alteration. The authors found that 50% of PDGFRA-mutant, ~35% of KIT/PDGFRA wild-type and ~10% of KIT mutant GISTs were irresponsive to imatinib. Considering the lack of an oncogenic mutation, it is not surprising the low imatinib response observed in KIT/PDGFRA WT GIST.Patients may show a primary resistance to the treatment , or, as often happens in GISTs, stop to respond at some point, after an initial response (secondary or acquired resistance during treatment)KIT/PDGFRA mutant GISTs, the mechanisms of primary resistance is quite unexpected. However, the most common PDGFRA mutation, D842V, is considered imatinib resistant; this is due to a conformational change located within the receptor activation loop which makes imatinib unable to bind it.With regard to the other KIT mutant GIST patients have been studied more extensively than other subtypes.Given the frequency, KIT exon 9, accounting for ~10% of GIST cases, are frequently characterized by duplications of six nucleotides  at position 502-503, located within the extracellular domain. It is thought that the consequence of this duplication is an alteration in the receptor conformation, which mimics the binding of the physiological ligand, the stem cell factor, thus promoting constitutive activation. In vitro studies have proven that mutations on KIT exon 9 reduce the sensitivity to imatinib. Furthermore, presence of exon 9 mutations has been reported as the strongest adverse prognostic factor for imatinib response, and increases the relative risk of progression and death by 171% and 190% respectively, with respect to KIT exon 11 GISTs. Results from different studies have shown that KIT exon 9 GISTs benefit from higher dose of imatinib, with significantly better progression-free survival (PFS),37,38. For this reason, this subset of patients is treated with 800 mg per day of imatinib, (instead of 400 mg), which is now considered the standard dose for this subgroup. In addition, for metastatic GISTs, it is strongly recommended that the treatment is continued indefinitely, as it has been observed that treatment outage is usually followed by quick tumor progression. ESMO-EURACAN Clinical Practice Guidelines for diagnosis, treatment and follow-up released in 2018, highlight, once again, the importance of biopsy with histological and mutational analyses to propose the 800 mg imatinib dose for less sensitive KIT exon 9 mutations.Mutations on KIT exon 11 GISTs are those patients who receive greater benefits from imatinib treatment compared with the other KIT mutational subtypes; indeed, these patients are twice as likely to respond to imatinib. In addition, KIT exon 11 show also higher response rate in terms of PFS and overall survival (OS). Primary mutations on KIT exon 13 characterize about 1% of GISTs; these genetic alterations promote changes within the receptors ATP binding pocket but the functional consequences have not been fully elucidate yet. The majority of data reported in literature show that mutations on KIT exon 13 confer sensitivity to imatinib, however a case report has described a rapid and aggressive tumor progression in a patient harboring a V654A alteration after imatinib and sunitinib treatment.PDGFRA, as mentioned before, carriers of D842V on exon 18 are usually primary resistant. PDGFRA D842V represents ~70% of all PDGFRA mutations in GISTs. Besides D842V, patients may harbor other alterations on exon 18, which can hit close hotspots, as the aminoacids 843 to 845, and commonly are represented by deletions; in this case, GISTs are sensitive to imatinib. Currently, the largest study so far conducted, investigating 289 PDGFRA mutant GISTs, showed a variable grade of imatinib sensitivity basing on the specific mutation. By virtue of these variable responses to imatinib observed in PDGFRA mutant GISTs, lately, different PDGFRA inhibitors have been entered into clinical studies. Among those showing better results, crenolanib was tested in a phase 2 trial and showed important preliminary clinical claims; these data have contributed to the initiation of a phase 3 randomized, placebo-controlled trial of crenolanib activity in PDGFRA D842V-mutant GISTs (NCT02847429).With regard to ,44.KIT mutations and rarely in those with primary PDGFRA mutations. Resistant mutations are most often found in the ATP-binding pocket of the kinase domain (exons 13 and 14) or in the kinase activation loop (exon 17 and 18). Despite the acquisition of secondary mutations, delayed resistance may be due to different mechanisms, including the KIT overexpression caused by genomic amplification, KIT loss of expression with activation of an alternative tyrosine kinase, and ABC transporters overexpression. Therapeutic management of GIST patients progressed on imatinib consider dose escalation from 400 to 800 mg/day and/or switch to the second and third line TKIs.As previously anticipated, progression after more than 6 months of initial clinical response is defined as secondary or acquired resistance. To date, secondary mutations have only been retrieved in patients with primary . Sunitinib is effective in imatinib resistant GISTs and it has been shown that its activity is affected by the specific primary and secondary mutation. In this context, exon 9 mutant patients usually have higher clinical benefits and objective response rates, compared with exon 11 mutants. In addition, both PFS and OS are generally significantly longer in KIT exon 9 mutant or KIT/PDGFRA WT GISTs with respect to KIT exon 11 mutant patients. Taking into consideration the acquired mutations, sunitinib seems to be more effective in case of alterations within KIT exon 13 and 14 (ATP binding pocket) rather than those within KIT exon 17 or 18.The second line sunitinib inhibits several TKRs, blocking multiple biological processes, as tumor growth, angiogenesis and metastasisKIT mutations and sensitivity to approved TKIs.KIT/PDGFRA WT GIST show poor response to imatinib and it has been reported that this GIST subset has a 76% higher risk of death if compared with KIT exon 11 mutants. In this regard, the reports in literature agree in finding a very low benefit for these subsets of GISTs under imatinib treatment. Furthermore, it seems that KIT/PDGFRA WT-SDH deficient GIST may have better response to sunitinib and regorafenib,48. Sometimes, metastatic tumors can be quite indolent so that a \u201cwait-and-see\u201d policy applies, while other patients could benefit from resection. However, considering the complex molecular landscape of this subset, genetic counseling and therapeutic management plan in a GIST treatment center should be strongly taken into account.It is not surprising that BRAF status, in vitro studies showed that alterations on BRAF gene confer imatinib resistance and, in general, to TKIs including sunitinib, regorafenib and sorafenib,51. It has been proposed that this subset of GIST may have benefits from treatment with BRAF inhibitors, as dabrafenib, even if data are quite limited.With regard to NF1 mutant GISTs are even more scarce and controversial. A first report showed a general good overall response in a long term follow-up, with only 5 out of 35 patients who died due to metastatic disease. On the contrary, two following case reports described NF1 mutant GISTs primary resistant or only initially responding to imatinib-54. Currently, a Phase II Trial of evaluating MEK1/2 inhibitor, selumetinib, is ongoing for NF1 mutant GIST patients (NCT03109301).The data on the prognosis of ,56. Therefore, it is conceivable that polymorphisms or gene expression regulation through methylation/miRNA mechanisms could represent key players in affecting the final clinical outcome. By virtue of this consideration, possible pharmacogenetic-62 and epigenetic-66 mechanisms of imatinib and sunitinib resistance have been broadly investigated in GISTs-71. Despite the extensive research, data are controversial and to date none of them supports the use of pharmacogenetic or pharmacoepigenetic tests to optimize treatment outcome in GIST patients.As reviewed, imatinib has represented a groundbreaking in GIST history. After its introduction, a huge research effort has been directed to the identification of driver mechanisms of acquired resistance, as well as, novel potential biomarkers for GIST treatment. In this context, the involvement of pharmacogenetic and epigenetic mechanisms in TKIs resistance has been investigated. In particular, with regard to pharmacogenetics, any drug goes through a specific pharmacokinetics itinerary that might be relevant for both drug efficacy and toxicityThis review aimed to point out the complex landscape of GISTs. This family of tumors is quite heterogeneous and any oncologist and clinical pharmacologist should consider every case individually, in order to deeply and accurately characterize the molecular events driving tumorigenis and treatment response. Currently, we know that GIST mutational status genotype has diverse impact on the therapeutic decision-making and this should be at the base of the clinical GIST management Wrote the manuscript: Ravegnini G, Angelini SRevised the manuscript: Hrelia PNot applicable.This work was supported by the Ministry of Education, University and Research of Italy (MIUR) (2015Y3C5KP_002) to Angelini S; the L\u2019Or\u00e9al-UNESCO for Women and Science Award to Ravegnini G.All authors declared that there are no conflicts of interest.Not applicable.Not applicable.\u00a9 The Author(s) 2019."}
+{"text": "A well-defined BH lesion filled with coagulated blood formed on day 0. A week after the treatment, fibroblast activation was induced at the treatment site, leading to the formation of extracellular matrix structure (ECM). The ECM was then disrupted for 7 to 28\u00a0days. Regenerated normal hepatocytes and newly formed blood vessels were found within the BH region with the absence of hepatic fibrosis. No significant morphological, histological and genetic changes around the BH lesion occurred. These results suggest that BH could be a safe and promising therapeutic tool for treating solid tumours without inducing any significant adverse effect such as the formation of liver fibrosis.Boiling histotripsy (BH) is a promising High-Intensity Focused Ultrasound technique that can be employed to mechanically fractionate solid tumours. Whilst studies have shown the feasibility of BH to destroy liver cancer, no study has reported on the healing process of BH-treated liver tissue. We therefore extensively investigated the evolution of the healing response of liver to BH in order to provide an insight into the healing mechanisms. In the present study, 14 Sprague Dawley rats underwent the BH treatment and were sacrificed on days 0, 3, 7, 14, and 28 for morphological, histological, serological and qPCR analyses. The area of the treated region was 1.44\u00a0cm HIFU thermal ablation has received FDA (U.S. Food and Drug Administration) approval for the treatments of uterine fibroids, pain palliation of bone metastases, osteoid osteoma, benign prostatic hyperplasia, prostate cancer and essential tremor5. The majority of heat deposition occurs in the HIFU focal volume with temperatures outside this focus being kept at noncytotoxic levels. The extent of necrotised tissue is therefore spatially confined to the HIFU focal region whilst sparing the intervening tissue6. The size and shape of the HIFU focal region is typically an ellipsoid or a cigar shape with 2\u20133\u00a0mm in lateral width and 8\u201310\u00a0mm in axial length along the wave propagation direction7.High intensity focused ultrasound (HIFU) is a non-invasive and non-ionising ultrasonic technique that has been used to thermally necrose solid tumours without damaging surrounding tissue. HIFU involves focusing an intense ultrasound beam into a small region of interest within the body. This results in localised tissue heating (>\u200955\u00a0\u00b0C) and protein denaturation at the HIFU focus, which can cause irreversible cell death through thermal coagulative necrosisP+) and negative (P\u2212) pressures at the HIFU focus can produce violent acoustic cavitation resulting in mechanical tissue fractionation10. Tissue debris remaining inside a BH lesion is likely to be absorbed as part of the physiologic healing response whereas HIFU thermal lesion becomes fibrous scar tissue8. Whilst BH has not been yet approved by FDA, numerous studies have shown that BH can be used to mechanically destroy different types of soft tissues  and cancer cells  without causing any significant thermal damage to the boundary of a BH lesion induced18. The overall shape of a BH lesion is tadpole like consisting of a head and a tail with the head closest to the HIFU source19. Localised shockwave heating-induced boiling bubbles and shock scattering-produced subsequent cavitation clouds are, respectively, responsible for the creations of the tail and the head of a BH lesion21. Detailed mechanisms of BH can be found elsewhere22. In case of HIFU thermal ablation, blood perfusion can lead to undertreatment of a targeted tissue due to heat sink effect, and thus normal tissue surrounding the HIFU focus can possibly be ablated via thermal diffusion24. On contrary, since BH treatment is not greatly affected by heat perfusion via blood flow and thermal diffusion25, BH can produce a sharply demarcated margin between treated (fractionated tissue) and untreated (intact) regions with a transition distance of less than a cell length of the order of a micrometre. Furthermore, the process of BH treatment can be monitored using conventional B-mode ultrasound technique, because bubbles are highly reflective to ultrasound. During a BH exposure, for instance, boiling bubbles and cavitation clouds can appear as a hyperechoic region whereas a BH lesion is visible as a hypoechoic region on B-mode images in vivo12. In addition, recent studies have reported that BH can also induce a stronger immune response than that obtained with HIFU thermal ablation as more non-denatured antigenic proteins which enhance immune reactions can be released after BH exposure27. Due to the aforementioned advantages of BH over HIFU thermal ablation, BH has now gained significant interests in the field of HIFU for treating solid tumours.In addition to HIFU thermal ablation, recent studies have clearly demonstrated the feasibility of employing HIFU to mechanically fractionate or break down solid tumours at the HIFU focus via acoustic cavitation. This promising HIFU technique known as boiling histotripsy (BH) which uses a number of millisecond long HIFU pulses containing shocks with high peak positive (28. According to the liver cancer treatment guidelines provided by the Barcelona Clinic Liver Cancer (BCLC) staging system29 and the Asia Pacific Association for the Study of the Liver (APASL)30, patients with early-stage liver cancer  can be treated with surgical liver resection or percutaneous ablation through radiofrequency or microwave, or trans-arterial radioembolization. These methods are invasive or minimally invasive. Whilst hepatic resection (hepatectomy) has been the mainstay of the treatment for early-stage liver cancer, the surgical procedures involved can however lead to certain complications such as bleeding, infection, blood clots and pneumonia. Noninvasive mechanical destruction of liver cancer using BH could potentially be a promising alternative or additional treatment option to minimise or even prevent these surgical risks. For BH to be used clinically, however, the efficacy and safety of BH including potential adverse tissue effects after BH treatment should be carefully evaluated. In fact, a large number of BH studies have clearly demonstrated the efficacy of BH for mechanically destroying solid tumours at the HIFU focus18, whereas no study has reported on the healing and repair process of the BH-treated tissue. This study therefore aims to provide an insight into the healing mechanisms and pathways triggered by BH through the observation of a long-term wound healing response of the liver to BH exposure.Liver cancer is the sixth most common cancer and the fourth leading cause of cancer-related death worldwide with 841,080 new liver cancer cases diagnosed in 2018P+ of 85\u00a0MPa, peak negative pressure P\u2212 of \u221214\u00a0MPa at the focus, a pulse repetition frequency of 1\u00a0Hz and a duty cycle of 1% was used to mechanically fractionate liver tissue. A total number of 25 BH lesions were created in the liver over the area of 1.4 cm2  rats were used to investigate a long-term wound healing process of the liver after BH treatment. Five SD rats were in sham group whilst 14 rats underwent BH experiments. A 2\u00a0MHz HIFU transducer coupled with a customised cone-shaped holder filled with degassed and deionised water was then placed on the animal\u2019s exteriorised liver  on day 7. This area shrinkage (from 1.4 cm2 on day 3 to 0.4 cm2 on day 7) is most likely to be due to wound contraction as a part of the healing process32. The length and width of the scar tissue were measured using a digital caliper, and the area was calculated by multiplying the length by the width. In the early stage of the normal wound healing process, fibroblasts secrete collagen which is the main component of ECM. During the experiments, significant amounts of \u03b1-smooth muscle-actin (\u03b1-SMA) expressing active fibroblasts were observed on days 3 and 7, as shown in Fig.\u00a0Morphological changes of the liver tissue treated with BH over time  are shown in Fig.\u00a0In our experiments, after the animal\u2019s sacrifice, the BH-treated liver tissue was cross-sectioned and stained with hematoxylin and eosin (H&E) or Masson\u2019s trichrome for histological observation. Partially fractionated and homogenised liver tissue filled with erythrocytes appeared immediately after the BH exposure on day 0 Fig.\u00a0C with in33. In the present study, qPCR assay for measurement of collagen expression  and gelatin zymography for detection of MMP-2 activation were performed on the BH-treated, BH-penumbra and BH-untreated regions  is a key representative inhibitor of collagen. Excessive collagen formation after injury is regulated by MMP-2 activation which breaks down the collagensons Fig.\u00a0. On day 36. In the wound healing process, the activation of caspase enzymes is the important biochemical change of apoptosis which results in cessation of collagen accumulation. In the present study, the induction of apoptosis in the BH-treated liver tissue was observed through triple immunohistochemistry (IHC) staining and the mRNA level of caspase enzymes. Three days after the BH treatment, only a few fluorescent signals of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic fibroblasts (\u03b1-SMA) and hepatocytes (Asgr1) were observed in the whole BH-treated tissue region  at 1\u00a0h and 1, 2, 3, 7, 14, and 28\u00a0days after the BH treatment. For comparison purpose, blood samples from the sham group  were also obtained. The mean serum ALT and AST levels in the sham did not significantly change (ALT of 57\u00a0IU/L and AST of 95\u00a0IU/L), whereas both serum ALT and AST levels of the BH-treated group drastically increased to 149\u00a0IU/L and 408\u00a0IU/L respectively at 1\u00a0h, as shown in Fig.\u00a0Blood samples were collected from the BH-treated rats , damaged hepatocytes can be replaced by proliferating hepatocytes through liver regeneration process including the cessation of fiber production by fibroblast apoptosis and collagen matrix breakdown by the up-regulated MMPs44. In the present study, two weeks after the BH exposure, the BH-treated liver tissue was filled with regenerated hepatocytes without the presence of hepatic fibrosis , the presence of hepatic fibrosis by excessive ECM deposition can lead to liver cirrhosis and hepatocellular carcinomasis Fig.\u00a0I-ii. Angsis Fig.\u00a0A\u2013C. In gtes Fig.\u00a0A and B. tes Fig.\u00a0.In conclusion, to the best of our knowledge, this is the first study reporting the long term investigation of the wound healing process of liver after BH treatment in vivo. Our experimental results clearly showed that BH can trigger normal wound healing cascade and tissue regeneration  without inducing hepatic fibrosis or forming permanent scar tissue at the treatment site. These can clearly suggest that BH technique could be a promising and safe therapeutic tool for liver cancer treatment.In the present study, BH was applied to produce multiple volumetric lesions on a rat\u2019s liver in vivo. Upon animal sacrifice at 0, 3, 7, 14 and 28\u00a0days after BH treatment, BH-treated liver tissue and blood samples were collected for morphological, histological, serological and quantitative real-time polymerase chain reaction (qPCR) analyses to investigate the changes of the extent of extracellular matrix (ECM) remodeling, mRNA expression of cells (hepatocytes and fibroblasts), apoptosis status and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels over time.Six weeks old male SD rats were obtained from Koatech . All rats were housed under a 12-h light/dark cycle with free access to water and food. All experimental protocols were approved by the Institutional Animal Care and Use Committee of Korea University College of Medicine . All the methods were performed in accordance with the relevant guidelines and regulations.47. Both peritoneum and xiphoid were bent and pushed aside using silk surgical sutures to expose the left lateral lobe of the liver. Sterile gauze was located between the liver and stomach to remove the blood and prevent potential gastrointestinal damage associated with BH exposure. After BH exposure, rats were closed by layers with 4-0 sutures and allowed free access to water and food upon wakening.After 2-weeks of acclimation, general anesthesia was performed with 3.5% isoflurane in a 2:1 N2O/O2 mixture. The mixture of gas was maintained in the anesthesia chamber via rat\u2019s inhalation through a 2.5% nasal cone. Rats were placed on the warm pad and subjected to the midline xipho-pubic laparotomy, as previously described in16. A photograph of the HIFU experimental setup employed in the present study is shown in Supplementary Fig. 2 (1.5\u00a0cm\u2009\u00d7\u20091.5\u00a0cm). A 12-\u03bcm thick acoustically transparent Mylar film  was attached to the end of the holder for coupling purpose. This transducer holder was designed so that the distance from the tip of the holder to the centre of the transducer surface was 60.2\u00a0mm, and whereby the HIFU focus was 3\u00a0mm below the surface of the liver. A laser pointer aligned with the HIFU transducer\u2019s axial axis was attached to the translation stage for targeting the HIFU beam on the exteriorised liver surface laterally. BH lesions were produced by raster-scanning the transducer focus over the area of 1.44 cm2 using 2\u00a0mm steps in the transverse and lateral directions (up to a maximum of 1.2\u00a0cm), as illustrated in Fig.\u00a023. After the BH treatment, the animals were placed back into the animal housing facility and were sacrificed on day 3 (n\u2009=\u20093), 7 (n\u2009=\u20092), 14 (n\u2009=\u20094), and 28 (n\u2009=\u20095). Upon euthanisation, the liver tissues containing BH-treated, BH-penumbra  and BH-untreated regions  on the degree of mechanical damage produced in the liver was initially investigated in vivo  for histological examination. The tissues were fixed in 4% PFA for 48\u00a0h and after dehydration in 70% ethanol, embedded in a paraffin block. Following the standard protocols48, the paraffin-embedded liver tissue was cut in 4.5\u00a0\u03bcm thickness and stained with H&E and MT. Next, stained sections were imaged using the EVOS m7000 imaging system (Thermo Fisher Scientific) and Zeiss Axio Scan Z1 (Carl Zeiss).Rats were humanely sacrificed after 3, 7, 14, and 28\u00a0days after BH exposure by using the COAfter deparaffinization and rehydration, liver sections were incubated with the following primary antibodies: anti-asialoglycoprotein receptor 1/HL-1 (ASGR1) antibody , and anti-\u03b1-SMA . Anti-goat IgG Alexa Fluor 488 and 594  were used for the secondary antibody. All fluorescence images were obtained with LSM 800 (Carl Zeiss) and Zeiss Axio Scan Z1 (Carl Zeiss).Apoptotic cells in BH-treated liver sections were analyzed using a TUNEL in situ apoptosis detection kit . The assay was conducted according to the manufacturer\u2019s instructions. Counterstaining was performed with 4\u2032,6-diamidino-2-phenylindole (DAPI) and anti-\u03b1-SMA (Abcam) or anti-ASGR1 antibody labeled with anti-goat IgG Alexa Fluor 594 .47. Blood samples were collected at the following intervals separately; 10\u00a0min before BH-treatment, 1\u00a0h, 24\u00a0h, 48\u00a0h, 72\u00a0h after BH-treatment, and upon sacrifice. Using a FUJI DRI-Chemiclinical Chemistry Analyzer , the levels of ALT  and AST (aspartate aminotransferase) in serum were measured.Prior to BH treatment, femoral artery cannulation was performed, as previously described in49. The sequence of template-specific primers were as follows: Caspase3 (5\u2019-TTTGGAACGAACGGACCTGT-3\u2019 and 5\u2019- GGCAGGCCTGAATGATGAAG-3\u2019), Caspaes9 (5\u2019-AAGCAGGATCCAGAAGCTGT-3\u2019 and 5\u2019- GAGATGGGTCCAGCTTCACT-3\u2019), COL1A1 (5\u2019-GATCTCCTGGTGCTGATGGA-3\u2019 and 5\u2019- GACCAGGGAAGCCTCTTTCT-3\u2019), COL3A1 (5\u2019-ACTGGTGAACGTGGCTCTAA-3\u2019 and 5\u2019- GGACCTGGATGTCCACTTGA-3\u2019), COL5A1 (5\u2019-CTTCGGGAGCAGATGGTGAA-3\u2019 and 5\u2019- CTGGAGGTCCTGGTAAACCC-3\u2019), GAPDH (5\u2019-CAAGGCTGAGAATGGGAAGC-3\u2019 and 5\u2019-GAAGACGCCAGTAGACTCCA-3\u2019), and \u03b1SMA (5\u2019-GCTATTCAGGCTGTGCTGTC-3\u2019 and 5\u2019- GTTGTGAGTCACGCCATCTC-3\u2019).Liver tissues for qRT-PCR were collected from regions labelled in Supplementary Fig. 50.Liver tissues for gelatin zymography were collected from regions labelled in Supplementary Fig. All data were expressed as mean\u2009\u00b1\u2009standard deviation. Shapiro\u2013Wilk test was performed to evaluate normality distribution. One-way analysis of variance (ANOVA) followed by post-hoc Tukey\u2019s correction was used for parametric analysis and the Kruskal\u2013Wallis test with a post-hoc Conover correction for non-parametric analysis. Data were analyzed using MedCalc version 20.008 . A p-value of\u2009<\u20090.05 was considered statistically significant for all analyses.This study was reported in accordance with ARRIVE guidelines.Supplementary Information."}
+{"text": "Background and objectiveIn the last decade, there has been significant evolution in thoracic surgery with the advent of robotic surgery. In this study, we aimed to evaluate the incidence of\u00a0postoperative\u00a0chronic pain (for six months and beyond) in robotic and video-assisted approaches to analyze the long-term effects of the two different techniques.MethodsThis was a retrospective study involving 92 patients who underwent various thoracic operations between\u00a0six months and two years preceding the study. Patients were classified into two groups based on the type of surgery: video-assisted (VATS) (n=51), and robotic-assisted (RATS) (n=41) thoracoscopic Surgery. We employed the EuroQol (EQ-5D-5L) questionnaire to assess the utility values in terms of five quality-of-life measures .ResultsIn the VATS group, the median age was 68 years while it was 57 years in the RATS group\u00a0(p=0.001). A higher proportion of patients in the VATS\u00a0group had anatomical lung resection (lobectomy) compared to the RATS group: 61.2 vs. 41.6% respectively (p=0.005). However, the groups were well-matched on other patient characteristics such as relevant past medical history, underlying disease pathology, and final disease staging , with no significant differences between groups observed regarding these traits. In the VATS group, 62.7% of patients were pain-free at the time of the questionnaire-based evaluation compared to 51.2% in the RATS group. Additionally, 25.5% vs. 39% of patients had mild pain in the VATS and RATS groups\u00a0respectively. Neither of these differences was statistically significant.ConclusionPatients who undergo RATS are known to have better recovery and less pain compared to those who have VATS in the immediate postoperative period. However, our results did not find RATS to be superior to\u00a0VATS in terms of long-term pain. Additionally, robotic surgery is associated with higher hospital costs. In light of these findings, further comparative studies between the two approaches are recommended, while strategies to reduce postoperative pain and financial cost should continue to be explored. The last decade has witnessed a significant evolution in thoracic surgery with the advent of robotic surgery. This development and the adoption of minimally invasive surgical procedures, initially with video-assisted thoracoscopic surgery (VATS) and then robotic-assisted thoracoscopic surgery (RATS), has resulted in a technological paradigm shift in the field of thoracic surgery -3.A key aim of minimally invasive surgery is to improve the patient\u2019s quality of life (QoL) postoperatively by facilitating faster recovery. An important part of this, especially in thoracic surgery, involves managing neuropathic chronic pain ,5. Some Although it is likely that\u00a0several mechanisms contribute to the development of chronic pain after thoracic surgery, intercostal nerve damage and subsequent dysfunction are believed to play a key role ,7. The rThe patient\u2019s psychosocial condition is also known to affect the perception and impact of chronic neuropathic pain. This includes factors such as the presence of a mental health disorder, a diagnosis of malignant disease, the lack of\u00a0social support structures, and lower socioeconomic status . It has In this study, we aim to evaluate and compare the incidence of postoperative\u00a0chronic pain\u00a0(defined as that persisting beyond\u00a0six months from the\u00a0surgery) between patients who undergo RATS and those who have VATS to gain insights into the long-term effects of the two different techniques.We conducted a retrospective study involving 92 patients who underwent various thoracic operations between six months and two years preceding the study. We classified patients\u00a0into two\u00a0groups based on the type of surgery . In our study, VATS utilized either a three- or four-ports approach, whereas RATS involved four ports with a further utility port. The operations were performed by two consultant surgeons , and patients were\u00a0managed in a specialist cardiothoracic surgery unit postoperatively.\u00a0The routine postoperative pain management strategy for all patients in this unit involved an intercostal nerve catheter inserted intraoperatively, opiate-based patient-controlled analgesia (PCA), and oral analgesic agents - titrated up as necessary once the PCA was taken down .We used the EuroQol (EQ-5D-5L) questionnaire\u00a0as part of\u00a0routine postoperative follow-up appointments to assess the utility values in terms of five quality-of-life measures . Performance status within each of these fields was scored from 1 to 5, with a score of 1 indicating task completion with no difficulty or discomfort, and a score of 5 suggesting that the patient cannot complete the task\u00a0or feels uncomfortable doing so.The EQ-5D-5L interview was conducted via telephone\u00a0with patients after their consent for the study participation was obtained. Data on patient\u00a0physical characteristics, lung cancer type and stage, and operation details were retrieved and collected from hospital medical records.Statistical analysis was performed on non-parametric data via either a Chi-squared or Fisher's exact test, as dictated by the assumption required for each test. Differences in continuous variables were evaluated using a Student's t-test. A p-value <0.05 was considered statistically significant for the purposes of our study. SPSS Statistics  was used to perform all comparative analyses.In the VATS group, the median age was 68 years, while it was 57 years in the RATS group\u00a0(p=0.001). A higher proportion of patients in the VATS\u00a0group had anatomical lung resection (lobectomy) compared to the RATS group: 61.2 vs. 41.6% respectively (p=0.005). The underlying diagnosed pathology varied\u00a0between the groups, with lung adenocarcinoma confirmed in 41.4% of the VATS group and 29.5% of the RATS group. A large proportion of patients in both groups had stage I lung cancer: 47.1% in the VATS group and 41.5% in the RATS group.Despite the groups not being prospectively matched and randomized, there were no significant differences observed in the majority of patient characteristics obtained, namely the presence of diabetes, hypertension, smoking history, heart or lung disease, or underlying pathology\u00a0and final disease staging Table .As shown in Table With the rapid evolution of minimally invasive thoracic surgery and the growing use of robotic surgery worldwide, many studies have aimed to identify if RATS has superiority over VATS.Three randomized controlled trials were performed to compare RATS and VATS . RVlob was the largest and included 320 patients. It found that RATS was associated with significantly more lymph node sampling and less intraoperative bleeding, but had significantly higher costs. Moreover, the study found no difference in the length of hospital stay or early postoperative pain between the two methods . The ROMIn contrast, a large meta-analysis\u00a0by Ma et al. involving 18 studies with 11,247 patients divided between RATS and VATS\u00a0showed the superiority of RATS over VATS in terms of the number of lymph nodes sampled, with no increase in operative time, as well as a shorter overall length of stay,\u00a0but again at a significantly higher cost .However, there is a paucity of studies regarding the quality-of-life parameters and risk of chronic pain between the two modalities. van der Ploeg et al. performed a retrospective study to compare postoperative pain between VATS and RATS. They observed that pain scores between the two different modalities did not differ following\u00a0surgery for non-small cell lung cancer . This alInterestingly, in our study, the rates of chronic pain and discomfort were higher in the RATS group despite the younger age of patients and fewer lung resections in that group. This was not a significant difference, and it is notable that rates of chronic pain were high in both groups; this is known to be a key indicator of morbidity in thoracic surgery, with multiple analgesic modalities usually employed as part of standard practice in the modern era . That coThis study has a few limitations. Primarily, this was a retrospective cohort analysis, and the groups were not prospectively matched or randomized; however, most patient characteristics did not differ significantly between the two groups. The time frame of quality-of-life measurement was not standardized, but it was at least six months postoperatively. Given that our patient numbers were small and\u00a0from a single institution, it is possible that these results could not be extrapolated to other centers with different patient demographics.RATS is known to lead to quicker recovery and less pain than VATS in the immediate postoperative period. However, our results did not find RATS to have superior pain control compared with VATS in the long term after surgery. Also, robotics is associated with higher hospital costs. It remains to be seen if the issues concerning the cost-effectiveness of RATS will be properly addressed. In the meantime, efforts to investigate the causes of postoperative chronic pain following minimally invasive thoracic surgery and the strategies to reduce the incidence of the same must be continued."}
+{"text": "Musa spp., Cavendish sub-group AAA) by the Taiwan Banana Research Institute (TBRI) has resulted in several cultivars resistant to Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), a destructive fungus threatening global banana production. However, the mutations in these somaclonal variants have not yet been determined. We performed an RNA-sequencing (RNA-seq) analysis of three TBRI Foc TR4\u2013resistant cultivars: \u2018Tai-Chiao No. 5\u2019 (TC5), \u2018Tai-Chiao No. 7\u2019 (TC7), and \u2018Formosana\u2019 (FM), as well as their susceptible progenitor \u2018Pei-Chiao\u2019 (PC), to investigate the sequence variations among them and develop cultivar-specific markers.The selection of tissue culture\u2013derived somaclonal variants of Giant Cavendish banana (A group of single-nucleotide variants (SNVs) specific to one cultivar were identified from the analysis of RNA-seq data and validated using Sanger sequencing from genomic DNA. Several SNVs were further converted into cleaved amplified polymorphic sequence (CAPS) markers or derived CAPS markers that could identify the three Foc TR4\u2013resistant cultivars among 6 local and 5 international Cavendish cultivars. Compared with PC, the three resistant cultivars showed a loss or alteration of heterozygosity in some chromosomal regions, which appears to be a consequence of single-copy chromosomal deletions. Notably, TC7 and FM shared a common deletion region on chromosome 5; however, different TC7 tissues displayed varying degrees of allele ratios in this region, suggesting the presence of chimerism in TC7.This work demonstrates that reliable SNV markers of tissue culture\u2013derived and propagated banana cultivars with a triploid genome can be developed through RNA-seq data analysis. Moreover, the analysis of sequence heterozygosity can uncover chromosomal deletions and chimerism in banana somaclonal variants. The markers obtained from this study will assist with the identification of TBRI Cavendish somaclonal variants for the quality control of tissue culture propagation, and the protection of breeders\u2019 rights.The online version contains supplementary material available at 10.1186/s12864-022-08692-5. Musa spp.), perennial herbaceous crops widely grown in subtropical and tropical regions, are consumed as a staple food in many tropical countries and are a popular fruit worldwide. The vast majority of banana cultivars were derived from the inter- or intra-specific hybridization of M. acuminata (A genome) and M. balbisiana (B genome) [Bananas ( genome) , 2. Most genome) , 4; ther genome)  or tissu genome) , 7. AlthFusarium oxysporum f. sp. cubense (Foc) [Fusarium wilt of banana, also known as Panama disease, is caused by the soil-borne fungal pathogen se (Foc) . The desse (Foc) , 9, whicse (Foc) \u201313. In 2se (Foc) . The effse (Foc) ; thus, tM. acuminata with resistance to Foc TR4 increased the Foc TR4 resistance of the Cavendish cultivar \u2018Grand Nain\u2019 in field trials [The improvement of Cavendish banana resistance to Foc TR4 has been conducted through transgenic or mutagenesis approaches because the triploid and sterile nature of commercial banana cultivars limits the success of conventional breeding. The expression of a resistance gene encoding a nucleotide-binding leucine-rich repeat protein from a wild diploid subspecies of d trials . Mutagend trials , 18. Simd trials , morphold trials . Althougd trials , 21, 22.d trials . Owing td trials .Oryza sativa) regenerants [Tissue culture can induce genetic and epigenetic changes in regenerated plants \u201327; for enerants . Regenerenerants , 30. DNAenerants , 31, 32.enerants , 30, 33,enerants , 35.The tissue culture\u2013derived Foc TR4\u2013resistant cultivars of Giant Cavendish bananas selected by TBRI are valuable resources for sustaining banana production under the threat of this devastating fungal disease; however, the genetic or genomic differences between these somaclonal variants and their susceptible progenitor have not yet been investigated. A lack of sequence information has retarded the development of molecular markers for these Foc TR4\u2013resistant cultivars and the exploration of underlying disease resistance mechanisms. Through the analysis of sequence variations in RNA-sequencing (RNA-seq) data, we successfully identified single-nucleotide variants (SNVs) specific to TC5, TC7, and FM and converted several SNVs into cleaved amplified polymorphic sequence (CAPS) markers or derived-CAPS (dCAPS) markers. An analysis of sequence variations also revealed chromosomal deletions in these three cultivars and chimerism in TC7. These findings demonstrate the effective use of RNA-seq data in the detection of sequence and structural variations in triploid bananas, which are relevant to the identification and further characterization of these banana somaclonal variants.Ananas comosus) [Raphanus raphanistrum subsp. sativus) [Hordeum vulgare) [The development of single-nucleotide polymorphism (SNP) markers from expressed sequence tags or RNA-seq data has been demonstrated in various crops, such as pineapple (comosus) , radish sativus) , and barvulgare) . Given tvulgare) . This coTo overcome this challenge, we performed an RNA-seq analysis of four samples of PC and TC5, three samples of FM, and two samples of TC7. Including replicates for each cultivar could reduce the detection of false cultivar-specific markers caused by SNVs in individual clones or sequencing errors. Notably, the RNA-seq analysis of PC, TC5, and TC7 included the replicates that were harvested and sequenced in different batches and years Table S. This woBy comparing with the DH \u2018Pahang\u2019 sequences, sequence variants were identified in each sample of the seven cultivars using GATK HaplotypeCaller  with a hPrevious analyses using whole-genome sequencing, genotyping-by-sequencing, and RNA-seq revealed a substantial degree of sequence heterozygosity in various diploid and triploid banana species and subspecies \u201346. NotaA total of 42 distinct SNVs between TC5 and PC were dispersed among the 11 banana chromosomes, with a hotspot in a region spanning coordinates 36,296,217\u201336,322,712 on chromosome 4  from three soil-grown PC and TC7 individuals using Sanger sequencing from genomic DNA. For each plant, we genotyped three leaves, one stem, one rhizome, and three roots, specifically focusing on two variant sites in the SNV-rich region I on chromosome 5 where TC7-2 showed a LOH. Based on the peaks in the sequencing chromatograms, we then calculated the minor allele ratio (mAR) as the ratio of the height of the minor allele to the total height of the major and minor alleles at each site. All PC samples for the four tissues exhibited a similar degree of heterozygosity, with a mAR\u2009\u2265\u20090.2 (type III) Fig.\u00a0. By contTo develop reliable SNV markers for the three TBRI Foc TR4\u2013resistant cultivars, we selected some cultivar-specific SNVs identified from the RNA-seq analysis for further validation using Sanger sequencing. Based on the sequence counts, 11, 10, and 8 SNVs were selected for TC5, TC7, and FM, respectively, for validation with the DNA isolated from samples that differed from those used for the RNA-seq analysis. With the exception of the four selected SNVs for TC7, the selected SNVs were successfully detected using the primers we designed and showed distinct alleles between the targeted cultivar and others in the sequencing chromatograms  [Since most bananas are hybrids both between and within species and subspecies , 2, theiinifera) , 52. WitSolanum tuberosum), which remain able to reproduce sexually, the bananas of a triploid genome must be propagated clonally, which abolishes the possibility of eliminating chromosomes with large deletions or extra chromosomal segments through meiosis. Moreover, the transposon activation and instability of DNA methylation induced by tissue culture may enhance the occurrence of chromosome breakage [Compared with other vegetatively propagated crops, such as grapevine and potato , and sugarcane (Saccharum sp.), are also propagated clonally. The successful development of molecular markers from RNA-seq data for the verification of three somaclonal variants of Cavendish banana in this study suggests the potential to apply similar approaches to somaclonal variants of other species.Experimental designs and analytic pipelines are important for the success of developing molecular markers for somaclonal variants. As highlighted by Michno and Stupar in the analysis of genomic variation in transgenic soybean , genomicIn addition to the identification of differentially expressed genes, as demonstrated in this study, RNA-seq data can be applied to the development of reliable molecular markers and the detection of chromosomal deletions for triploid and tissue culture\u2013derived banana cultivars. The SNV markers developed in this study have good specificity and reproducibility to distinguish TBRI Cavendish somaclonal variants of very high genetic similarity.Musa spp., AAA group) used for RNA-seq or genotyping in this study were obtained from the Taiwan Banana Research Institute . Shoots of unrooted or rooted plantlets (1\u20132 months) in tissue culture jars were harvested for RNA-seq analysis. Details of the samples used in RNA-seq and the 11 samples used in the CAPS/dCAPS marker analysis are included in Tables SThe Cavendish bananas . The RNA-seq libraries were prepared from the extracted RNA using either the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) or the TruSeq RNA Library Prep Kit v. 2 (Illumina), following the manufacturers\u2019 instructions. Paired-end sequencing was performed on an Illumina HiSeq 2500 or 4000, or a NovaSeq 6000, following the standard protocols, to produce reads of 126 or 150 nt  using HISAT2 v. 2.1.0 [RNA-seq data were generated for five TBRI cultivars  in this study, while RNA-seq data of \u2018Grand Nain\u2019 and \u2018Williams\u2019 were downloaded from the NCBI database , 1\u00a0M KCl, 10 mM EDTA (pH 8.0)), following the protocol described previously  or usinghttp://primer3.ut.ee/) [-1) and 7\u20137.8\u00a0\u00b5l of ultrapure water. For all of the primer pairs listed in Table STo confirm the SNVs identified from the RNA-seq data, PCR primers were designed using Primer3   to amplihttp://helix.wustl.edu/dcaps/dcaps.html) [Cultivar-specific SNVs confirmed using Sanger sequencing were selected for the development of CAPS or dCAPS markers. The primers used for the validation of TC5-specific SNVs using Sanger sequencing were also employed in the CAPS analysis. The dCAPS primers, which introduced a restriction enzyme site to the amplification products of the sequences shared among four cultivars but not the sequence specific to one cultivar, were designed using dCAPS Finder 2.0 (ps.html) . The priAdditional file 1: Figure S1-S10 and Table S1-S4."}
+{"text": "Recently, mental health has received increasing attention, particularly preoperative anxiety, which constitutes a bad emotional experience for surgical patients. Many experts have studied preoperative anxiety in terms of its related risk factors, interventions, and postoperative effects; however, there has been no systematic analysis of published articles. This paper presents a bibliometric review of documents related to preoperative anxiety published between 2001 and 2021. A detailed data analysis of 1,596 publications was conducted using CiteSpace and VOSviewer. Since the 20th century, the field of preoperative anxiety has gradually developed; research began around 2000 and has made a huge leap forward since 2016. Developed countries, led by the United States, were the first to conduct research, but preoperative anxiety research in developing countries like Turkey and China has gradually increased and led to an irreplaceable contribution. Intervention has remained the main topic of preoperative anxiety research, and measures have developed from premedication to the provision of education and information. Moreover, the use of advanced equipment such as virtual reality has emerged with great popularity. Based on previous research, the application of virtual reality combined with pediatric patients will become a new research direction. Preoperational anxiety, or preoperative anxiety, is a common reaction experienced by patients admitted to a hospital for surgery . It can With medical treatment developments, the estimated total global surgical volume reached levels of up to 312.9 million major operations in 2012 , 5. AmonNonetheless, the influence of preoperative anxiety on patients cannot be ignored. Studies have shown that preoperative anxiety can increase cardiac mortality and adverse reactions during the induction of and recovery from anesthesia \u201311. MorePreoperative anxiety reflects the patient's psychological structure, which can be affected by other people. Too many interventions can be adopted. Pregabalin acting as an antianxiety drug is useful and safe for preoperative and intraoperative anxiety control in patients undergoing surgery . Music iFortunately, in recent years, several research papers about preoperative anxiety have been published. Researchers have investigated medical or nonmedical interventions and the influence and evaluation of preoperative anxiety. It was confirmed that nursing interventions have a positive impact in reducing preoperative anxiety, but the low number of studies and the heterogeneity of the sample size means further research is needed regarding this topic . MeanwhiWe selected literatures with systematic review and meta-analysis to understand the current status of preoperative anxiety research. However, the studies in these articles mainly focus on: an overview of the effects of the preoperative anxiety, the certain of a intervention to reduce the preoperative anxiety and researches for the specific population such as adults, children or for the specific surgery such as\u201chip or knee replacement\u201d, \u201cbrain surgery\u201din this filed. These articles can help us understand the degree of research in a certain field of preoperative anxiety, but there is still a lack of an overall grasp of preoperative anxiety research: how preoperative anxiety research have changed in the past, what specific studies have been involved, how these studies were distributed in time and space, what the knowledge framework looks like, what co-cooperation with other studies and what are the hot trends for future research, etc. These can help us to have an overall grasp of the degree of preoperative anxiety research and provide guidance for the future research direction. However, there is lack of such review articles.Considering the research gaps mentioned above, the study addressed the following research question and objectives:\u201cDo we need to create an overview of status of publications and a clear picture of scientific exchanges?\u201dThe answer to this query is yes. The present study reports a bibliometric review of all papers on preoperative anxiety from 2001 to 2021, based on data obtained from the Web of Science Core Collection with the aid of two tools (CiteSpace and VOSviewer). Given the emphasis on preoperative anxiety, it is necessary to create an overview of status of publications and a clear picture of scientific exchanges in the field because of its significance in helping to master the knowledge before and making research direction decision in the future. Considering the research gaps and intended contribution, the study had the following objectives based on previous literature: publication trends, hotspot shifts, intellectual milestones, and research fronts in the field of preoperative anxiety.The rest of the paper is organized as follows: Section 2 describes the Materials and Methods. Identifying the articles refined by search strategy. Introducing analytic strategy and the two software programs: Citespace5.8 and VOSviewer1.6.17. Section 3 was the presentation of results base on research objectives: publication trends, hotspot shifts, intellectual milestones, and research fronts in the field of preoperative anxiety. Section 4 was a discussion of the results, followed by the conclusions in Section 5 \u201322.The relevant publications were extracted from the Web of Science (Classic) Core Collection using the following search strategy, and the detailed procedures are drawn in a PRISMA flow chart attached as TOPIC: TS\u2009=\u2009(*preoperati* near/3 anxiet*) The use of \u201c*\u201d means any group of charactersRefined by: DOCUMENT TYPES:(ARTICLE)Timespan: 2001\u20132021.Language: EnglishTo reduce data bias, we used the Web of Science (Classic) Core Collection, one of the most comprehensive and authoritative databases , to perfWe began our analysis with a comprehensive overview of preoperative anxiety publications. First, we drew a chart illustrating the number of annual publications. Using VOSviewer, we noted the distribution of research on preoperative anxiety across national and regional research institutions over time. Meanwhile, we used CiteSpace to visualize author co-citation relationships and the cooperation across disciplines in preoperative anxiety.CiteSpace used to identify the bursts of keywords is a valuable indicator of most active research topics . Using VIntellectual milestones are the most influential research studies in a particular field. By reading these articles, we acquired a deeper understanding of the progress of research into preoperative anxiety. In the CiteSpace co-citation cluster analysis, references with betweenness centrality >0.1 had purple circles. we focused on articles with high betweenness centrality, or nodes with thick purple circles; these articles are the bridges that form correlations between articles. Intellectual milestones are regarded as an important theoretical basis to guide the formation of new research results.A research front is the foundation of scientific research; trends in research fronts reflect the direction of scientific research. Through burst detection of the cited literature in the co-citation network, articles can be ranked in terms of the strength of their citation bursts, illustrating citation frequency increases over a short time. References with the strongest citation bursts meant that the research had recently received a high level of attention and had a large influence on the field. In the meantime, these influential articles may also form the basis of future research. The co-citation network is based on the analysis of the co-citations of downloaded references; each citation is replaced by a small node and the network is formed by connecting lines between related citations, wherein nodes with red circles represent bursts. Generally, the thicker the red circles, the stronger the bursts and the more likely the citation is to become a research front. Nonetheless, using only the co-citation network to detect the scientific research front has some limitations. For example, ongoing studies are not counted and these studies may reflect the research fronts to some extent. ClinicalTrials.gov was selected to identify ongoing studies on preoperative anxiety by searching studies with recruiting, not yet recruiting, or active, but not recruiting.We conducted a preliminary analysis of preoperative anxiety publications from a macroscopic perspective. The Web of Science (Classic) Core Collection generated 1,596 publications, as shown in Hotspots represent the topics that most frequently appear in relevant studies; in other words, hotspots are areas that receive the most attention from scholars, which highlights the focus of preoperative anxiety research. In CiteSpace, burst detection revealed the keywords whose frequencies increased abruptly over time, such that the burst of keywords is a valuable indicator of most active research topics . Figure\u00a0We used VOSviewer to construct the term co-occurrence network and acquire more information between different keywords. Intellectual milestones are significant research studies. They are the result of quantitative to qualitative change based on previous research. Meanwhile, intellectual milestones in one field can have great impacts with respect to opening another field of research. these articles are the bridges that form the correlation between two unconnected clusters, indicating the \u201cturning point\u201d in CiteSpace, called \u201cintellectual milestones\u201d . In the As shown in The first article , Kain ZNvia non-drug interventions. The paper by Yip , which can be applied to different patients and surgeries, especially in pediatric patients.Today is an information society, the development of computer technology will push the artificial intelligence applied to all fields, medicine is no exception. Virtual reality (VR) is a computer technology that provides the feeling of being immersed in a simulated three-dimensional (3D) world where the user may interact with the virtual environment . VR techOur research still has some limitations. First, our data were extracted from Web of Science, which means that relevant research from other databases may have been missed. Additionally, the methods of almost all bibliometric analyses are based on the refinement of articles, ignoring other types of documents. For example, by analyzing the research results of reviews, we can also have an overall understanding of the research on preoperative anxiety, the research status and shortcomings of preoperative anxiety, and the future research directions. Moreover, our search strategy was in English, excluding any non-English research; this is particularly important since many developing countries  have achieved great progress in preoperative anxiety research, and their exclusion may introduce bias.We conducted a bibliometric review of preoperative anxiety articles and analyzed publication trends, hotspots, intellectual milestones, and research fronts. We get the overview of preoperative anxiety publication. There were 1,596 publications during 2001\u20132021. There was a sharp increase from 2015 to 2016, peaking at 225 articles in 2021. United States had the highest number of preoperative anxiety studies followed by some developed countries: Canada, Germany, etc. Yale University appeared to play a significant role as an institution followed by the University of California, Irvine. Caumo W, Unknown, and Kain ZN could be regarded as the most influential authors in this filed. Kain ZN had the huge influence on this field with his famous studies focus on preoperative anxiety in pediatric, which was regarded as intellectual milestones. The keywords had changed over time, the application of medicine mostly occurred in the past, whereas non-premedication and informational interventions are new hotspots. Research on preoperative anxiety in pediatric would remain a hotspot for a long time in the future. Besides because of the development of the artificial intelligence, the application of VR technology combined with pediatric may lead the field in a new future direction."}
+{"text": "First of all, in order to meet the limit condition of the chaotic phase transition of the system output, the frequency value of the characteristic signal is reconstructed and transformed based on the variable scale theory. Then, in order to solve the influence of the initial phase of the characteristic signal on the detection accuracy, a detection model based on the array Duffing system is presented, and a frequency-detection scheme with all-phase coverage is given. Finally, another array Duffing system is designed for the parameter estimation of the characteristic signal. The critical value of chaotic phase transition is determined by adjusting the amplitude of the driving signal of the array Duffing system, and then the amplitude and phase parameters of the characteristic signal are synchronously estimated. The experimental results show that the proposed method can effectively extract the weak characteristic signal within the strong noise, and the SNR of the characteristic signal can be as low as \u221221 dB. Through the attitude calculation for the extracted characteristic signal, it can be seen that the proposed method can improve the accuracy of the inclination of the drilling tool significantly, which proves the feasibility and effectiveness of the method proposed in this paper.In the process of dynamic Measurement While Drilling (MWD), the strong vibration and rapid rotation of the Bottom Hole Assembly (BHA) lead to multi-frequency and high-amplitude noise interference in the attitude measurement signal. The weak original signal and extremely low signal-to-noise ratio (SNR) are always the technical difficulties of dynamic MWD. To solve this problem, this paper uses the chaotic effect of the Duffing system, which takes the expression (\u2212 The meaning of dynamic measurement while drilling (MWD) is to obtain real-time measurement data about the drill bit\u2019s posture during the drilling process. Then, the obtained measurement data are transmitted to the ground through wireless transmission in real time, which provide reference opinions for the next steps of the staff operations. The acquisition of these measurement data is achieved by converting the analog measurement signals of various sensors in the measuring instrument into digital signals, and then calculating them based on physical models. Then, with the help of the drilling fluid (mud) inside the drill string, it is transmitted to the ground. Finally, the ground receives the mud pressure waveform through a pressure sensor, and decodes the waveform to obtain parameters.During the signal transmission process, vibrations such as cutting the rock stratum, and the collision between the drill string and the borehole wall can cause strong mechanical vibrations in the drilling tool assembly. Therefore, the pressure wave of the mud pulse signal detected by the pressure sensor contains a large amount of noise signals, resulting in an extremely low signal-to-noise ratio (SNR) of the downhole attitude measurement signal ,2. The rThe digital filter based on time\u2013frequency analysis was first applied to the noise suppression of MWD signals. Tu  developsDigital filtering is a signal processing method based on noise suppression. Its realization is based on the premise that the spectrum of the characteristic signal does not overlap with the spectrum of noise interference. Under such conditions, it can retain useful signals and filter out irrelevant noise components during the filtering process . HoweverIn order to improve the accuracy of attitude measurement for the drilling tool, some scholars refer to the attitude measurement principle in the field of integrated navigation. The advanced filtering algorithm is applied to the downhole sensor signal to eliminate the noise interference maximum and realize its optimal estimation. Ref.  proposedAiming at the above problems, refs. ,15,16 esWith the rapid development of science and technology, some emerging signal processing methods have been applied to weak signal detection in dynamic MWD ,19,20. RIn recent years, the profound study for nonlinear science has provided a new way for the researcher to understand, analyze and solve the problem of weak signal detection. The discovery of nonlinear dynamic phenomena such as chaos, fractal and stochastic resonance poses a powerful challenge to traditional signal processing methods ,24,25,26As a typical nonlinear model that can produce chaotic effects, the second-order Duffing system can realize the transformation of the output state under the conditions of appropriate parameters ,30. WhenIn conclusion, this paper presents a weak signal detection method for dynamic MWD based on the chaotic effect of the array Duffing system. Using the transition of the output state of the array Duffing system, the frequency detection and parameter estimation of the downhole attitude measurement signal are carried out, and then the complete sensor signal is obtained. Finally, real-time and accurate measurement of attitude parameters for drill tool is realized.The use of chaotic systems to detect weak signals was first proposed between 1990 and 1992. Based on the sensitivity of chaotic systems to initial conditions, the weak signal is taken as the initial condition of the chaotic system, and the chaotic system whose dynamic behavior is extremely sensitive to the weak signal is established. Putting the chaotic system in a specific state introduces the weak signal into this system, and then the weak signal covered by noise based on changes in the phase trajectory of the chaotic system is detected. This is the basic principle of using chaotic systems to detect weak signals. Therefore, establishing a chaotic system sensitive to weak signals is the primary condition for signal detection.Chaos is a unique solution of some nonlinear equations. In other words, it is a unique motion form of some nonlinear systems. Chaotic motion has randomness, but the equations describing its motion are deterministic, such as the famous Duffing equation, Lorenz equation, etc. This article takes the Duffing equation as the research object to detect the dynamic MWD signals. The typical Duffing equation is as follows.x is the solution of the equation, also known as the system output. k is the damping coefficient. According to the experimental results in ref. . Assume the angular frequency of the characteristic signal \u03c9 = N \u2260 1 rad/s, then the characteristic signal is updated to the following form.Assume the characteristic signal N to 1 rad/s on the frequency axis. At this time, it is possible to detect whether the signal s(t1) exists under the action of Equation (3), and then determine the frequency value of the characteristic signal according to the relationship of \u03c9 = N. However, it is impossible to directly reconstruct the signals collected by downhole sensors through the linear transformation shown in Equation (4) when processing actual measured signals. Therefore, the scale transformation of the numerical calculation step is considered to realize the reconstruction of the frequency value. The specific steps are as follows.It can be seen from Equation (4) that the angular frequency of the MWD signal is compressed from (1)fs, the step size of the numerical calculation should be as follows.Assuming the sampling frequency is (2)The transformation coefficient R is introduced, and the step size of numerical calculation is updated to the following form.N times, and the corresponding signal angular frequency is compressed N times. In other words, an MWD signal with a sampling frequency of fs and an angular frequency of \u03c9 is transformed into a signal with a sampling frequency of fs/N and an angular frequency of \u03c9/N through variable scale processing.At this time, the time interval of the signal is increased by (3)Input the above signals into the Duffing detection system and make the calculation step meet Equation (6) to complete the parameter reconstruction of the MWD signals, and then the frequency value of the characteristic signal is identified by the transition of the output state of the Duffing system.From the above analysis, it can be seen that scale transformation does not change the value of the characteristic signal, but only changes the frequency (or time) scale of the signal, compressing (or enlarging) it on the time axis and reordering the values, which will not affect the output result of the Duffing system.According to the above principle of the variable scale Duffing system for frequency detection, no matter how the frequency value of the characteristic signal changes, it is reduced to 1 rad/s. In other words, the frequency of the characteristic signal is consistent with the angular frequency of the driving signal through scale transformation processing. Therefore, a fixed set of system parameters is always used in the detection process. At this time, the determination of the critical amplitude of the system is independent of the frequency value of the characteristic signal. Thus, problems (1) and (2) proposed in a(t) as 0.01cost and 0.01cos(t + \u03c0), respectively, and other parameters remain unchanged. The output state of the Duffing chaos system are shown in Based on the analysis above, it is always assumed that the initial phase angle of the characteristic signal and the driving signal is 0. However, for the actual MWD signal, the initial phase is not always equal to 0. Therefore, it is necessary to study the influence of the initial phase of the characteristic signal on the frequency detection of the Duffing system. In Equation (3), take the characteristic signal As can be seen from \u03b3 is still the amplitude of the characteristic signal, \u03b2 and \u03b8 represent the initial phase angle of the driving signal and the characteristic signal, respectively. The value range of \u03b8 is from \u2212\u03c0 to \u03c0.To solve this problem, this section establishes an all-phase frequency detection scheme based on the array Duffing system. Because the angular frequency of the characteristic signal will eventually be adjusted to 1 rad/s by scale transformation, in order to simplify the analysis, the angular frequency of the characteristic signal is directly set to 1 in this section. In addition, the amplitude of the driving signal is still set to 0.72. Therefore, the Duffing system frequency detection model after considering the initial phase angle is as follows.If the first two terms at the right end of Equation (7) are simplified, the following form can be obtained.A and B are the two right sides of a right triangle, and C is the oblique side. A, B and C are represented by the following equations.Assume that \u03b4 = B/C, cos\u03b4 = A/C, then Equation (7) can be further simplified as follows.If sin\u03b4 in Equation (10), the value of \u03b4 has little effect on the amplitude parameters of the Duffing system, only having an effect on the initial position of the trajectory solution. What really affects the detection result is the amplitude term of the trigonometric function in Equation (10). Therefore, the expression of the amplitude term is established as follows.It can be seen from Equation (10) that the characteristic signal and the driving signal are combined into a trigonometric function term. According to the position of \u03b3 of the characteristic signal is known, the identifiable initial phase range of the characteristic signal can be solved according to Equation (11). The amplitude of the MWD signal is usually greater than 0.1, and the minimum is not less than 0.02. Therefore, the amplitude of the characteristic signal is set to 0.02 and substituted into Equation (10). The following relationship can be obtained.The right end of Equation (11) is the minimum amplitude that can cause the output state of the Duffing system to change. Therefore, when Equation (11) holds, the total amplitude of the input signal will exceed the critical value, and the output state of the system will change from chaos to large-scale period. Further, the characteristic signal is identified by the change in phase state. If the amplitude \u03b8-\u03b2 is from \u221260.7\u00b0 to 60.7\u00b0. The simulation results also prove that when \u03b2 = 0 and the other conditions remain unchanged, the variation range of \u03b8 is from \u221260\u00b0 to 60\u00b0. Only when \u03b8 is within this range can Equation (7) realize the change in output state of the Duffing system. It is basically consistent with theoretical calculation. In addition, when the amplitude of the characteristic signal is greater than 0.02, the variation range of \u03b8 increases correspondingly according to the calculation results of Equation (12). Therefore, the above variation range of \u03b8 is applicable to all characteristic signals with an amplitude not less than 0.02.By solving Equation (12), the variation range of term \u03b8 while \u03b2 = 0.From the above analysis, when other parameters of the system are determined, the initial phase angle of the characteristic signal must be within a certain range to effectively obtain the frequency of the characteristic signal, that is, there is a detectable window for the initial phase angle of the characteristic signal. The detection window is shown in \u03b2 of the drive signal is set as \u2212120\u00b0, the corresponding detection window of \u03b8 is from \u2212180\u00b0 to \u221260\u00b0. When \u03b2 is set as 120\u00b0, the corresponding detection window of \u03b8 is from 60\u00b0 to \u2212180\u00b0. Therefore, in this section, the tradition Duffing system, which was shown in Equation (7), will be expanded to an array Duffing system which is composed of three Duffing equations with different initial phase angles of driving signals. Then, the all-phase frequency detection of characteristic signals is realized. The array Duffing system for all-phase frequency detection is as follows.It can be seen from When the amplitude of the characteristic signal is greater than 0.02, it is substituted into the array Duffing equations, respectively. Because the initial phase of the signal detected by these three equations covers the whole range. Therefore, as long as the output state of one of the equations changes, the input signal can be identified as a weak characteristic signal with the same frequency as the drive signal, and then the frequency value can be determined. It can be seen that the influence of the initial phase angle of the characteristic signal on its frequency detection can be effectively solved through the array Duffing system.\u03b2 is equal to 0, the effect of the system output phase transition can also be achieved. At this point, it is necessary to further increase the amplitude of the driving signal. Taking this article as an example, when the sum of the amplitudes of the driving signal and the signal to be measured is greater than 0.75 and the initial phase angle of the driving signal \u03b2 is equal to 0, regardless of the initial phase angle of the signal to be measured, the frequency value of the signal to be measured can be detected through the system output phase change. However, in application, this method requires continuous adjustment of the amplitude of the driving signal to ensure that the amplitude of the two signals added is greater than 0.75. Therefore, the calculation amount of this method is relatively large and the calculation results are influenced by the amplitude of the signal to be measured. Based on the above analysis, the proposed all-phase frequency detection based on the array Duffing Chaos system in this article is a more suitable method for the dynamic MWD signal.It should be pointed out that according to the Lyapunov exponent analysis method, when the initial phase angle of the driving signal It can be seen from the above analysis that the frequency value of the MWD signal in the strong noise background can be identified using the array Duffing equations combined with the scale transformation. However, in order to calculate the real-time attitude information of the drilling tool, it is also necessary to determine the amplitude and phase value of the characteristic signal, so as to recover the complete signal waveform. To solve this problem, an amplitude and phase synchronization estimation method based on the array Duffing system will be presented in this section.1 represents the driving signal amplitude when the output state of the Duffing system just changes from chaotic to large-scale periodic, and then Equation (11) can be rewritten as follows.\u03bb1 could be determined by observing the output status of the Duffing chaos system.According to the analysis results in the previous section, when Equation (11) holds, the output state of the Duffing system will change from chaotic to large-scale periodic. Assuming that \u03bb\u03bb1 is determined, Equation (14) is a binary equation with an amplitude \u03b3 and phase \u03b8 of characteristic signals as variables. Therefore, another binary equation about amplitude and phase could be established in a similar way, and then the amplitude and phase values of the characteristic signal can be obtained by solving the binary function equations. It should be noted that since the variation range of \u03b8 is from \u2212\u03c0 to \u03c0, the absolute value of \u03b8 is solved through the binary function equation instead of \u03b8. To further determine the value of \u03b8, one more equation needs to be added. Therefore, aiming at the problem of parameter estimation to be solved in this section, a chaotic detection model based on array Duffing system is established as follows.After Firstly, the initial phase of the drive signal is set to 0, 0.5\u03c0 and \u03c0, as shown in Equation (15). Then, the characteristic signal is input into the array Duffing equations in Equation (15), respectively, and the amplitude of the driving signal is adjusted step by step. Finally, the output state of the Duffing system is observed and the amplitude of the system is recorded when the phase transition occurs.\u03bb1, \u03bb2 and \u03bb3. Therefore, the array Duffing equations for the amplitude \u03b3 and phase \u03b8 of the characteristic signal can be expressed as follows.When the output state of the array Duffing system changes from the chaos to large-scale period, its driving signal amplitude is expressed as The following results can be obtained by solving the above equation.The equations above is the estimation formula for the amplitude and phase of the characteristic signal. It can be seen that the array Duffing system used for parameter estimation in this section and the array Duffing system used for frequency detection in the previous section are derived based on Equation (10).But the difference is that in Based on the analysis above, the specific process of the chaotic effect-based improved array Duffing systems for dynamic MWD signal detection is as follows.k is set to 0.5, the angular frequency of the drive signal is set to 1 rad/s, the initial value of the system (x(0), x\u2019(0)) is set to , and the amplitude of the drive signal \u03bb is set to 0.72.Step 1 Determine the parameters of Duffing system. For example, in Equation (3), the damping ratio Step 2 Input the noise characteristic signal into Equation (7), and set the initial phase angle of the driving signal to be 0, \u00b12\u03c0/3 , from which the array Duffing system for frequency detection is obtained.R, the characteristic signal with sampling frequency of fs and angular frequency of \u03c9 is updated to the signal with sampling frequency of fs/R and angular frequency of \u03c9/R. Then, the array Duffing equation for frequency detection is solved with the calculation step T1 = R/fs, and the phase trajectory of the output state of the array Duffing system is obtained.Step 3 By introducing the transformation coefficient R and observe the output state of the above array system. As long as one of the phase trajectories jumps from the chaotic state to large-scale periodic state, it means that the value of the transformation coefficient at this time is the frequency value of the characteristic signal.Step 4 Adjust the transformation coefficient Step 5 After determining the frequency value of the characteristic signal, the array Duffing system for parameter estimation is designed according to the initial phase angle of the driving signal. At this time, the initial phase angles of the system drive signal are 0, \u03c0/2 and \u03c0, respectively. The frequency of the characteristic signal is reconstructed according to the transformation coefficient, that is, the frequency value is scaled on the time axis, so it does not affect the initial phase angle.\u03bb1, \u03bb2 and \u03bb3 in turn.Step 6 Input the characteristic signal with noise after the previous step into the array Duffing system for parameter estimation, as shown in Equation (15), and observe the phase trajectory of output state of the Duffing system. The amplitude of the driving signal of the array Duffing system is trimmed to determine the corresponding driving signal amplitude while each Duffing equation jumps from the chaotic state to the large-scale periodic state, and is marked as \u03bb1, \u03bb2 and \u03bb3 is substituted into Equation (17) to obtain the amplitude and phase of the MWD signal.Step 7 Finally, The MWD signal detection method based on the improved array Duffing systems was verified by laboratory conditions and field measurement data in this section. Compared with original measurement data, the FIR filter and standard Duffing systems were conducted to evaluate the performance of the proposed improved array Duffing systems comprehensively.Under the laboratory conditions, the hardware-in-the-loop simulation (HILS) experimental platform is used to simulate attitude measurement signal in noise under the strong vibration environment in the pit, so as to comprehensively evaluate the performance of the chaotic effect based on array Duffing system in frequency detection and parameter estimation for weak signal detection. Furthermore, the inclination calculation is used for the sensor signal after chaos detection. The feasibility and effectiveness of the proposed method are proved by comparing the calculation results with the set values.The main equipment of the HILS experimental platform are shown in (a)Testing for feasibilitya(t) = 0.02cos(10t \u2212 2\u03c0/3). On the other hand, a random interference signal n(t) with a variance of 0.04 is generated by the six-dimensional space vibration experimental platform, whose variation range of frequency is from 0 to 10 Hz. It includes the part that is overlapped with the frequency of the characteristic signal. Therefore, the input signal of the Duffing system is obtained by the superposition of the X-axis accelerometer signal and noise signal as follows.By setting the control panel of the clinometer calibration, the real-time attitude angle of the drilling tool is simulated, and the output signal of the built-in three-axis accelerometer sensor is recorded by the storage oscilloscope. The X-axis accelerometer signal is selected as an example to carry out the simulation experiment in this section. It is assumed that the output signal of the X-axis at a certain time is expressed as I(t) is about \u221223 dB, which is consistent with the SNR condition of dynamic MWD. At this time, the characteristic signal is completely annihilated in the noise signal, and it is difficult to identify the frequency and other parameter of the characteristic signal by spectrum analysis. Therefore, the proposed variable scale array Duffing system is used to detect the frequency value of the characteristic signal based on its chaotic effect. The frequency detection model based on the array Duffing system is built using MATLAB/Simulink R2014a version, and the characteristic signal and noise signal are used as the model input. The initial parameters and simulation parameters of the model are shown in According to the calculation, the SNR of input signal I(t) is not considered and the initial phase angle of the drive signal is set to 0, the output result of the array Duffing system is chaotic, as shown in I(t), the scale transformation coefficient R is introduced at the same time, and the calculation step and sampling frequency are changed according to R. The array Duffing system is simulated and verified with the adjusted simulation parameters, and the output state of the system is observed when the initial phase angle of the drive signal is set to 0, 120\u00b0 and \u2212120\u00b0. The partial simulation results are shown in According to the simulation results, when the input signal It can be seen from Further analysis of the detection results can lead to the following conclusions.(1)Since the angular frequency of the characteristic signal is different from the driving signal, the scaling method is used to reduce the angular frequency of the characteristic signal to 1 rad/s, which can effectively solve the problem of large frequency parameters of the MWD signal. Moreover, when the output state of the Duffing system is the large scale period, the corresponding value of scale transformation coefficient is the angular frequency of the characteristic signal.(2)On the premise that the scale transformation coefficient is correct, for the characteristic signal with amplitude greater than 0.02, the output state of a Duffing equation must be changed. For this example, the output state of the Duffing equation with an initial phase angle of \u2212120\u00b0 changes after the input signal. These results are consistent with the theoretical analysis.(3)It can be seen from the simulation results that the high-intensity noise signal does not affect the output state of the array Duffing system, but only makes the phase trajectory of the Duffing system more rough.(b)Testing for SNR thresholdI(t) is about \u221224 dB.In order to further explore the SNR threshold that the proposed method can handle when detecting frequency, the intensity of the noise signal is reset to increase its variance to 0.05, and all other parameters are the same as the previous section. At this time, the SNR of the input signal According to the detection results in the previous section, the scale transformation coefficient and the initial phase angle of the array Duffing system are directly set to 10 and \u2212120\u00b0, respectively.Then, the characteristic signal with different SNRs is input into the frequency detection model for simulation, and the change trend of the Duffing system output state with time is shown in y(x\u2019) is similar to x, which changes periodically with time, ranging from \u22121.3 to 1.3. If the two output responses x and y are plotted on a two-dimensional graph, it is obviously in the so-called large-scale periodic state.It can be seen from the simulation results that when the SNR is \u221223 dB, the system output response x changes periodically with time, ranging from about \u22121.6 to 1.6. In other words, the trend of change has obvious regularity. The other output response x still changes periodically with time in the first 2200 s, and the change trend is basically the same as that at \u221223 dB. However, the change trend showed partial disorder after 2200 s, with a range far less than \u00b11.6. The change trend of the other output y is similar, and disorder occurs after 2200 s. At this point, if the output response x and y are plotted on a two-dimensional graph, it is in the so-called chaotic state.When the SNR drops to \u221224 dB, the system output Therefore, when the amplitude of the characteristic signal is set as 0.02, the SNR threshold that the proposed method can identify its frequency is about \u221223 dB. In addition, through a large number of experimental verification, the following conclusions can be drawn: the lower the amplitude of the characteristic signal, the lower the SNR threshold that the proposed method can recognize.I(t) is still used as the input variables to input the array Duffing system for parameter estimation. The amplitude and phase value of the characteristic signal are estimated by the change in the system output state. The initial parameters and simulation parameters of the model are the same as those in the previous section. The implementation process of parameter estimation is described in Steps 5 to 7 in When the frequency value of the characteristic signal is determined, the signal I(t) is input into the three equations of the array Duffing system, and the critical values \u03bb1, \u03bb2 and \u03bb3 of the driving signal amplitude are determined by observing the output state of the Duffing system.During the simulation experiment, the signal I(t) is input into array Duffing equation I, and the SNR of the characteristic signal is the same as that of the previous section, which is still \u221223 dB. When its driving signal amplitude is adjusted to 0.7414, the system output state is shown in First of all, the signal \u03bb1 to be solved. Therefore, in order to determine the critical value, the intensity of noise signal n(t) is changed in the simulation experiment. By reducing the intensity of the noise signal, the SNR of the characteristic signal is increased from \u221223 dB to \u221221 dB. With other parameters unchanged, the value 0.7414 is still set as the critical value. At this time, the output state of the Duffing system is shown in However, the approximate large-scale periodic state shown in \u03bb2 is set to 0.7183 and the critical value \u03bb3 is set to 0.7447, the output state of array Duffing equation II and III is between chaos and large-scale period, with an obvious periodization trend, as shown in \u03bb2 is 0.7183 and the critical value \u03bb3 is 0.7447.The determination process of the critical value of Duffing equation II and Duffing equation III is the same as that of Duffing equation I. When the critical value In order to further discuss the influence of noise intensity on the output state of the array Duffing system, the change curves of output x and y of the array Duffing equation in the time domain are shown in x of the three array Duffing equations changes periodically with time in the first 2700 s of the simulation time, with a range of \u22121.6 to 1.6. However, after about 2700 s, the trend of change is transient chaos, and the range of change is far less than \u00b11.6. On the other hand, it can be seen from It can be seen from y with time under different SNR conditions is shown in x, but the change range is different. Therefore, the influence of noise on parameter estimation based on the chaotic effect is greater than that of frequency detection. Specifically, the SNR threshold of the detectable signal during frequency detection is \u221223 dB, which is lower than the SNR threshold of the detectable signal during parameter estimation, which is \u221221 dB.The curve of another output item \u22122. In this situation, the depth of the chaos state in the system is deeper. On the contrary, when the driving signal jumps to 0.73, the stability of the large-scale periodic state of the system is higher. In other words, the amplitude of the driving signal is relatively far from the critical amplitude of the system, and the system has strong anti-noise ability.According to the frequency detection model based on the array Duffing system, the amplitude of the driving signal is set to 0.72, and the adjustment accuracy is only 10\u22124. At this time, the degree of chaos or large-scale periodic state of the system is shallow. In other words, the amplitude of the driving signal is closer to the critical value, and then the anti-noise ability of the system is poor. Therefore, for the same noise intensity, irregular and disordered changes occur at the end of the simulation time. This is the main reason why the SNR threshold of parameter estimation is higher than that of frequency detection.However, for the array Duffing system with parameter estimation, when the output state changes, the solved driving signal amplitude is 0.7414, 0.7183 and 0.7447, respectively, and the adjustment accuracy is 10a(t).Therefore, considering the frequency detection and parameter estimation comprehensively, the SNR detection threshold based on the chaotic effect of the array Duffing system is \u221221 dB for the characteristic signal \u03bb1 = 0.7414, \u03bb2 = 0.7183 and \u03bb3 = 0.7447, respectively. By substituting \u03bb1 and \u03bb2 into Equation (17), the estimated value of the amplitude of the characteristic signal is 0.0188, and the first solutions of the initial phase angle are about \u00b1128\u00b0. Then, by substituting \u03bb3 into Equation (17), the second solutions of the initial phase angle of the characteristic signal are about \u221252\u00b0 or \u2212128\u00b0. By combining the solutions of the two sets of initial phase angles, the final estimated result of the initial phase angle is \u2212128\u00b0.Based on the analysis above, when the output state of the array Duffing system changes, the amplitudes of the three driving signals are Further statistics show the detailed error results for the parameters estimation, as shown in It can be seen from the statistical results in In this section, the sensor signal after chaos detection is solved for the attitude of the drill bit, and the result is compared with that of the original measurement signal, FIR filter and standard Duffing system to evaluate the performance of the proposed method comprehensively.First of all, the attitude parameters of the drilling tool while working vertically are simulated by the clinometer calibration, and the inclination is set to 5\u00b0. Then, the output signal of the three-axis accelerometer sensor is recorded by the storage oscilloscope and superimposed with the noise signal generated by the six-dimensional space vibration experimental platform. Thus, the attitude measurement signal under the background of strong noise is obtained. Finally, according to the chaos detection method described in According to the experimental results in The solution results of the inclination are shown in But on the contrary, the Duffing detection system is used to process the MWD signal, and the maximum inclination error is almost the same regardless of whether the SNR of the measured signal is \u221210 dB or \u221220 dB. The solution results are very close to the reference value during the whole simulation process. It can be seen that the change in noise intensity has no obvious influence on the solution result of inclination. This is mainly because the array Duffing system is not affected by noise intensity while detecting weak signal. According to its detection principle, increasing the noise intensity will make the output track of Duffing system rough, but will not affect the accuracy of frequency detection and parameter estimation. The simulation result indicates that the Duffing system indeed has good immunity to noise. In addition, the improved Duffing system proposed in this article results in smaller calculation errors, and both the maximum error and RMSE are better than the solution results of the standard Duffing system.The above simulation results and analysis demonstrate that the weak signal detection method based on the chaotic effect of the array Duffing system is indeed immune to noise signals and sensitive to weak characteristic signals. As long as the SNR does not exceed the detectable threshold of chaos detection, the proposed method can effectively improve the accuracy of the inclination solution.In order to further verify the performance of the proposed weak signal detection method for dynamic MWD signal, field-drilling data were used for testing and analysis. The experimental data came from the field-drilling process of a well in northern Shaanxi. The field-acquisition process and installation position of the triaxial accelerometer sensors are shown in The CS-3LAS sensor, which was developed by the Zhongxing measurement and control company, was chosen as the accelerometer in this test. The accelerometer is suitable for the specific requirements of downhole drilling. The specific parameters of the sensors are shown in In order to further evaluate the overall performance of the proposed method, the data after the improved Duffing system was implemented were used to obtain the real-time inclination of the drilling tool. There was no interference of vibration acceleration in the attitude measurement of the stopping of the drilling, which ensures the accuracy of the attitude parameters, such as inclination. Therefore, it was used as a reference value to verify the detective performance of the improved Duffing system. The solution results of the inclination obtained by the proposed algorithm are given in From the statistical results in The contributions of this paper are as follows.(1)An improved array Duffing system is developed by combining the technique of scale transformation to solve the problem that the frequency value of the MWD signal is too large, and it also make the detection window cover all phases to solve the influence of the initial phase angle of the MWD signal on frequency detection. Experimental results indicate that the proposed method can effectively detect the frequency value of the MWD signal in strong noise interference.(2)Three equations with different initial phases of the driving signal are combined to form another array Duffing system, which synchronously estimates the amplitude and phase of the MWD signal, and then recovers the complete MWD signal. Experimental results demonstrate that the proposed estimation method has high accuracy and the SNR can be as low as \u221221 dB when the amplitude of the MWD signal is greater than or equal to 0.02.(3)Simulation and field-drilling test results and comparison analysis validate that the proposed methodology can effectively curb the adverse impacts of multi-frequency and high-intensity vibration noise for weak signal detection, leading to higher solution accuracy for the inclination with dynamic MWD."}
+{"text": "Globally, it has been reported that different social determinants of health -structural, sociodemographic, economic, living conditions and cultural factors- may affect opportunities to adhere to prevention measures against SARS-CoV-2. The objective of this study was to explore the perceptions around barriers and facilitators for adherence to COVID-19 prevention measures among the adult population residing in three large cities in Chile from a social determinants of health perspective.Qualitative paradigm, multiple case-study design. Online semi-structured interviews were conducted with men and women aged 18 and over from different socioeconomic groups residing in three large cities. For participant recruitment and selection, purposive contacts were made based on community and social media networks, followed by snowball sampling. Saturation was reached at 61 participants, after which a thematic analysis was carried out with the support of AtlasTi software. The Ethics Committee of the Universidad del Desarrollo in Chile approved this study.The main perceived barriers to adherence to COVID-19 preventive measures are linked to structural social determinants of health such as income, occupation, gender, access to basic supplies, and housing. Perceived facilitators are the fear of contagion and the incorporation of measures into daily habits. The social communication of preventive measures by health authorities is perceived as punitive, affecting adherence once the fear of contagion decreased in the country. It is also perceived that the recommended preventive measures are disconnected from communities\u2019 cultural practices and people\u00b4s identity, as well as affected by gender inequities and socioeconomic conditions that stakeholders in the country do not sufficiently address.Study findings suggest that adherence to preventive measures, such as social distancing, mask use, and hand washing, could be promoted through their incorporation into the daily life habits of people and communities. These measures should consider the structural social determinants that generate multiple barriers to adherence, like poverty, occupational risks, and overcrowding. Socio-cultural dimensions of health and everyday risks need further understanding among the different communities in the country, allowing for differences in viewpoints and practices based on gender, age, place, and social identity. The health crisis generated by the SARS-CoV-2 pandemic has implicated significant challenges for societies, States, and health systems. Efforts to curb the pandemic have focused mainly on implementing a range of non-pharmacological prevention measures, such as hand washing, physical distancing, use of masks, quarantines, contact tracing and border control. However, to make the compliance of these measures sustainable over time and to assess their relevance for future health crises, several authors emphasise the need to identify barriers and facilitators for adherence to preventive and control measures. This is key to ensuring that public health interventions implemented in future health crises such as global pandemics come from robust evidence demonstrating their effectiveness in real-life settings .International evidence indicates that to analyze the compliance of COVID-19 preventive health measures, it is necessary to consider the Social Determinants of Health (SDH), defined as the situations and contexts of life and work that can influence health . At the Regarding educational factors, positive associations are reported between a higher level of education , 21\u201324, Most studies about the transmission and contagion prevention measures of SARS-CoV-2 have been focused in North America, Europe and Asia. The implications of the pandemic in South America have been less attended . In 2021Shortly after the World Health Organization (WHO) declared the COVID-19 epidemic a global pandemic in March 2020, the Chilean government decreed a State of Constitutional Exception for Catastrophe, which lasted for approximately one year. The aim was to have greater control over people\u2019s transit and enforce compliance with quarantine and other social distancing measures. Among the different measures adopted, all of them aimed at curbing the transmission of SARS-CoV-2, some addressed the provision of essential services to low-income families, others promoted labor benefits to enable working from home, social benefits, closure of educational establishments and their later reopening, to mention the most significant ones. Regarding the containment measures for COVID-19, the \u201cStep by Step Plan, we take care of ourselves\u201d was designed and implemented, establishing mobility restrictions according to the phases of the plan . These pOne significant measure adopted within the healthcare system was the establishment of the Chilean Health Network that coordinated public, private, and institutional establishments to increase the number of hospital beds to treat COVID-19 patients . In thisDespite the efforts made by the government, the measures did not impact the population in the same way. In Santiago, it has been reported that poorer boroughs have both higher incidence and higher mortality rates related to COVID-19 compared to more affluent boroughs . LikewisFrom the perspective of risk communication, it has been suggested that it is important not only to deliver the necessary and timely information so that citizens can make evidence-based decisions related to COVID-19 prevention, but also to understand their perceptions, beliefs and concerns about the pandemic and to incorporate this information into ongoing prevention strategies . In thisQualitative, descriptive, and exploratory study , carriedThe sample was constituted based on theoretical and feasibility criteria, considering areas of residence and socioeconomic group (SEG), based on household income. Theoretical sampling refers to the a priori establishment of categories of participant profiles according to the relevant dimensions of the SDH approach, which for this particular study were the following: age group, sex, socioeconomic level, and type of health insurance. The inclusion criteria for the sample were being over 18 years of age, residing in any of the three cities included the study, and speaking Spanish fluently (relevant for foreigner participants). The exclusion criteria were two: (i) that the potential participant had some physical or mental disability  that limited his/her ability to decide to participate or to answer the interview, and (ii) no access to the Internet to conduct the interview. Study participants were recruited according to their borough of residency from the three large cities included in the study. Several boroughs were selected in each city to obtain a wide representation of SEG in each urban area. Selecting several boroughs in each city is due to the fact that in Chile there is a strong socioeconomic segregation between boroughs of residence. For the same reason, and to approach the experiences of adherence to Covid-19 prevention measures of different socioeconomic profiles, we emphasized the inclusion of different boroughs in each city addressed by the study. The SEG\u2019s included in this study were defined from a descriptive borough-level analysis we conducted from the national representative CASEN survey in Chile (2017), ranking them according to their average income poverty index. Through this process, 6\u20138 boroughs from each of the cities of Santiago, Valpara\u00edso and Concepci\u00f3n were selected.For participant recruitment and selection, purposive contacts within each borough were made  based on community and social media networks established by the research team in previous projects, after which snowball sampling followed. Also, through connections with social organizations in each city, especially those linked to survival in the pandemic period, like community pots to feed the most vulnerable boroughs and municipal offices. People contacted through these strategies (and who met the inclusion criteria) were asked to participate by email or whatsapp. Those who expressed interest were then invited to receive a phone call from the project coordinators to learn more about the project and schedule an interview date. This process took place between March and April 2021. The initial sample size considered following the pre-established theoretical criteria was 60 people in all three selected cities . This initial sample size was established considering that qualitative research seeks depth of information rather than a statistical representation of its sample , so it wSemi-structured interviews were conducted . The infThe information gathered through the semi-structured interviews was transcribed verbatim into Word. Each interview was assigned a code to protect participant confidentiality. The interview codes were created based on the following general structure: the interview number, the initial letter of the city, the sex of the person (H-M for man and woman in Spanish), the GSE of belonging and finally, the age of the person interviewed. The information was analyzed between July and August 2021. An inductive thematic analysis strategy of the verbatim of interviews was used, a qualitative analysis method that allows identifying thematic patterns from the data collected , with thTo assess the rigor of the study , the following strategies were followed: (a) triangulation of information coming from different types of participants ; (b) constant audit trail, by maintaining a field diary to record the ideas and experiences of the research team. This information complemented semi-structured interviews and contributed to a more coherent and trustworthy data analysis and interpretation.The study was designed and implemented by the universal ethical principles of scientific research: respect for people, beneficence, non-maleficence, and justice. The study contemplated an online informed consent process, ensuring that participants received relevant information about the objectives and procedures of the study and certifying their voluntary participation before the informed consent was signed. The informed consent was signed remotely by filling out a secure and encrypted online form on the Alchemer virtual platform. If the person accepted to participate, he/she could save a PDF of the informed consent that was automatically downloaded from the system. All the participants signed the informed consent. An informative PDF document was also provided to participants, with a detailed explanation of the study and their participation, as well as the contact information of the principal investigator. The data analysis process was carried out with anonymized information, preventing individual people from being recognized. Additional methodological details and general results of this study are available and freely accessible in the recently published final report  and poli51% of the study participants were women and 49% were men. 31% belonged to the most affluent socioeconomic group (SEG) ABC1 to C1B; 36% to the C2-C3 group; and 33% to the poorest D-E group (roughly a third each). 34% of the participants lived in Santiago, 33% in Valpara\u00edso and 33% in Concepci\u00f3n. Table\u00a0Participants were able to describe their perceptions of main barriers and facilitators for following the COVID-19 preventive measures during the pandemic, such as social distancing, quarantines, use of masks, and hand washing; as well as control measures like COVID-19 testing and traceability. Perceptions around each of these COVID-19 preventive and control measures are presented below, according to selected dimensions of SDH as the theoretical perspective of analysis.\u201cThere are people that do not have the chance to buy through delivery, it doesn\u00b4t reach their homes. So they are not being thought of, the measures are being thought from a desk, so more territorial work is needed\u201d .\u201cIn the neighborhoods where the supply is, the market\u2026the main streets where they sell food and all that, that are like two or three, that is always full, always full. So, there is very little safeguard for that\u201d .Study participants identified several barriers to adhere to social distancing measures. They mentioned the impossibility of complying with the recommended social distance when using public transport, especially during peak hours. Similarly, they argued that it is more difficult for the poorest groups to comply with this measure than for the well-off population. They added the difficulty of social distancing when buying basic living groceries, considering that families in the lower socioeconomic groups had to leave their homes to buy in person in overcrowded settings  and without the supervision of the compliance with prevention measures:achoclonarse (staying close together). We greet each other with kisses, if you are talking with your friends, you are seated next to your friend, there is no distance, you are always touching each other. It\u2019s a culture where we don\u2019t have much distance\u201d .\u201cThe culture here is of Another barrier identified was one that the participants recognized as a typical cultural habit, in which every social gathering involved physical contact like kissing and hugging as a standard social practice:\u201cI believe that with people on the street, one does tend to maintain social distance, because you do not know about the other person or who they hang out with. But maybe with friends, one relaxes (\u2026) at least in those moments I didn\u2019t comply with, like being a meter away from my friend. I was careful with whom I got together with, like having more knowledge that the person wasn\u2019t going out everywhere\u201d .Young adults pointed out that, in general, they kept their distance from strangers in public spaces. However, they said they did not follow this measure when they met friends or acquaintances, since they were believed to be reliable in their self-care in relation to COVID-19:\u201cThe bad communication that the authority has had about this\u2026keep distances \u201cjust because\u201d and if we see you, you get a fine. There is no education work, which has ultimately caused people to distance themselves from the government and its measures. What you have to generate is precisely the opposite, empathy with the population, try to raise awareness, in a good way, knowing that if you don\u2019t comply, this will happen. Some people have the privilege of being informed, like me, but the lady who lives around the corner, that uses the mask underneath, because the elastic fell off and she hasn\u2019t bought another\u2026she doesn\u2019t know where to get that information, the lady barely watches television, and on television they show every morning that there are three thousand deaths and that\u2019s all, but there is no clear explanation of the measures\u201d .Along with the above, the interviewees stated that the way in which the health authorities implemented social distancing did not facilitate adherence. They argued that individuals and communities should be better educated about the reasons for social distancing beyond simply informing that everyone should follow it:According to the people interviewed, the main facilitating aspect for adherence to this measure was the fear of contagion and death, which led some to distancing themselves from crowds and public spaces.\u201cNow, thinking about the issue of gender, regarding the topic of care, now that I think about it\u2026as well as the role of care and worrying about one\u2019s own and others\u2019 health, women also carry a bigger burden, so perhaps there is also a concern on the part of women to follow the prevention measures\u201d .As a facilitating aspect for adherence to staying at home, participants positively highlighted the gradual implementation of the Step-by-Step Plan. This national strategy was applied in Chile in accordance with local realities at the borough level. However, they perceived quarantines in several boroughs were excessively long and limited individuals\u2019 compliance over time. Also, it was perceived that the quarantine experience meant having to readjust daily life, for instance having to accommodate several tasks such as working online, domestic work, childcare, and school support for children, caring for children with special needs, and caring for the elderly. Furthermore, they pointed out the significant burden that this strategy brought for women, who had to multiply their working hours to carry out all the care-related tasks. Hence, participants emphasized that one significant undesirable consequence of quarantines was that they generated an unequal negative impact of COVID-19 for women. This gender inequity against women was observed across all age groups, territories, and socioeconomic groups:\u201cThere is no way that the people who work on the street can sustain themselves without that, without earning a living. At least not for a long time and the COVID bonus isn\u2019t much money either. So, I am honestly stressed with the phase one (quarantine) situation, it is really hard emotionally speaking and rationally, I don\u2019t find it so logical\u201d .Another barrier to complying with quarantines identified by the participants was the economic one, linked to the need to work for subsistence even during recommended confinement. This made quarantines unrealistic, inefficient, and stressful. This meant, in many cases, a perceived loss of trust in the effectiveness of the measure at the community and population levels:\u201cYes, the curfew makes sense because it\u2019s legal within a state of constitutional exception. But what does a curfew at nine or ten imply. The difference is that businesses close earlier, the buses leave earlier\u2026what happens, the buses instead of getting crowded at nine, they get crowded at seven. That\u2019s what has happened in Valpara\u00edso, for example. People living in poverty feel that their life has been a punishment. Furthermore, you work eight hours and when you leave you want to go to the supermarket, and it\u2019s closed. You want to go to the neighborhoods store, closed\u201d .Linked to quarantines, the curfew was another measure questioned by the population interviewed. People expressed that it was an excessively long and meaningless measure in terms of curbing the pandemic. One participant expressed the following:\u201cIn the beginning the use of face masks was very confusing because there wasn\u2019t an established version agreed between the government and the College of Physicians, because they said only infected people had to wear it. It was because of some kind of shortage of masks, but also the pharmaceutical companies took advantage and raised too much the price of the masks, it was too expensive\u201d .Participants mentioned that the initial information regarding this measure provided by health authorities and healthcare teams was confusing, which made it challenging to establish face masks as a central prevention measure at the community level. Furthermore, participants mentioned the high prices of masks, which was also a barrier to its use and frequent replacement every few hours. Therefore, the use of non-disposable masks was preferred for their lower cost and durability, yet providing less or no protection against COVID-19:\u201cBecause you are supposed to use the masks, the one that is useful, it\u2019s useful twice a day, so not everybody will be able to buy that or have that sort of protection\u201d .The most vulnerable groups mentioned the high price of good quality masks -which they identify as surgical masks-, explaining that they could not afford them. In addition, since they are disposable and require permanent replacement, they involve greater expense than the use of reusable masks, which are perceived as less safe:\u201cThe other day, for example, I went to a gathering and it was like if COVID did not exist. No one, no one wore the mask. When I go to the market, I go with the mask on and everything, but I also see a lot of people, mainly the vendors, without a mask. The salespeople in the shops in my neighborhood work without a mask on\u201d .Misusing the mask in public spaces, such as when the nose and mouth are exposed, was also recognized as a barrier. It was pointed out that the information available from health authorities regarding the use of masks needed to fully address the risks that their mishandling entails. Participants, for instance, identified street markets, public transportation, and informal settlements as places where large crowds misused masks, or simply did not use them at all:\u201cIn the camp they were giving out lunches from a common pot and the truth is that the people who came to get lunch were not wearing a mask. Sure, since they live next door, they don\u2019t worry because they live close by\u201d .In addition, the interviewees recognized that many communities in the country did not wear the mask given the notion of collective life that prevails in their neighborhoods, where public space is conceived as an extension of private space and, therefore, not using face masks was normalized since it is not perceived as risky:Among other elements identified as barriers to adherence to the use of masks were diminishing fear of getting COVID-19 over time, the discomfort caused by wearing a mask, and the perception of safety when socializing with family or friends. As a facilitating factor for adherence, the interviewees mentioned how quickly people included masks in their daily lives, together with the fear of contagion, which encouraged its use.\u201cWell, there has always been (the hand washing), but I don\u2019t know if it\u2019s the Chilean people, but I think it\u2019s not an established culture, very few people washed their hands before sitting at the table before the pandemic\u201d .Most study participants stated that since the beginning of the pandemic, they incorporated frequent handwashing into their routines, a personal hygiene practice that, before the pandemic was not part of their daily habits:\u201cWe have replaced the constant hand washing with hand sanitizer. I feel that you are told to use hand sanitizer and they promote hand sanitizer, but they don\u2019t tell you that it\u2019s more important to wash your hands, because hand sanitizer does not replace water or soap\u201d .Among the main barriers to adherence to this hygiene routine was the gradual loss of fear of COVID-19 and the fact that people were replacing handwashing with soap and water with the use of hand sanitizer:\u201cWhen I was in Santiago I realized that the situation regarding the availability of hand sanitizer is very different, since it is available in public spaces, unlike here (Concepci\u00f3n) where there are no dispensers or anything\u201d .In terms of territorial inequalities regarding the availability of resources to prevent COVID-19, people from Valpara\u00edso and Concepci\u00f3n reported that while in Santiago there was access to hand sanitizer in most public spaces, it was not as frequent in other regions:\u201cIn the  region there are places without water, the Petorca area\u2026so telling them to wash your hands for thirty seconds\u2026 Sometimes in the videos they show how the water runs, it doesn\u2019t matter, that is, that is impossible for them\u201d .Another barrier, identified mainly in Valpara\u00edso, was the lack of access to drinking water in some more geographically remote boroughs of the city, which affected the population living in informal settlements:\u201cI believe that the facilitator, precisely, I think it is fear and not wanting to get infected, people are forced to wash their hands, use hand sanitizer, carry their own bottle, arrive home and wash their hands\u201d .Fear of contagion was identified as the main facilitator to compliance with handwashing recommendations. They mentioned the inclusion of this measure into their everyday routines and a greater awareness of the importance of handwashing to avoid infections, especially among older adults, since they remembered previous health crises in the country, such as cholera:\u201cWhen I arrived there, people were very sick, they couldn\u2019t breathe, and people like that were just going to do the PCR test. So when I arrived I walked into a room in the primary health care facility here in Lo Prado, and it was a closed room, there was only one window open, and they put me there and suddenly four people entered, and those people really were in bad shape\u201d .In relation to the PCR testing for COVID-19, most interviewees said they were in favor of taking the test, and several confirmed having taken it both for presenting symptoms and for having been in close contact with a confirmed case COVID-19. On the one hand, the participants reported that access to PCR testing was faster and more accessible through the private health system; however, they identified the high cost of the test in the private system as a barrier for those who did not have a work contract considering that at the beginning of the pandemic, some workplaces paid for the tests. On the other hand, they reported that in the public system, access to PCR was slow and testing centres were often overcrowded, making them a potential source of infection according to the interviewees:\u201cI think that with social media, on the internet it\u2019s easy to find information. I think it is difficult, mainly for people who do not have access to the internet, especially those people who do not have internet literacy\u201d .Another barrier of the public health sector about taking PCR tests identified by the participants was the requirement to present symptoms to be eligible for one, which left those suspected of being asymptomatic without testing. One positive aspect of the public health sector that participants highlighted was the reach-out activities carried out around the country providing free tests. Nevertheless, they warned about the need to improve access to the information regarding the places, days and times where they were carried out, since the main information channels identified by the participants were social networks, excluding those who do not use them:\u201cIt happened a lot that people didn\u2019t go to get the test, or they took it when they were very sick, or if it came out positive, they hid it from all the other people with whom they lived because they were the only source of income, or the others had to work and things like that\u201d .Participants also identified fear as a barrier to taking the test due to the risk of being discriminated against for having COVID-19 and not being able to work for daily wages. In this context, they observed that, given the need to work to make money to live, people with a positive PCR left their homes anyway:Many participants said they had taken the test, some more than once, both for having symptoms and for being in close contact with someone infected. In addition, experiences related to COVID-19 testing were positive.\u201cIt\u2019s complicated to be a close contact (to someone with COVID-19) today, it takes away your freedom, they force you to take a PCR test, they keep you \u201cmonitored\u201d, it isn\u2019t a \u201cbig brother\u201d either, but they keep you there. That is why many people wait until they feel really bad to have the test, because in the end, it takes away your freedom. So in general, people don\u2019t talk too much or they don\u2019t give out 100% of their close contacts\u201d .Although most participants considered traceability crucial for controlling COVID-19, they reckoned that it was difficult to implement given the need for coordination between the different state institutions involved. In turn, they detected a lack of rigor from the general population in communicating when they had been in close contact with a COVID-19 positive case. In this sense, some participants even mentioned that they were not sure if, when the time came, they would give the name of all the people with whom they had been in contact with, given the restriction of freedoms that this implied in providing this information to health authorities:As a facilitating factor for traceability, despite reservations towards freedom limitations, the interviewees expressed that this measure is essential to curb the pandemic, leading to adherence among some.In all, the interviewee\u00b4s experiences regarding the different health measures discussed in this study allow us to understand how the social determinants of health are involved, especially concerning barriers to adherence. Belonging to a lower socioeconomic group, facing job insecurity and needing to leave home to generate a daily income, being a woman, not having access to basic supplies such as drinking water, and overcrowding were the main factors affecting the participants\u2019 compliance with different preventive and control measures against COVID-19. In addition to this, structural socio-cultural elements like community practices of sharing personal items, kissing and hugging, as well as social interactions in public spaces in low-income boroughs  proved their relevance to how individuals understood and related to COVID-19 preventive and control measures. Furthermore, gender inequity surfaced due to some of these measures, as women faced the challenges of multitasking and work overload over time. Regarding the healthcare system, although there were efforts to increase the number of hospital beds through the Integrated Health Network, some other aspects were sidelined. As participants emphasize, the access to COVID-19 testing was unequal between the private and public healthcare systems. While in the private system access was fast, and results were ready in a short period of time, in the public system the process was more bureaucratic and slower, with multiple requirements to be eligible for a test such as having symptoms. This also made public health care facilities overcrowded, which people perceived as a barrier due to the fear of getting infected by being close to crowds.The main results of the study are summarized in Table\u00a0The present study explored perceived barriers and facilitators for adherence to the COVID-19 prevention and control measures promoted by the health authorities in Chile, among the population aged 18 and over, in three large cities in 2021, from the SDH perspective. According to the study findings, people men and women from different age groups, socioeconomic levels and with different health insurance reported multiple barriers to adhering to preventive COVID-19 measures. Likewise, in accordance with the literature, the findings show that the wealthiest socioeconomic groups are more likely to adhere to these measures, unlike the most vulnerable groups -especially those who do not have a formal job and must go out to the street for daily wages , 19, 30.The perception that the State has favored measures that are perceived as punitive, including prolonged quarantines and curfews, also affect adherence to protection measures. For this reason, the study participants expressed the need for a change in the authorities\u2019 strategies, in order to face the deep fatigue and emotional exhaustion generated by the pandemic. Furthermore, prevention and control strategies should consider the diverse realities faced by the Chilean population as well as the SDH that specifically affects specific groups, considering that not everyone faces equal conditions to deal with a health crisis of this magnitude. These gaps lead to inequities in the response to the pandemic, with vulnerable groups being the most exposed to contagion and death from COVID-19. Consistent with the above, the SDH approach that considers some of the dimensions identified in this study as minimum dimensions of approach, such as age, sex, living in an urban or rural area, socioeconomic level, and type of health insurance, is central to efforts to promote greater adherence to prevention and testing measures. According to the existing evidence at international level, other dimensions of the SDH that could be considered are ethnicity and migration , which wIt is important to highlight that only some facilitators for adherence to prevention measures by the study participants were identified. Fear of contagion and death is recognized as the primary motivation for compliance, which reveals the distance between the standards proposed by the health authorities and the self-care practices of the population as responses to health crises such as the COVID-19 pandemic.As a general recommendation, we suggest that strategies focus on facilitating the integration of prevention measures in the daily habits of individuals and communities through participatory actions involving populations and authorities from a rights-based, intercultural and gender approach that takes into consideration and respects the cultural and social diversity of the country. This is highly significant because of the uncertainty surrounding the medium-term evolution of the COVID-19 pandemic and the challenge that new health crises may raise at the local and global levels.This study presents both strengths and limitations. First, regarding strengths, it is the first study of this kind in Chile and using the qualitative paradigm allows for an in-depth exploration of the phenomenon from the voice of critical actors. Additionally, using the SDH approach adds analytical depth and unveils the structural dimensions of the inequities perceived. Second, with regards to limitations, carrying out the interviews during mandatory quarantines made it impossible to access the territories and the people interviewed in person, limiting a direct appreciation of the impact of the preventive measures of the pandemic in the contexts studied. Another limitation is that the sample was preferably made up of residents of urban areas, leaving rural sectors less represented in the study.The results of this study have direct implications for public health as they are relevant for the construction and adjustments of health policies and actions in the face of crises such as the COVID-19 pandemic. Furthermore, promoting future qualitative research on the topic would allow the collection and systematization of further valuable information about strategies to face the pandemic to bridge adherence gaps and ensure the acceptability of the measures taken for the diverse groups in our society."}
+{"text": "Proteins often undergo large conformational changes when binding small molecules, but atomic-level descriptions of such events have been elusive. Here, we report unguided molecular dynamics simulations of Abl kinase binding to the cancer drug imatinib. In the simulations, imatinib first selectively engages Abl kinase in its autoinhibitory conformation. Consistent with inferences drawn from previous experimental studies, imatinib then induces a large conformational change of the protein to reach a bound complex that closely resembles published crystal structures. Moreover, the simulations reveal a surprising local structural instability in the C-terminal lobe of Abl kinase during binding. The unstable region includes a number of residues that, when mutated, confer imatinib resistance by an unknown mechanism. Based on the simulations, NMR spectra, hydrogen-deuterium exchange measurements, and thermostability measurements and estimates, we suggest that these mutations confer imatinib resistance by exacerbating structural instability in the C-terminal lobe, rendering the imatinib-bound state energetically unfavorable. Atomic-level descriptions of protein\u2013small molecule binding processes that involve a large conformational change of the protein have been elusive. Here, the authors report unguided molecular dynamics simulations of such a process\u2014Abl kinase binding the cancer drug imatinib. By way of example, we previously reported unbiased MD simulations of the drug dasatinib binding to Src kinase2, a process that occurs without any substantial conformational changes of the protein3. A well-known example of a process that is known to require a substantial conformational change (involving 20 residues with displacements averaging >15\u2009\u212b) is the cancer drug imatinib binding to its target, Abelson tyrosine kinase (Abl). Previously, some intermediate conformations in this binding process were inferred from simulations starting from imatinib bound with Abl or the Abl homolog Src (using biasing forces to induce imatinib unbinding and binding)5, but simulations of the binding process without using biasing forces have not been reported.Structural studies indicate that many proteins undergo large conformational changes when they bind to small-molecule ligands, yet achieving a detailed understanding of these large changes remains a challenge. Crystal structures, for example, are in some cases available for the unbound and bound states of a protein, but they do not provide an atomic-level view of the entire pathway. Molecular dynamics (MD) simulations, on the other hand, can provide a continuous view of binding7. CML results from expression of the constitutively active tyrosine kinase Bcr-Abl, a fusion of the breakpoint cluster region (Bcr) protein and the kinase domain of Abl (Abl kinase). Imatinib binds and inhibits the kinase activity of Bcr-Abl, and imatinib therapy can give CML patients a normal life expectancy if provided at an early stage of the disease. Unfortunately, patients whose disease is more advanced at the time of diagnosis often develop mutations in Abl kinase that render them resistant to imatinib. These mutations are almost exclusively located in the kinase domain8, suggesting that imatinib binding is little affected by the other domains, and resistance predominantly arises from altered dynamics of the kinase domain. Achieving a more detailed understanding of Abl-imatinib binding could potentially suggest strategies to combat imatinib resistance or inform new directions in kinase-targeting drug discovery.In 2001, imatinib was approved for the treatment of chronic myeloid leukemia (CML), making it the first small-molecule kinase inhibitor to receive FDA approval as a cancer therapy9. The N-lobe consists of five \u03b2-strands and a helix (the \u03b1C helix), whereas the C-lobe consists of multiple helices and includes an activation loop (A-loop or AL) and a myristoyl-binding site that is important for Abl regulation  motif\u2014that plays a critical role in defining the active and autoinhibitory states of the kinase. In the catalytically active state, the DFG motif adopts a \u201cDFG-in\u201d conformation, which allows ATP binding, and the A-loop adopts an \u201copen\u201d conformation, which allows substrate binding; we will refer to this as the DFG-in/AL-open conformation. In the autoinhibitory conformation, the DFG motif is \u201cflipped\u201d to adopt a \u201cDFG-out\u201d conformation, while the A-loop remains open (the DFG-out/AL-open conformation). These conformational states and their relative populations have been systematically characterized using NMR11. Imatinib binding at the ATP-binding site causes Abl to undergo a significant conformational change in which the A-loop adopts a closed conformation that precludes substrate binding, while the DFG motif is locked in the DFG-out conformation  conformation; Supplementary Fig.\u00a013. This conformation is distinct from the active conformation to which dasatinib binds , with an ATP-binding site located between them14. The simulations are also in agreement with a nuclear magnetic resonance (NMR) study showing that the binding process involves a hybrid mechanism of conformational selection and induced fit; it begins with a conformational selection step (in which Abl kinase adopts a DFG-out conformation to which imatinib binds), followed by a large conformational rearrangement that is induced by the binding of imatinib (an induced-fit mechanism)15. In addition to providing confirmation of the binding scheme inferred from experiments, the simulations provide a detailed description of key states that were not elucidated by the NMR studies, and also of transitions in the binding process.Here, we present unbiased MD simulations of the process of Abl-imatinib binding. These simulations capture the conformational change from the DFG-in/AL-open conformation to the DFG-out/AL-closed conformation, and successfully reproduce the known Abl-imatinib complex , though to a lesser degree. Interestingly, there are a number of residues clustered in this destabilized region of the C-lobe that confer imatinib resistance when mutated in patientsFigure\u00a014, suggesting that the binding process does not begin from the DFG-out/AL-closed conformation of the native Abl-imatinib complex. Moreover, it is likely that the DFG-out/AL-closed conformation is rarely accessible by Abl in the absence of imatinib, as it has only been captured in crystal structures of Abl bound with imatinib or imatinib analogs. We thus started the imatinib binding simulations from either the DFG-in/AL-open (active) or DFG-out/AL-open (autoinhibitory) conformations of apo Abl kinase . Further, it has been shown that protonation promotes flipping to the DFG-out conformation3. In 13 of these 20 simulations  in which three imatinib molecules were arbitrarily placed in the vicinity of unphosphorylated Abl kinase in the apo DFG-in/AL-open (active) conformation  (see Methods). Asp381 of the DFG motif was protonated because its pKa is high due to the local electrostatic environment  the ATP-binding site and its extension between the N- and the C-lobes; (2) the interface with the SH2 domain used in Abl autoinhibition10; (3) the myristoyl-binding site  of Abl-imatinib binding starting from State 1, the autoinhibitory DFG-out/AL-open conformation of the apo kinase. These are termed G1 simulations because we concluded that they captured the step of imatinib binding following the DFG flip. Prior to initiating these simulations, three imatinib molecules were arbitrarily placed in the vicinity of Abl kinase. Asp381 was protonated in these simulations as well because its pKa in the DFG-out/AL-open conformation was estimated to be >11obe Fig.\u00a0. Intriguobe Fig.\u00a0, suggestobe Fig.\u00a0. We thus17, and each subsequent set of simulations was initiated from a snapshot generated from the preceding set. Based on the current understanding of imatinib binding, we believe these snapshots are representative of key intermediate states of binding  conformation of Abl kinase and entering the ATP-binding site, we next initiated a series of consecutive simulations , an imatinib molecule entered the ATP-binding site. Although imatinib remained in the ATP-binding site to the ends of the simulations  in a pose that runs approximately parallel to the native pose, this conformational state  We thus surmised that Asp381 deprotonation occurs as this residue becomes solvent exposed. Deprotonation likely destabilizes the autoinhibitory conformation and would help induce the transition to the imatinib-bound conformation in the simulations. In one of these simulations, the imatinib molecule adopted a more buried pose, with the hinge hydrogen bonds formed and the middle moiety of imatinib buried by the DFG motif  simulations from this pose. We deprotonated Asp381 in the G2 simulations, as Asp381 is solvent-exposed and likely deprotonated in the native imatinib-bound state18, but it is protonated when bound with Abl  from the native-like pose (State 4), with the N34 atom of the piperazine moiety protonated; this moiety was located adjacent to Glu286 and Glu282 of the \u03b1C helix, and Asp381 of the DFG motif. In 8 of the 10 simulations, both the conformation of Abl and the binding pose of imatinib remained largely stable. In the other two G4 simulations, the A-loop closed, with the \u03b29 strand at the N-terminal end of the A-loop being displaced by the piperazine moiety and rotating ~90\u00b0  and in terms of the A-loop conformation  starting from State 1  without imatinib present. As expected, the DFG-out/AL-open conformation remained stable in all these simulations. For comparison, we performed another 20 simulations, which were set up identically to the initial 25 except that Asp381 was deprotonated, and we found that a transition to the DFG-in/AL-open conformation occurred in two of these simulations. We did not observe closing of the A-loop in any of our simulations starting from the DFG-out/AL-open conformation, regardless of the protonation state of Asp381. Together, these simulations suggest that spontaneous closing of the A-loop is likely to be rare without imatinib binding, and that protonation of Asp 381 stabilizes the DFG-out conformation , produced an aggregate simulation of imatinib binding in which imatinib first selectively recognizes the autoinhibitory conformation of Abl and settles in the ATP-binding site. Imatinib then induces closing of the A-loop, suggesting that Abl-imatinib binding is a hybrid process of conformational selection and induced fit.20. This disruption of the interactions between the two lobes is reflected in our simulations of imatinib binding, in which we observed the A-loop become wedged between these lobes during A-loop closing. , we next conducted a more detailed analysis of the 20 G0 simulations starting from the active, DFG-in/AL-open apo Abl kinase conformation (State 0). We observed that these simulations can also arrive at State 1 and 2 Fig.\u00a0, and froIn two of the 13 G0 simulations in which a spontaneous DFG flip occurred Fig.\u00a0, an imat24. Previous MD simulations of the transition of EGFR kinase from its active conformation to its inactive one, however, have shown that the conformational change involves \u201ccracking\u201d of the C-lobe26. The term cracking was introduced to refer to local loss of native residue-residue interactions, compensated by increased local entropy, in intermediate states of a large conformational change in a protein. In C-lobe cracking, native residue-residue contacts of the C-lobe are substantially disrupted, giving rise to high local conformational fluctuations, which entropically compensate for the C-lobe instability25. Analysis of our Abl-imatinib binding simulations shows that similar C-lobe cracking occurred with imatinib binding after the imatinib arrived at the intermediate pose of binding (State 3), and especially just before and after closing of the A-loop , one binding event was observed in which dasatinib arrived at the native binding pose and remained there to the end of the simulation , of which five were apo and five were imatinib-bound. In all 10 simulations, the C-lobe initially exhibited a degree of conformational fluctuation similar to what we had previously observed in the imatinib-bound conformation of our binding simulations. In the imatinib-bound simulations, this behavior continued throughout the simulations and the A-loop remained closed, but in three of the five apo simulations the A-loop switched to the open conformation Fig.\u00a0, strongl27. When treated during the more aggressive stage of blast crisis, however, the disease in most patients ultimately becomes drug resistant29. The majority of relapsed patients harbor mutations within the Bcr-Abl kinase domain8. Many of these mutations are located at or near the ATP-binding site, of which the T315I  mutation is the most common, with 240 entries reported in the Catalogue of Somatic Mutations in Cancer 30. Structurally, it is not surprising that these mutations can disrupt imatinib binding, as they remove favorable interactions or introduce unfavorable interactions between the kinase and the drug. In addition to the mutations near the ATP-binding site, there is a group of distal imatinib-resistance mutations clustered in the C-lobe  begins to deviate from linearity with increasing concentrations of imatinib, indicating a rate-limiting step associated with conformational change in the binding. In the present study, we found that imatinib binding to Abl with resistance mutations at the cracking site has similar binding kinetics at low imatinib concentrations  Abl occurs in a two-step process, as indicated by stoped-flow measurements of the kinetics of binding31 to estimate the imatinib-resistance mutations\u2019 effects on the thermodynamic stability of Abl kinase and found that the mutations mostly reduce its stability; further, the C-lobe imatinib-resistance mutations tend to destabilize the kinase more than other resistance mutations . We used FoldX softwareons Fig.\u00a0.33. We also used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to probe the effects of the M472I mutation (as an example of a C-lobe mutant)  and the simulations.We also performed HDX-MS experiments on the A344V mutant  could impact the effects of imatinib or dasatinib in cellular proliferation assays. We found that two of the cracking site mutations (M351T and F486S) conferred substantial imatinib resistance in cellular assays 35, and analyzed the allosteric coupling between the GNF-2-binding site and the imatinib-binding site using N-body Information Theory analysis36 , so we cannot exclude the possibility that, for Abl kinase, this is a rare conformation that is stabilized by imatinib.) This pathway of imatinib binding thus appears to involve both conformational selection and induced fit\u2014a hybrid model that is consistent with findings from previous studies15\u2014and our simulations provide new structural information about the intermediate states of imatinib binding.In this study, we conducted simulations of imatinib and Abl kinase, starting from an unbound state and without using any biasing force, and arrived at a bound structure that is highly consistent with crystal structures of the Abl-imatinib complex. The simulations describe an atomic-level pathway of imatinib binding in which imatinib first selectively recognizes the autoinhibitory conformation of Abl kinase and enters the ATP-binding site. The protein then undergoes a large conformational change in which the A-loop closes, after which the system settles into the native Abl-imatinib structure. Given that this closing of the A-loop coincides with imatinib arriving at the native pose, and that the DFG-out/AL-closed conformation has not been observed in crystal structures of Abl without imatinib or an imatinib analog bound, we believe the conformational change represents an induced-fit step.  provides a proof of principle that this challenge can be overcome.5. These simulations started from the imatinib-bound structure, and used various biasing forces to promote unbinding and binding. In this study, we started our simulations from the unbound state, and arrived at the bound structure without using any biasing forces. The timescale of the simulated binding appears to be broadly consistent with Abl-imatinib binding kinetics. Our work differs from the previous studies in that we determined key intermediate states, or \u201cmilestones,\u201d in the binding process from which additional simulations were launched to advance the process\u2014an approach that resembles adaptive sampling methods39. Adaptive sampling methods, however, use an algorithmic approach to identify key intermediate states, whereas we used domain-specific knowledge, visual inspection of the simulations, and trial-and-error. At present, automated methods for identifying key intermediate states yield results of varying accuracy in the case of large conformational changes39, but further development of these methods could help obviate the need for laborious trial-and-error approaches in the future.Abl-imatinib binding has been previously studied using simulations4. We also observed that the closing of the A-loop that occurs with imatinib binding is accompanied by cracking in the C-lobe, and some degree of local structural instability remained in the imatinib-bound state. A number of mutations that are known to confer resistance to imatinib are clustered within a region of the C-lobe, but the mechanism by which these mutations confer resistance has been unknown. Based on our analyses, we propose from the linkage between the A-loop conformational change and C-lobe instability that these mutations confer imatinib resistance by reducing C-lobe stability and disfavoring the closed conformation of the A-loop that is associated with imatinib binding. The destabilizing effect may result from the disruption of the two conserved hydrophobic spines19 that connect the N-lobe and C-lobe, and anchor catalytically important regions of the kinase to a conserved and highly hydrophobic \u03b1F helix40. The imatinib-resistance mutations in the C-lobe are either part of or adjacent to this hydrophobic architecture, and our results suggest that these mutations affect imatinib binding by way of this network. This notion is consistent with the recent finding that many imatinib-resistance mutations increase Abl kinase dynamics and reduce the residence time of imatinib binding41. From a therapeutic perspective, our findings suggest that combining imatinib with small molecules that bind at the myristoyl-binding site (such as GNF-5) may counter the destabilizing effects of imatinib-resistance mutations at the C-lobe, and potentially overcome imatinib resistance caused by these mutations.In the binding path from our simulations, imatinib enters the binding site from the hinge side of Abl kinase rather than from the C-helix side, as previously suggested42. The DFG Asp residue was protonated and three imatinib molecules were placed at random locations not in contact with the kinase. The system was solvated in a cubic simulation box with periodic boundary conditions . Water molecules were represented explicitly and Na+ and Cl\u2013 ions were added to neutralize the system and achieve physiological levels of 150\u2009mM each. The systems were parametrized with the a99SB-disp force field43, imatinib was parametrized using the general Amber force field44, and the systems were equilibrated on Desmond45 using a mixed NVT/NPT schedule. MD simulations were performed on the special purpose machine Anton 246 in the NVT ensemble with T\u2009=\u2009310\u2009K using the Nos\u00e9\u2013Hoover thermostat48. The simulation time step was 2.5\u2009fs, and the r-RESPA integration method49 was used, with long-range electrostatics evaluated every three time steps. Electrostatic forces were calculated using the u-series method50 with a 1.37-nm cutoff for the electrostatic pairwise summation; a 0.9-nm cutoff was used for the van der Waals interactions.The systems for the G0 simulations were prepared based on a crystal structure of Abl kinase in which the DFG motif is in the active \u201cDFG-in\u201d state and the A-loop is in the open state 17. The DFG Asp residue was protonated and three imatinib molecules were placed at random locations. The systems were solvated in a cubic simulation box with periodic boundary conditions . The simulations were prepared, equilibrated, and run following the same methods used for the G0 simulations. Twenty independent unbiased simulations were conducted. In 11 of the 20 simulations, imatinib found the binding pocket ket Fig.\u00a0.We then randomly selected a G1 simulation snapshot in which imatinib was in the binding pocket and in the correct orientation. At this point, we deprotonated the DFG Asp, which was preventing imatinib from becoming fully embedded in the binding pocket. Five independent simulations (G2)\u00a0were extended from this pose with a similar system setup and protocol. Another five independent G3 simulations were extended from the last frame of one of the G2 simulations, with no changes to the system. .Once imatinib was fully embedded in the binding pocket, we observed that N34 of imatinib (the methylation site of the N-methylpiperazine group) was in a solvent-exposed environment, in close proximity to Glu286, that might allow for protonation of N34. We thus protonated N34 and extended five independent simulations (G4) from this point, following the same methods used for the G0 simulations.17. DFG residue Asp381 was protonated. The systems were solvated in a cubic simulation box with periodic boundary conditions . The simulations were prepared, equilibrated, and performed following the same methods used for the G0 simulations. 25 independent unbiased simulations were conducted, and the A-loop remained open throughout all of the simulations.The system was prepared based on the same crystal structure used for the G1 simulations 51. Dasatinib was removed from the binding pocket and three dasatinib molecules were placed at random locations. The system was solvated in a cubic simulation box with periodic boundary conditions . MD simulations were performed following the same methods used for the G0 simulations. Twenty independent 10-\u00b5s simulations were run.The system for the dasatinib-Abl binding simulations were prepared based on the crystal structure of Abl kinase bound with dasatinib (PDB ID: 2GQG)17. Four systems were prepared based on the imatinib-bound WT Abl structure (PDB ID: 1OPJ): (1) WT bound with imatinib; (2) and (3) M472I and F486S mutants bound with imatinib; and (4) apo WT in the DFG-out/AL-closed conformation. The systems were solvated in cubic simulation boxes with periodic boundary conditions . Five independent 20-\u00b5s simulations were run for each WT system and 10 independent 10-\u00b5s simulations were run for each mutant system. An additional five independent simulations of 10 \u00b5s each were performed with the system in which WT Abl kinase was bound with imatinib. We also simulated WT and the M472I and F486S mutants of Abl each simultaneously bound with imatinib and GNF-2 in the crystal pose from PDB ID: 3K5V35. The prepared systems contained ~70,000 atoms with a cubic simulation box of ~82\u2009\u00c5 per side, and 10 independent 10-\u00b5s simulations were run for each construct. The simulations were performed following the same methods used for the G0 simulations.The systems for the simulations starting from the DFG-out/AL-closed conformation were prepared based on the crystal structure of Abl kinase bound with imatinib, in which the DFG motif is in the autoinhibitory state and the A-loop is closed 42. Three systems were prepared: (1) WT, (2) M472I, and (3) F486S Abl kinase. The systems were solvated in cubic simulation boxes with periodic boundary conditions . Three independent 20-\u00b5s simulations were run for each system. The simulations were performed following the same methods used for the G0 simulations.The systems for the simulations starting from the DFG-in/AL-open conformation were prepared based on the crystal structure of the active conformation of Abl kinase (PDB ID: 2F4J)12 to construct the crystal lattice for our MD simulations. The protein was crystallized in an F 222 space group with \u03b1\u03b2\u03b3 angles of 90\u00b0, which allowed us to satisfy the 90\u00b0 angle requirements of the MD simulation box. We generated the crystal symmetry pairs using PyMOL Molecular Graphics System  to build the full unit cell (containing 32 copies of the kinase). The system was solvated in simulation boxes with dimensions a\u2009=\u2009112.89\u2009\u00c5, b\u2009=\u2009147.37\u2009\u00c5, and c\u2009=\u2009153.44\u2009\u00c5, and with periodic boundary conditions to match the unit cell dimensions with varying distance between the protein system and the first shell of the water molecules. Water molecules were represented explicitly and Na+ and Cl\u2013 ions were added to neutralize the system and achieve concentrations of 150\u2009mM each. The system, containing ~320,000 atoms, was equilibrated on Desmond45 using a mixed NVT/NPT schedule. Next, the system was simulated for 100\u2009ns on Anton 246 in the NPT ensemble with T\u2009=\u2009277\u2009K and a 2-fs step size . The cell dimensions were monitored throughout the simulations and a system that sustained the reported crystal dimensions was picked to progress to the 10-\u03bcs production run, which was performed in the NVT ensemble (still at 277\u2009K and with a 2-fs step size).We used the crystal structure of Abl kinase in complex with imatinib (PDB ID: 1IEP)52, and the images of protein structures were made using the PyMOL Molecular Graphics System .The simulation trajectories were visualized and analyzed using Visual Molecular Dynamics (VMD) software31. FoldX 4.0 was used to predict the effect of mutations on protein stability through the approximation of Gibbs free energy (\u0394G). FoldX uses an empirical force field that is trained directly on experimental data. Prior to calculating free energies, we used the FoldX 4.0 Repair PDB command to optimize side-chain orientations for WT Abl kinase (PDB ID: 2F4J)42 atom coordinates53. The resulting coordinates were then processed to calculate the change in folding free energy of the mutants relative to the WT protein (\u0394\u0394GFoldX\u2009=\u2009\u0394Gmut\u2009\u2013\u2009\u0394GWT). A negative \u0394\u0394GFoldX  suggests that the mutant protein is more stable than the WT protein.FoldX is a computational tool used to predict the effects of mutations on protein stability, folding, and protein dynamics54 was cloned into pET28 and modified to yield an N-terminal, TEV-cleavable His-tag55. The kinase domain mutations were introduced into the human c-Abl kinase domain by site-directed mutagenesis using the QuikChange II kit  and verified by DNA sequencing.The kinase domain of human c-Abl , with human Abl 1a residue numberingEscherichia coli BL21DE3 cells with full-length YopH phosphatase from Yersinia pseudotuberculosis56 and GroEL/Trigger factor, as previously described57. E. coli cells were grown in 2xYT media  at 37\u2009\u00b0C for 4\u2009h to a cell density of OD600\u2009=\u20090.6. The temperature was subsequently reduced to 16\u2009\u00b0C before cells were induced with IPTG  and grown overnight. Kinases were purified using Ni+2-affinity chromatography , anion-exchange chromatography , and size-exclusion chromatography . Proteins in 20\u2009mM Tris , 250\u2009mM NaCl, 5% glycerol, and 1\u2009mM DTT were concentrated, frozen in liquid nitrogen, and stored at \u221280\u2009\u00b0C. Protein purity was at least 95%, as determined by SDS-PAGE and Coomassie Blue staining.Kinases were coexpressed in Denaturation of WT Abl kinase and its mutants was monitored by changes in protein fluorescence using a Jobin Yvon Fluoromax-4 (Horiba) spectrofluorimeter exciting at 290\u2009nm  and scanning emission at 320\u2013400\u2009nm , with an integration time of 0.1\u2009s. Each protein was individually added to a final concentration of 1\u2009\u03bcM in 1\u2009mL of buffer containing 20\u2009mM Tris (pH 8.0), 250\u2009mM NaCl , 5% glycerol, and 1\u2009mM DTT. The samples were incubated for 1\u2009min at each 1\u2009\u00b0C from 10\u201380\u2009\u00b0C before the fluorescence at each temperature was recorded. Protein fluorescence as a function of temperature was fit to a Boltzmann sigmoidal equation to obtain V50, or melting temperature.58. In brief, the consumption of ATP is coupled by way of the pyruvate kinase/lactate dehydrogenase enzyme reactions to the oxidation of NADH, which can be monitored through the decrease in absorption at 340\u2009nm. Reactions contained 100\u2009mM Tris (pH 8.0), 10\u2009mM MgCl2  0.5\u2009mM ATP  1\u2009mM phosphoenolpyruvate, 0.21\u2009mg/mL NADH , 75\u2009U/mL pyruvate kinase, 105\u2009U/mL lactate dehydrogenase, and 0.5\u2009mM substrate peptide 59. Reactions were initiated through the addition of kinase at a final concentration of 33\u2009nM, and the decrease in absorbance was monitored over 10\u2009min at 30\u2009\u00b0C in a spectrophotometer (SpectraMax 340PC384 and BioTek Neo2). The background activities of the proteins at different drug concentrations were determined in experiments without the substrate peptide, and subtracted from the kinase activities with the substrate peptide. To measure the IC50 for imatinib, imatinib  was serially diluted threefold in 100% DMSO and added to the reactions to produce an 8-point dose-response curve ranging from 0\u20131 \u03bcM. The activity measured by the slope of NADH oxidation (mOD/min) was plotted verses the logarithm of the concentration of drug and fit to a sigmoidal equation with the hill slope set to 1.0.Kinase activity was monitored using a continuous spectrophotometric assay, as described previouslyBCR-ABL cDNA was sub-cloned into pEYK3.1, a retroviral vector, to construct the pEYKBA plasmid60. The pEYKBA plasmid was mutagenized by site-directed mutagenesis using the QuikChange II kit (Agilent) to create point mutations in the kinase domain of BCR-ABL. The pEYKBA plasmids were transfected into 293\u2009T cells  using FuGENE HD (Promega). At 24\u2009h after transfection retroviral supernatants were recovered as previously described61. The interleukin 3 (IL-3)-dependent hematopoietic pro-B cell line Ba/F362 was transduced with the retroviral supernatant supplemented with 8\u2009\u03bcg/mL of polybrene (Sigma) and IL-3-enriched media. Ba/F3 cells were deprived of IL-3 at 2\u2009days post-infection to select for cells that had taken up the pEYKBA retrovirus. The Ba/F3 cells were a kind gift from Mohammad Azam . They are commercially available at DSMZ .Full-length p210 4 Ba/F3 cells expressing mutant Bcr-Abl proteins were plated in each well of 96-well plates in RPMI medium containing 10% FBS and lacking IL-3 . Imatinib (Ark Pharm) was included in the media in increasing concentrations . Dasatinib  was included in the media in increasing concentrations . Viable cell numbers were assessed after 48\u2009h using the WST-1 reagent (Roche), according to manufacturer\u2019s specifications. Assays were performed in quadruplicate. A450 readings were plotted versus the logarithm of the concentration of drug and fit to a sigmoidal equation with the hill slope set to 1.0. The concentration of drug resulting in 50% maximal inhibition is reported as cellular IC50.0.5\u2009\u00d7\u20091023. A 50 pmol/\u00b5L stock solution of Abl kinase  was prepared in 20\u2009mM Tris (pH 8.0), 150\u2009mM NaCl, 1\u2009mM DTT, and 5% glycerol in H2O. Deuterium exchange was initiated by dilution of each protein with 15-fold D2O buffer (pD 8.0) at room temperature. At each deuterium exchange time point, from 10\u2009s to 4\u2009h, an aliquot from the exchange reaction was removed and labeling was quenched by adjusting the pH to 2.5 with an equal volume of quench buffer (150\u2009mM potassium phosphate in H2O). Quenched samples were immediately injected into the LC/MS system for mass analysis.Hydrogen exchange was performed as previously described63. Briefly, the samples were digested online using a Poroszyme immobilized pepsin cartridge  at 15\u2009\u00b0C for 30\u2009s, then injected into a custom Waters nanoACQUITY UPLC HDX ManagerTM and analyzed on a XEVO G2 mass spectrometer . The average amount of back-exchange using this experimental setup was 20%\u201330%, based on analysis of highly deuterated peptide standards. Deuterium levels were not corrected for back-exchange and are thus reported as relative64. All experiments were performed in duplicate. The error of measuring the mass of each peptide averaged \u00b10.12\u2009Da in this experimental setup. The HDX-MS data were processed using PLGS 3.0 and DynamX 3.0 . Additional experimental details are provided in Supplementary Table\u00a065. Detailed protocols for sample processing and data processing are provided in the Supplementary Methods.Each sample was analyzed as previously described55. Briefly, Abl kinase containing a TEV-cleavable N-terminal His6-tag was co-transformed into E. coli BL21 (DE3) cells with plasmids expressing YopH phosphatase, GroEL, and Trigger factor. 15N Abl kinase samples for NMR experiments were produced in 100% 2H2O  M9 minimal media containing 1\u2009g/L of 15NH4Cl  and D-glucose as the sole nitrogen and carbon sources, respectively. The cells were grown at 37\u2009\u00b0C to O.D600 ~0.6\u20130.8, cooled to 16\u2009\u00b0C, treated with 1\u2009mM IPTG to induce expression, and allowed to continue growing overnight.Abl kinase (residues 251\u2013533 of human c-Abl) was cloned, expressed, and purified in bacteria with additional co-expression of the GroEL chaperone and YopH phosphatase2H2O was added to the final NMR samples for a lock signal. ATP-competitive ligands (dasatinib and imatinib) and the myristoyl-binding-site ligand  were added in excess to saturate the binding sites and stabilize particular Abl kinase states. DMSO was maintained at 5% for NMR samples containing ligands solubilized in DMSO.Proteins were purified by Ni-NTA affinity chromatography followed by anion-exchange chromatography. The purified sample was concentrated by ultrafiltration and buffer exchanged into 50\u2009mM Na/K pH 6.5, 50\u2009mM NaCl, and 5\u2009mM DTT. 10% 1H)-15N heteronuclear NMR experiments were acquired at 25\u2009\u00b0C on a Bruker Avance III HD spectrometer operating at a 1H frequency of 850\u2009MHz and equipped with a Triple Resonance (TCI) 13C-enhanced 5-mm cryogenic probe. Abl inhibitor  1H-15N samples were prepared at concentrations of 200\u2013350\u2009\u00b5M. Standard Bruker pulse sequence for 1H-15N TROSY-HSQC, trosyf3gpphsi19.2, was used71. All spectra were acquired with the following parameters: Scans\u2009=\u200964, Size of FID (F2:1\u00a0H/F1:15\u2009N)\u2009=\u20092048/128, Spectral Width (F2:1\u00a0H/F1:15\u2009N)\u2009=\u200915.9791 ppm/ 36 ppm. Sample stability prior and post backbone dynamics were assessed by acquiring 1D 1H spectra. The backbone assignments were downloaded from BMRB and transferred onto the Abl kinase WT with imatinib spectrum for the further analysis72. The chemical-shift differences were analyzed with the software CCPNMR, and graphed with Prism GraphPad 8.0.2.All backbone . Protein fluorescence decay upon drug binding was fit to a single exponential equation to obtain the observed rate constant (kobs)3. The kobs was plotted against drug concentration, and the association and dissociation rates were calculated based on the relationship: kobs\u2009=\u2009kon[drug] + koff. A range of imatinib concentrations was used from 6\u2009\u03bcM to 200\u2009\u03bcM. The assay was limited to a maximal imatinib concentration of 200\u2009\u03bcM due to imatinib solubility.We monitored inhibitor binding of imatinib and dasatinib by changes in protein fluorescence using a spectrofluorimeteric ligand-binding assay, as described previouslyFurther information on research design is available in the\u00a0Supplementary Information fileDescription to Additional Supplementary InformationSupplementary Movie 1Supplementary Movie 2Reporting summary"}
+{"text": "It was recently reported  that theAbelson tyrosine kinase (Abl) ATP-site inhibitor imatinib also binds toAbl's myristoyl binding pocket, which is the target of allosteric Ablinhibitors. This was based on a crystal structure of a truncated Abl kinasedomain construct in complex with imatinib bound to the allosteric site aswell as further isothermal titration calorimetry (ITC), NMR, and kinase activity data. Although imatinib's affinity for the allosteric site is significantly weaker (10\u202f However,the oncogenic Philadelphia chromosomal translocation leads to the expressionof the highly active fusion protein Bcr-Abl and subsequently to chronicmyeloid leukemia (CML) . The ATP-site inhibitors imatinib (Gleevec), nilotinib (Tasigna), and dasatinib (Sprycel) constitute the front-line therapy against CML. The recently FDAUnder healthy conditions, Abl regulation is achieved by a set ofinteractions within its regulatory core consisting sequentially of the SH3 domain,SH2 domain, and kinase domain (KD), which is preceded by anSurprisingly, we observed in P1 that binding of the ATP-site inhibitor imatinib disassembles thecore into an arrangement where SH3 and SH2 domains move withhigh-amplitude nanosecond motions relative to the KD . Theadditional binding of the allosteric inhibitor GNF-5 to the myristoylbinding pocket then led to a reassembly of Abl's core . Theseconclusions were derived from multiple pieces of evidence (see below) and based onbackbone resonance assignments obtained earlier on the KD alone as wellas further triple-resonancebackbone assignment experiments on the regulatory core in P1. Theassignment experiments showed that the ATP-site inhibitors imatinib,nilotinib, and dasatinib induce the strongest chemical shift changes at theATP site , whereas GNF-5 induced the strongest chemical shift changes in the vicinity of themyristoyl pocket in agreement with the expected binding modes of allinhibitors.Multiple evidences for the disassembly of the Abl core by inhibitor binding to the ATP site were obtained from In publication P2 weextended these findings in a systematic way to 14 ATP-site ligands,comprising all FDA-approved Bcr-Abl inhibiting drugs. Compelling evidence byIn a recent publication , Kalodimos and coworkers described a crystal structure of a truncated Abelson kinase domain  and purified as described previously in P2.The Abl regulatory core fragment Abl2.2Similar to our previous studies, the isotope-labeled AblAll NMR experiments were performed at 303\u202fK on a Bruker Ascend 600\u202fMHzspectrometer equipped with a TCI triple-resonance cryoprobe.Theoretical Abl imatinib binding curves were calculated by solving therespective mass action law equations for a four-state model  using Mathematica . These equations are the following:33.1Kalodimos and colleagues argue that we had not stated the imatinibconcentrations in our work. While this is true for publication P1, where we had only stated that AblP1 clearly showed thatnot only imatinib but also nilotinib disassembles the regulatory core. Theevidence was given by In publication P2 weobserved the inhibitor-induced disassembly in an identical manner for allinvestigated type-II inhibitors, i.e., imatinib, nilotinib, ponatinib,rebastinib, and bafetinib. Of these, imatinib, nilotinib, and ponatinib weretested in P3 for allosteric pocket binding, with imatinib having micromolaraffinity, ponatinib much weaker affinity than imatinib, and nilotinib no observable binding. The observed core disassembly by all investigatedtype-II inhibitors in P1 and P2 contradicts the hypothesis of P3 that thedisassembly is caused by binding to the allosteric pocket.It is doubtful whether the imatinib In P3, Kalodimos and colleagues argue that our explanation of animatinib-induced push via the closed A-loop towards the SH3 domain, leading to core disassembly, is not valid, since they observed a closed A-loop in the assembled state in recent work on an apo SH3-SH2-KD fragment, which theycompared to obtained solution NMR structures of the KD alone. However, the apo conformation of the A-loop in SH3-SH2-KD is irrelevant when imatinib fills the ATP pocket and exerts additional forces. Moreover, the A-loop in the apo form is certainly not in a singleconformation but in dynamical exchange. We have reported a dynamicalequilibrium of the A-loop already in Vajpai et al.\u00a0(2008b) even in the presence of inhibitors as well as in P1  where many resonances of the A-loop were broadened beyond detection due to conformational exchange for both the apo form and the GNF-5 complex. In contrast, binding of imatinib rigidifies the A-loop to the \u201cclosed\u201d conformation of the crystal structure. It also needs to be indicated that the solution structures of the Abl3.2To further characterize the binding of imatinib to Abl, we have carried outa titration of imatinib to the Abl regulatory core construct  to address this criticism in aneditorial correspondence between us and the authors of P3 remained unheeded.With the exception of the sequence used for the crystal structure, it is often not specified which Abl constructs were used for the measurements. For example, there is no indication of the amino acid sequence of the Abl KD, AblIn particular, it would have been very important to compare directly a construct harboring the entire The conclusions of P3 heavily depend on the ITC data. However, syringe concentrations of imatinib, other ATP-site blocking inhibitors, myristic peptide, GNF-5, and the Abl constructs themselves are not properly indicated for the crucial ITC titrations shown in their Figs.\u00a01, S1, S2, S3, S5, and S8. Thus, it is impossible for others to reproduce these data.Their Fig.\u00a04B shows a small region of a methyl spectrum of the Abl regulatory core construct , which is presumably the apo form according to theconcentration arrow at the top of the figure, does not agree with the bluespectrum on the right, which is annotated as apo Abl by the color legend atthe top. Furthermore, the color code for the orange spectra shown in Fig.\u00a05B, C is not explained. Full spectra for these titrations should also have been shown in the Supplementary Information.The authors of P3 argue that they developed a new method to differentiate Abl allosteric inhibitors from activators by monitoring resonances of JMB without indicating the erratum as the so far (8\u00a0May 2022) only reaction to the present note).In addition, P3 contains many typos and other inconsistencies, which make the findings extremely hard to follow. Such problems are, for example, a wrong labeling of amide Publication P3 contains a number of further problematic points that weakenthe evidence of the described experiments and make their results hard toreproduce. We are surprised that these problems escaped the refereeing andeditorial process. A request to the 4In summary, previous data and the new evidence presented here unequivocallyshow that binding of imatinib to Abl's myristoyl pocket does not cause theobserved disassembly of its regulatory core. Rather, the disassembly must becaused by imatinib binding to the ATP site and forces transmitted from thereto the interface between the KD and the SH3-SH2 domains as explained in P2.Careful documentation of experimental procedures and taking into account allobservations pertinent to a system as complicated as Abl is the only wayforward to an in-depth understanding of its function and towards the goal ofrational intervention.10.5194/mr-3-91-2022-supplementThe supplement related to this article is available online at:\u00a0https://doi.org/10.5194/mr-3-91-2022-supplement."}
+{"text": "We examined whether the risk of incident atrial fibrillation (AF) in a large, biracial, prospective cohort is lower in participants who adhere to heart-healthy dietary patterns and higher in participants who adhere to less heart-healthy diets.Between 2003 and 2007, the REasons for Geographic and Racial Differences in Stroke (REGARDS) cohort study enrolled 30,239 Black and White Americans aged 45\u00a0years or older. Dietary patterns  and the Mediterranean diet score (MDS) were derived based on food frequency questionnaire data. The primary outcome was incident AF at the follow-up visit 2013\u20132016, defined by either electrocardiogram or self-reported medical history of a physician diagnosis.This study included 8977 participants  free of AF at baseline who completed the follow-up exam an average of 9.4\u00a0years later. A total of 782 incident AF cases were detected. In multivariable logistic regression analyses, neither the MDS score (odds ratio (OR) per SD increment\u2009=\u20091.03; 95% confidence interval (CI) 0.95\u20131.11) or the plant-based dietary pattern  were associated with AF risk. Additionally, an increased AF risk was not associated with any of the less-healthy dietary patterns.While specific dietary patterns have been associated with AF risk factors, our findings fail to show an association between diet patterns and AF development.The online version contains supplementary material available at 10.1007/s00394-023-03159-z. Atrial fibrillation (AF) is the most frequently encountered cardiac arrhythmia in clinical practice and more often seen in older populations. The overall prevalence of AF in the United States is 1% to 2% . The risAdherence to the Mediterranean or Dietary Approach to Stop Hypertension (DASH) diets are advocated by major medical societies due to their established effectiveness in reducing incident cardiovascular disease (CVD) \u201310. The Similarly, prior study has also not evaluated whether the less-healthy Western dietary patterns that do not incorporate elements of a heart-healthy diet may place an individual at an increased risk for AF. Prior research in the REasons for Geographic And Racial Differences in Stroke (REGARDS) study demonstrated that a Southern dietary pattern, one that is high in added fats, fried food, eggs and egg dishes, organ meats, processed meats, and sugar-sweetened beverages, was associated with an increased risk of coronary heart disease and sudden cardiac death , 17. We Details of the methods of the REGARDS study have been published . BrieflyDiet was assessed at baseline with the Block 98 food frequency questionnaire (FFQ), a validated semi-quantitative FFQ that assessed usual dietary intake of 110 food items  , 20. ForSplit sample replication was used to (1) derive the dietary patterns using exploratory factor analysis, and (2) test the patterns using confirmatory factor analysis , 21. PatTwo patterns were considered to have health promoting properties based on their food composition and the general nutritional epidemiologic literature. Specifically, both the alcohol/salads and plant-based patterns had high factor loadings for foods such as vegetables, leafy greens, nuts, seeds, and fish, which are beneficial for health. We also identified three patterns, convenience, sweets, and southern, as unhealthy because they had higher factor loadings for foods that are known to be associated with unfavorable health and disease outcomes .The Mediterranean diet score (MDS) was derived according to published methods used in REGARDS based on the method of Trichopoulou and colleagues . In brieAF was identified at baseline and a subsequent follow-up visit approximately 10\u00a0years later by (1) a visit ECG and (2) self-reported history of a physician diagnosis during the CATI survey. The ECGs were read and coded at a central reading center  by analysts who were blinded to other REGARDS data. Self-reported AF was defined as an affirmative response to the following question: \u201cHas a physician or a health professional ever told you that you had atrial fibrillation?\u201d This question was as reliable a predictor of incident stroke events as AF detected by ECG .Participant characteristics at baseline were used as covariates. Age, sex, race, household income, education, and smoking status were self-reported. Body mass index (BMI) and waist circumference were measured at the baseline examination. Physically active was defined as\u2009\u2265\u20094\u00a0days of exercise (enough to work up a sweat) per week. Hypertension was defined as systolic blood pressure\u2009\u2265\u2009130\u00a0mm Hg, diastolic blood pressure\u2009\u2265\u200980\u00a0mm Hg, or self-reported current use of anti-hypertensive therapy. Dyslipidemia was defined as total cholesterol\u2009\u2265\u2009240\u00a0mg/dL, low-density lipoprotein cholesterol\u2009\u2265\u2009160\u00a0mg/dL, high-density lipoprotein cholesterol\u2009\u2264\u200940\u00a0mg/dL, or self-reported current use of lipid-lowering therapy. Diabetes mellitus was defined as fasting glucose\u2009\u2265\u2009126\u00a0mg/ dL, non-fasting glucose\u2009\u2265\u2009200\u00a0mg/dL, or self- reported current use of anti-diabetic medications. CRP measurement used a high-sensitivity particle-enhanced immunonephelometric assay on the BNIII nephelometer  with an interassay coefficient of variation of 2\u20136%. CVD included the presence of coronary heart disease  or prior stroke which was ascertained by participant\u2019s self-report.Descriptive statistics for demographic, socioeconomic, lifestyle, anthropometric, and medical history variables at the baseline assessment according to quartiles of consumption of each dietary pattern and MDS categories were calculated using the chi-square test (for proportions) and analysis of variance (for continuous variables).p\u2009<\u20090.05. SAS version 9.4  was used for all analyses.Logistic regression was used to calculate the odds ratio (OR) [95% confidence interval (CI)] for prevalent (baseline visit) and incident (follow-up visit) AF for each of the five dietary patterns as well as the MDS per standard deviation (SD) increment in adherence. Models were built as follows: model 1: age, sex, race, education, household income, and region; model 2: model 1 plus total energy intake, lifestyle factors , CVD risk factors , and CRP. Statistical significance for all comparisons including interactions was defined as Due to inherent limitations with the MDS, namely the designation of any dairy intake as adverse, and the availability of other scoring systems, we repeated the analyses using the Mediterranean-DASH Diet Intervention for Neurodegenerative Delay (MIND) diet score. Among the MIND diet components are 10 brain healthy food groups  and 5 unhealthy food groups . This diet score has been associated with slower cognitive decline and reduced cardiovascular disease \u201326.There were 18,967 REGARDS participants with complete covariate data and dietary assessment at baseline and 8,977 of these were included in the prospective analyses because they were without prevalent AF and completed a follow-up examination. Baseline characteristics stratified by MDS category and dietary pattern quartiles are shown in Table Risk of incident AF according to the various dietary patterns and MDS are reported in Fig.\u00a0Measures of adiposity and hypertension were most responsible for attenuation of association between Southern pattern and incident AF . The convenience and alcohol and salads dietary patterns were not associated with incident AF risk. In cross-sectional analyses, no associations were observed for any of the dietary patterns or MDS with prevalent AF Below is the link to the electronic supplementary material."}
+{"text": "Dual targeting to immune checkpoints has achieved a better therapeutic efficacy than single targeting due to synergistic extrication of tumour immunity. However, most dual targeting strategies are usually antibody dependent which facing drawbacks of antibodies, such as poor solid tumour penetration and unsatisfied affinity. To meet the challenges, we engineered a cell membrane displaying a fusion protein composed of SIRP\u03b1 and PD\u20101 variants, the high\u2010affinity consensus (HAC) of wild\u2010type molecules, and with which prepared nanovesicles (NVs). Through disabling both SIRP\u03b1/CD47 and PD\u20101/PD\u2010L1 signalling, HAC NVs significantly preserved the phagocytosis and antitumour effect of macrophages and T cells, respectively. In vivo study revealed that HAC NVs had better tumour penetration than monoclonal antibodies and higher binding affinity to CD47 and PD\u2010L1 on tumour cells compared with the NVs expressing wild\u2010type fusion protein. Exhilaratingly, dual\u2010blockade of CD47 and PD\u2010L1 with HAC NVs exhibited excellent therapeutic efficacy and biosafety. This study provided a novel biomaterial against tumoural immune escape and more importantly an attractive biomimetic technology of protein delivery for multi\u2010targeting therapies. RIPA lysis buffer, penicillin\u2010streptomycin were obtained from Solarbio . Anti\u2010Flag\u2010agarose beads and Flag peptide were obtained from Bimake . UltraFection 3.0, Human IFN\u2010\u03b3 Elisa Kit, Human IL\u20106 Elisa Kit and Human IL\u20108 Elisa Kit were purchased from 4A BIOTECH . Recombinant Human IFN\u2010gamma was purchased from PeproTech . PC5.5\u2010anti\u2010Human\u2010CD45, PE\u2010anti\u2010Human CD4 and FITC\u2010anti\u2010Human CD8 antibodies were purchased from BD Pharmingen . Eastep Super\u00ae Total RNA Extraction Kit was purchased by Promega . Annexin V, 633 Apoptosis Detection Kit and Calcein\u2010AM/PI Double Staining Kit was purchased from Dojindo Laboratories . One\u2010step TUNEL Apoptosis Assay Kit and Hoechst 33342 were obtained from Beyotime . Atezolizumab was obtained from Selleck , and Magrolimab was bought from AtaGenix . Anti\u2010Perforin, anti\u2010Granzyme B, anti\u2010Mannose Receptor, anti\u2010CD68, anti\u2010CD81 and anti\u2010calnexin antibodies were purchased from Abcam . Anti\u2010CD9, anti\u2010CD63, anti\u2010PD\u2010L1, anti\u2010CD47 and anti\u2010\u03b2\u2010actin antibodies were purchased from Proteintech . Anti\u2010flag was obtained from Cell Signaling Technology . EdU Imaging Kits (Cy3) was purchased from APExBIO . PrimeScript\u2122 RT reagent kit with gDNA Eraser and TB Green Premix Ex Taq\u2122 were obtained from Takara (Japan).2.All cells were preserved by Cancer Hospital Chinese Academy of Medical Sciences . HEK293 cells, MDA\u2010MB\u2010231 cells and A549 cells were cultivated in Dulbecco's modified eagle medium , Leibovitz's L\u201015 medium  and Ham's F\u201012K medium  containing 10% fetal bovine serum (FBS) and 1% penicillin\u2010streptomycin, respectively. The cells were incubated in a 37\u00b0C\u2010incubator containing 5% COFemale SPF BALB/c\u2010nude mice  were purchased from Beijing Huafukang Bioscience Co. Ltd. All animals were bred in a specific pathogen\u2010free environment. All animal procedures were in accordance with all relevant guidelines of the Institutional Animal Care and Use Committee of Cancer Hospital of Chinese Academy of Medical Sciences (No. NCC2021A278).2.2We transfected HEK293 cells with an expression plasmid to construct engineered cells. The cells were collected and washed with PBS via centrifugation, and the supernatant was discarded. The cell pellet was gently resuspended in an appropriate amount of PBS containing PMSF . Ultrasonication was performed for 1\u00a0min  using an ultrasonic cell pulveriser . The supernatant was collected for further centrifugation at 20,000\u00a0g for 30\u00a0min at 4\u00b0C to remove the nucleus and intracellular membrane, and then extruded and filtered with a 0.22\u00a0\u03bcm filter to acquire cell\u2010membrane nanovesicles. The resulting nanovesicles were quantified using a Pierce BCA Protein Assay Kit and dissolved in PBS and stored at 4\u00b0C for further study.2.3n\u00a0=\u00a03) at 25\u00b0C and the results were counted as mean\u00a0\u00b1\u00a0SD. All samples were respectively dropped on copper grids and stained with 1% uranyl acetate. The morphology of vesicles was examined using a transmission electron microscope (TEM) .The size distributions of Blank, Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs were detected using a Nanoparticle Tracking Analysis . We collected potential of vesicles by dynamic light scattering (DLS) . All measurements were performed three times  assay was employed to examine the interaction of tumour cells with the fusion WT NVs. In brief, 100\u00a0\u03bcg/mL Flag\u2010fusion WT or Flag\u2010fusion HAC NVs were added and incubated with tumour cells (10\u00a0cm dish) for 12\u00a0h. After that the cells were washed three times with PBS to remove the unbound NVs. And then, the cells were lysed in 1\u00a0mL RIPA lysis buffer containing phosphatase inhibitor cocktail. The cell lysis was then centrifuged at 15,000\u00a0\u00d7\u00a02.5Equal amounts (20\u201350\u00a0\u03bcg) of proteins were size\u2010fractionated by 7.5%\u221210% (w/v) SDS/PAGE. The antibodies used were anti\u2010CD9, anti\u2010CD81, anti\u2010TSG101, anti\u2010calnexin, anti\u2010PD\u2010L1, anti\u2010CD47, anti\u2010\u03b2\u2010actin and anti\u2010Flag.2.66 MDA\u2010MB\u2010231 or A549 cells were harvested and incubated with 200\u00a0\u03bcg/mL Blank, Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs or 20\u00a0\u03bcg/mL Atezolizumab and Magrolimab for 30\u00a0min, respectively. After that the cells were washed three times with PBS to remove the unbound NVs. Then, adding fluorescent\u2010labelled specific antibody , the incubation was continued for 30\u00a0min. All samples were analysed by Flow Cytometry .For antibody competition assay, 1\u00a0\u00d7\u00a010+/CD45+ T and CD4+/CD45+ T cells were evaluated using flow cytometry. Tumour tissues were efficiently dissociated to collect single\u2010cell suspensions with a GentleMACS Dissociator  according to the manufacturer's instructions. Briefly, we firstly removed fibrous and necrotic areas of tumour samples and cut them into small pieces. Subsequently, the tumour pieces were transferred into gentleMACS C Tubes containing the enzyme mixture, respectively. The C Tubes were loaded onto the gentleMACS Dissociator carefully, then ran the selected program. Using a 70\u00a0\u03bcm mesh cell strainer to collect filtered cell suspensions into a 50\u00a0mL centrifuge tube. The cell suspensions were washed with RPMI 1640 medium and PBS at 300\u00a0\u00d7\u00a0g for 7\u00a0min, respectively. The following immune cells were stained with PC5.5\u2010anti\u2010Human\u2010CD45, PE\u2010anti\u2010Human CD4 and FITC\u2010anti\u2010Human CD8 antibodies for 20\u00a0min. All samples were analysed by Flow Cytometry .For tumour infiltrated T cells detection, the percentage of CD82.73/well) were seeded into a 96\u2010well plate 24\u2009h before experimentation. Cells were treated with 100\u00a0\u03bcg Blank, Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs for 12\u00a0h, 24\u00a0h, 48\u2009h, respectively. After treatment, CCK\u20108 was added to the 96\u2010well plate and incubated at 37\u00b0C for 1\u2009h. The absorbance of each sample was read at 450\u2009nm.Equal numbers of MDA\u2010MB\u2010231 cells  was used according to the manufacturer's instructions. Hoechst 33342 solution was used to counterstain cell nuclei. Proliferative cells were determined under the confocal microscope .2.95 MDA\u2010MB\u2010231 cells were seeded in a 24\u2010well plate. Afterward, 100\u00a0\u03bcg Blank, Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs were added to the plate. After incubation for 24\u00a0h, the cells were washed gently with PBS three times. After that, the cells were stained with Calcein\u2010AM/PI for 20\u00a0min at dark. Using a fluorescence microscope to investigate the relative viability of MDA\u2010MB\u2010231 cells after different treatments .1\u00a0\u00d7\u00a0102.105 MDA\u2010MB\u2010231 cells were seeded in 6\u2010well plates and incubated for 24\u00a0h to attach. Then the cells were incubated with 100\u00a0\u03bcg Blank, Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs. After 48\u00a0h, the cells were collected and stained following the manufacturer's instructions. Finally, apoptosis in different treatments were evaluated by flow cytometry .To further investigate the antitumour activity of nanovesicles in vitro, we carried out Annexin/PI staining assay. 2\u00a0\u00d7\u00a0102.115 MDA\u2010MB\u2010231 cells were seeded in 6\u2010well plates and cultured until 90% confluent. After that, cells were scratched using a 200\u00a0\u03bcL pipette tips and then treated with NVs. Finally, the images of scratched areas were obtained at the appointed times using an inverted microscope. Besides that, A549 cells were pretreated with recombinant human interferon\u2010gamma (100\u00a0ng/mL) for 12\u00a0h, then detected the change of the wound width as above method.We used the scratching method to study the effect of Flag\u2010fusion HAC NVs on cell migration. 2\u00a0\u00d7\u00a0102.12For fluorescence microscopic analysis, MDA\u2010MB\u2010231 cells were first incubated with indicated concentrations of Blank, Flag\u2010fusion WT or Flag\u2010fusion HAC NVs. After that, THP\u20101 derived macrophages and MDA\u2010MB\u2010231 cells were stained with CellTracker\u2122 Red and CellTracker\u2122 Green, respectively. Fluorescence labelled THP\u20101 derived macrophages and MDA\u2010MB\u2010231 cells were then co\u2010cultured for 24\u00a0h. For FACS analysis, the MDA\u2010MB\u2010231 cells phagocytosis by THP\u20101 derived macrophages cells was evaluated as the percentage of green fluorescent positive macrophages. And for fluorescence microscopic analysis, phagocytosis was measured as green fluorescence signalling related to the formation of phagosomes in macrophages.2.137 PBMC were cultured in T cell specific medium for 72\u00a0h, and activated in the plate coating anti\u2010CD3/anti\u2010CD28 antibodies  for 48\u00a0h. MDA\u2010MB\u2010231 cells (1\u00a0\u00d7\u00a0106 cells/mL) were first incubated with Blank, Flag\u2010fusion WT or Flag\u2010fusion HAC NVs at the concentration of 100\u00a0\u03bcg/mL and then co\u2010cultured with the resulting activated CD8+ T cells at the ratio of 1:1 for 48\u00a0h, after which the supernatants were collected for measurement of IFN\u2010\u03b3 production by enzyme\u2010linked immunosorbent assay (ELISA).1\u00a0\u00d7\u00a0102.14Activated T cells and macrophages were cultured as mentioned above, following by incubating with Blank, Flag\u2010fusion WT or Flag\u2010fusion HAC NVs at the concentration of 100\u00a0\u03bcg/mL for 24\u00a0h, respectively. The expression levels of function related proteins in activated T cells and macrophages were detected by qPCR.2.153 cells per well and grew in a 37\u00b0C incubator with 5% CO2. The medium was replaced every two days with fresh culture medium for a week to form multicellular spheroids with an appropriate size. Afterward, the tumour spheroids were incubated with the same amount of DiO\u2010labelled different vesicles for 6\u00a0h, and visualised permeation of tumour spheroids on Confocal Laser Scanning Microscopy by sequential scan .MDA\u2010MB\u2010231 cells were seeded into a 96\u2010well ultralow attachment plate at 3\u00a0\u00d7\u00a0102.163, mice were randomly divided into three groups. PBS, DiR\u2010labelled Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs were intravenously injected into mice, respectively (10\u00a0mg/kg). After 24\u00a0h post\u2010administration, the mice were sacrificed and major tissues  were harvested, the fluorescent distribution images were captured using an IVISs Lumina system .To examine the biodistribution of Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs in MDA\u2010MB\u2010231 tumour\u2010bearing mice, Flag\u2010Fusion WT and Flag\u2010fusion HAC NVs were labelled with DiR. When the volume of the orthotopic MDA\u2010MB\u2010321 tumour reached 300\u2212400 mm2.176 MDA\u2010MB\u2010231 cells were implanted into the right breast pads of BALB/c nude mice. When the tumour size approximately 40 mm3, mice were randomly divided into four groups (n\u00a0=\u00a06). (1) Blank NVs (10\u00a0mg/kg); (2) Flag\u2010Fusion WT NVs (10\u00a0mg/kg); (3) Flag\u2010Fusion HAC NVs (10\u00a0mg/kg); (4) Atezolizumab + Magrolimab (1\u00a0mg/kg). Approximately 5\u00d7106 PBMC were intravenously administrated once a week, and different treatments via the tail intravenous injection three times a week. Tumour volumes and body weights were measured and recorded every 2 days. The tumour volumes were calculated as V\u00a0=\u00a0(length\u00d7width2) / 2. On the 15th day of treatment, all mice from each group were sacrificed and the tumours were resected, taken photographs and weighed. Major organs  and blood were also harvested.Approximately 2\u00a0\u00d7\u00a0102.18The whole blood was collected through mice's eyeball removal in each group before euthanasia for the routine blood examination. To evaluate the level of liver and renal function after different treatments, we determined the levels of alanine transaminase (ALT), aspartate transaminase (AST), creatinine (CRE) and blood urea nitrogen (BUN) in the plasma. Meanwhile, the major organs were collected and fixed with 4% paraformaldehyde for 48\u00a0h, stained paraffin\u2010embedded tissue with haematoxylin\u2010eosin (H&E) for histopathological analyses. The sections were observed with a microscope .2.19Sectioning and immunohistochemical (IHC) staining of formalin fixed, paraffin\u2010embedded mice xenograft tumour specimens were performed by the standard protocols. All sections were 5\u00a0mm thick. Briefly, sections were deparaffinised through xylenes and graded ethanol, and antigen retrieval was performed in Tris/EDTA buffer at pH 9.0. For cell apoptosis, tumour tissues were evaluated using the one\u2010step TUNEL kit according to the manufacturer's instructions. For multiple immunofluorescence staining, PANO 4\u2010plex IHC kit (PANOVUE) was used according to the manufacturer's instructions. The samples were then observed under a fluorescence microscope. CD4, CD8 and CD45 antibodies were purchased from Proteintech , and CD68, CD86 and CD206 antibodies were purchased from Abcam .2.20Total RNAs of tissues were extracted using Eastep Super\u00ae Total RNA Extraction Kit, and quantified the concentration of tissue RNA via NanoDrop One . One\u00a0microgram of total RNA was reverse transcribed into cDNA using a PrimeScript\u2122 RT reagent kit with gDNA Eraser. Then RT\u2010qPCR was performed with TB Green Premix Ex Taq\u2122 . The primer sequences were as follows:2.21t tests. Multi\u2010group comparisons were analysed by one\u2010way or two\u2010way ANOVA with Tukey's post hoc test. p\u00a0<\u00a00.05 was considered statistically significant .The data represent the means\u00a0\u00b1\u00a0SD of at least two independent experiments. Statistical analyses were performed by GraphPad Prism 8. The differences between two groups were assessed by unpaired two\u2010tailed Student's 33.1Given that mutations of specific amino acids on the extracellular immunoglobulin (Ig) domain of PD\u20101 and SIRP\u03b1 have been found to enhance their affinity for ligands dramatically in vitro  were harvested and prepared from cultured HEK293 cells by sonication and extrusion Figure\u00a0 Andalou. Transmi3.2We then explored the affinity of fusion HAC NVs and their ligands PD\u2010L1 and CD47 on tumour cells. We firstly performed flow cytometry to detect the expression levels of PD\u2010L1 and CD47 on triple\u2010negative breast cancer (TNBC) cells MDA\u2010MB\u2010231 and 100\u00a0ng/mL IFN\u2010\u03b3 pre\u2010treated lung adenocarcinoma cells A549. We verified that PD\u20101 and CD47 were highly expressed on these cells as previously reported  database, and found that the expressions of CD47 in breast tumour cells  were significantly higher than that in normal cells , while PD\u2010L1 was only significantly expressed in MDA\u2010MB\u2010231 cells with high malignance Figure . To test3.3Several studies reported that PD\u2010L1 and CD47 also played a vital role in regulating immune\u2010unrelated biological processes in tumour cells, moreover, antagonising PD\u2010L1 or CD47 could inhibit the tumour cell proliferation and migration and induce apoptosis  derived T cells. The concentration of IFN\u2010\u03b3 in the culture supernatants were measured by enzyme linked immunosorbent assay (ELISA), which was about 619.6, 1313 and 1706 pg/mL after treatment with blank NVs, fusion WT NVs and fusion HAC NVs, respectively. A significant increase of IFN\u2010\u03b3 was measured after the fusion HAC NVs treatment, indicating potent T\u2010cell activation Figure\u00a0. In addiTo investigate whether fusion WT NVs and fusion HAC NVs have any direct effect on immune cells, we treated macrophages and T cells with blank, fusion WT or fusion HAC NVs, and found that there was no significant change in the expression of immune cell function related proteins in the fusion WT NVs and fusion HAC NVs groups compared with that in control group Figure\u00a0,i. These3.5After confirming the abilities of fusion HAC NVs in immune modulation, the antitumour effect of these NVs in vivo was further investigated. An important feature of NVs is their ability to be naturally recruited by tissues and involved in physiological processes  of 10\u00a0mg/kg fusion WT NVs or fusion HAC NVs were performed three times a week. In control groups, 10\u00a0mg/kg blank control NVs or 1\u00a0mg/kg Atezolizumab and 1\u00a0mg/kg Magrolimab combination (mAbs) were administrated i.v. Figure\u00a0. We founs Figure\u00a0,e. The es Figure\u00a0.In addition, the least ratio of Ki67 positive cells and the highest TUNEL fluorescence intensity suggested that fusion HAC NVs could reduce the proliferation Figure\u00a0,g and pr3.6+ immune cells. Compared with the control group injected with saline, we found that more than 20% of human derived immune cells were present in mice peripheral blood after 3\u00a0weeks of PBMC injection in the fusion HAC NVs group as the experimental group. This indicated that the human immune reconstitution in the mice model was successful  can be divided into two main subtypes namely M1 anti\u2010tumour macrophages and M2 pro\u2010tumour macrophages (Zhou et\u00a0al., n Figure\u00a0,f. Moreon Figure\u00a0,h. The gn Figure\u00a0. These rFurthermore, the mice received i.v. injections of NVs and mAbs were studied for in vivo toxicity. Neither death nor an obvious body weight difference was observed between the different groups over 15 days, demonstrating that no detectable side effects were induced by fusion HAC NVs Figure . Plasma 4With the exhilarating efficacy of immunotherapy in melanoma, lung cancer, breast cancer and other malignant tumours, the ICB therapy against PD\u20101 or PD\u2010L1 has become a clinical hot spot (Garon et\u00a0al., Interestingly, both PD\u2010L1 and CD47 are highly expressed in tumour cells and can be synchronously regulated by transcription factor MYC (Casey et\u00a0al., To improve ICB blocking efficiency, recent studies have used directed evolution by yeast\u2010surface display to select the ectodomain of PD\u20101 or SIRP\u03b1 as a high\u2010affinity competitive antagonist of its ligand. The beneficial mutated clones showed a 400\u2013500\u2010fold increase in affinity for hPD\u2010L1 and about a 50000\u2010fold increase in affinity for hCD47 compared with the corresponding wild\u2010type consensus as measured by surface plasmon resonance (Maute et\u00a0al., Till now, several cell types were used to produce NVs, including tumour cells, erythrocytes and stem cells (Meng et\u00a0al., In addition to immune suppression, PD\u2010L1 plays not only a role in the interaction with PD\u20101 on lymphocytes, but also as an important molecule involved in tumour proliferation, which is considered as a marker indicating tumour aggressiveness (Stefan Kraft et\u00a0al., The NVs engineered from cell line obtain tumour\u2010 or tissue\u2010specific accumulation, easy translocation through physical barriers and extracellular matrix, excellent biosafety and stability (Andaloussi et\u00a0al., Luyao Zhang: Data curation; formal analysis; methodology; investigation; writing\u2014original draft; writing\u2014review and editing. Xu Zhao: Formal analysis; methodology; investigation; software; writing\u2014original draft; writing\u2014review and editing. Yanan Niu and Xiaoya Ma: Investigation; validation. Wei Yuan: Formal analysis; writing\u2014review and editing; supervision; funding acquisition. Jie Ma: Formal analysis; writing\u2014review and editing; supervision; funding acquisition. All authors have agreed to publish the manuscript.The authors declare that they have no competing interests.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file."
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