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+{"text": "The compounds 1 and 2 are characterized by X-ray crystallography; they have hydrogen-bonded dimeric structures. Similar reaction of 1,4-naphthoquinone with 3-picolylamine and 4-picolylamine gives the corresponding 2-amino 1,4-naphthoquinones; two products are characterized by X-ray crystallography. The reaction of 1,4-naphthoquinone with 4-aminothiophenol and 1,4-naphthoquinone with 4-aminophenol are compared. The former leads to C-S and the latter to C-N bond formation. The reaction of 1,4-naphthoquinone with 4-aminothiophenol in an NMR tube is studied to explain that 2-(4-anilinothiolato) 1,4-naphthoquinone derivative to be the sole product in the reaction.Reaction of 1,2-naphthoquinone with primary amines gives a 2-amino-1,4-naphthoquinone derivative which is equivalent to 1,2 to 1,4 carbonyl transposition. For example the reaction of 1,2-naphthoquinone with 4-methoxyaniline gives 2-(4-methoxyanilino)-naphthoquinone-1,4-(4-methoxyanil) ( Amino quinones are used as medicines, \u20133 herbicn-butylamine, with 1,2-naphthoquinone are shown in 1 and 2 are characterized by determining their crystal structures and also by other spectroscopic techniques. The formation of products 1 and 2 is interesting as in the case of 1,4-benzoquinone and 1,4-naphthoquinone we did not observe condensation reaction between either of the carbonyl group with amines under ambient conditions , and the other to a double bond (C4-N2 1.297 \u00c5). In the crystal lattice, the compound 1 forms dimeric assembly held by hydrogen bonding interaction via N-H. . . O . The compound 2 has also similar features as that of the 1 and it has C=O distance C4-O1 as 1.231\u00c5. It also has two different carbon-nitrogen bond distances N2-C3 as 1.353 \u00c5, N1-C1 as 1.286\u00c5 corresponding to a C-N and a C=N respectively. In the solid state the molecules of 2 are held by hydrogen bonding interactions between N-H. . . O leading to dimeric structures with dD-H, 0.889 \u00c5, dH-A 2.287 \u00c5, dD-A 3.151 \u00c5, <D-H. . . A 163.98\u00b0 respectively.The solid-state structures of the compounds Formation of the imine derivative at the 4-position in these reactions may be attributed to the concomitant formation of 4-amino derivative along with the imine at 2-position. Tautomerism of the imine and amine group leads to the desired product. Valence tautomerism is commonly observed in metal complexes of 1,2-quinonic compounds. \u201322 In oup-methoxy aniline with 1,4-naphthoquinone leading to the 2-(p-methoxy-anilino) 1,4-naphthoquinone along with its crystal structure. [In a sense, both the reactions of 1,2-naphthoquinone and 1,4-naphthoquinone with primary amines are equivalent, as both reactions lead to formation of 2-amino 1,4-naphthoquinone derivatives as illustrated in ructure.  We extenructure.  Thus, we3 and 4 are shown in 3 and 4. Among the weak interactions N-H. . . O, C-H. . . O and C-H. . . \u03c0 interactions play roles in the self-assembly formation as illustrated in The products from these reactions are characterized by different spectroscopic techniques and also by X-ray crystallography. The crystal structures of 3 C6-H. . . O1 , C11-H. . . O1 , N2-H. . . O2  interactions and these interactions are shown in are C16-H. . . O1 , C3-H. . . O1 , N2-H. . . O2 , C13-H. . . O2  (refer 4 which forms self-assembly through Some of the important weak bonds responsible for hydrogen bonding assemblies in \u00b0) refer . In addi1H NMR  in deuterated methanol by reacting them in an NMR tube. The reaction products are compared with the purified isolated product . Surpris1H NMR , IR and  product  whilst tIn conclusion the reaction of 1,2-naphthoquinone as well as 1,4-naphthoquinone with primary amines are compared. The amino-substituted derivatives formed from both systems are amino-substituted at the 2-position, which in the case of 1,2-naphthoquinone is similar to incorporation of a carbonyl group at the 4-position by 1,2 to 1,4 transposition of carbonyl group. The reaction of 2-aminophenol with naphthoquinone leads to C-N bond formation whereas similar reaction of 1,4-napthoquinone leads to C-S bond formation. The advantage of these reactions is mildness and versatility. C-N as well as C-S bond formation can be achieved in quinonic compounds under mild conditions. However, limited mechanistic understanding of the reactions is the identification of a suitable system for obtaining homogeneous solutions with primary amines.File 1Supporting information. Experimental procedure and spectral data of products and the crystallographic information.File 21\u20136.CIF. Crystallographic information files for compound"}
+{"text": "Members of a protein family often have highly conserved sequences; most of these sequences carry identical biological functions and possess similar three-dimensional (3-D) structures. However, enzymes with high sequence identity may acquire differential functions other than the common catalytic ability. It is probable that each of their variable regions consists of a unique peptide motif (UPM), which selectively interacts with other cellular proteins, rendering additional biological activities. The ability to identify and localize such UPMs is paramount in recognizing the characteristic role of each member of a protein family.We have developed a reinforced merging algorithm (RMA) with which non-gapped UPMs were identified in a variety of query protein sequences including members of human ribonuclease A (RNaseA), epidermal growth factor receptor (EGFR), matrix metalloproteinase (MMP), and Sma-and-Mad related protein families (Smad). The UPMs generally occupy specific positions in the resolved 3-D structures, especially the loop regions on the structural surfaces. These motifs coincide with the recognition sites for antibodies, as the epitopes of four monoclonal antibodies and two polyclonal antibodies were shown to overlap with the UPMs. Most of the UPMs were found to correlate well with the potential antigenic regions predicted by PROTEAN. Furthermore, an accuracy of 70% can be achieved in terms of mapping a UPM to an epitope.Our study provides a bioinformatic approach for searching and predicting potential epitopes and interacting motifs that distinguish different members of a protein family. Multiple protein sequence comparison can provide a valuable protein signature and, thus, contribute to the fields of structural biology and molecular evolution ,2. In geIn our study, the highly conserved human ribonuclease A (RNaseA) superfamily was initially tested to define the default input parameters. The RNaseA superfamily is composed of eight RNases including RNase1, 2, 3, 4, 5, 6, 7, and 8 with similar sequences and biological functions . Its jth primary unique descriptor is denoted as Pn . ZI, j] represents a set of collection from the jth unique pattern, which allows (n-m) mismatched symbols for matching processes, and ZI, j] is its kth tolerant pattern. The C is the total number of possible combinatorial patterns set for reserved m symbols from a unique pattern with length n, and \u3008ZI, j]\u3009 \u220a  is the number of appearance of the kth tolerant unique pattern shown in family set Z. It is trivial that the value of \u3008ZI, j]\u3009 is greater than or equal to 1, since at least one sequence from set Z containing the primary unique pattern Pn  must exist. If all the k tolerant unique patterns ZI, j] can be matched in each sequence in Z, then \u3008ZI, j]\u3009 = N for all k, and it follows that R = 0%. This describes a special condition in which each possible tolerant pattern of the primary unique descriptor ZI, j] can be discovered in all other sequences of Z. Therefore, its representative percentage of primary uniqueness will be decreased to 0%. On the other hand, if all of the combinatorial tolerant patterns from Zi, j] cannot be found from other family sequences, then \u3008ZI, j]\u3009 = 1 for k \u220a  and it follows that by R = 100%. This describes another extreme case in which the program provides all of the possible tolerant patterns for approximate matching, and the primary unique pattern of ZI, j] still possesses 100% unique representative percentage. These calculated quantitative percentages represent the level of uniqueness and range from 0% to 100%. All of the primary unique patterns with different representative percentages will be sent to the next module to perform merge operations based on their neighbouring conditions and threshold settings for the representative levels of uniqueness.where we define the set of all primary unique descriptors with pattern length ZI can be merged if they possess overlapping symbols in ZI and both are satisfied the criteria of the minimum representative percentage. Given any two segments u1and u2 with length n1 and n2, the merged result is denoted by w and constructed byThe merging operation is proposed to enhance the discrepancies in a family set and emphasize the neighboring relationships instead of the traditional concatenation operation. Two matched primary unique patterns in sequence l is the number of overlapping symbols between u1 and u2, and l is strictly greater than 0. If u1 and u2 do not contain any overlapping symbols between them, then the merging operation will not be performed, and both will retain their original relationship. It is obvious that if u1 and u2 are merged with respect to ZI, the length of the merged segment w is strictly less than the concatenated string uv. To reinforce the strength of uniqueness of the merged patterns, a strict merging operation is also provided to make sure each subsegment of the merged pattern still possess unique properties. Assume two primary unique patterns with same length n are merged. The strict merging requirements are satisfied only when the lengths of the overlapping symbols are equal to the primary pattern length minus one or the length of the merged segment is equal to n+1. Therefore, strictly merged unique patterns are constructed using fundamental unique descriptors and reserve the most unique characteristics in sequences. After obtaining all candidate merged segments, the system provides a trimming function which returns a substring from each merged segment with the symbols stripped off the beginning and the end. This function is achieved by evaluating the beginning and ending (n-1) symbols respectively from merged unique pattern set. The (n-1) symbols will be trimmed off when they are matched with another merged unique. Through these three designed modules, UPMs of each sequence from a protein family set will be allocated efficiently. After reinforced processing, these merged UPMs satisfy the tolerance and representative level of uniqueness criteria. The RMA is available free of charge for academic use [where emic use ,45 and aemic use .The protein sequences could be selected according to the following criteria: (1) If the protein of interest belongs to a protein family classified by the sequence similarity, each member of this protein family is selected from GenBank and saved as FASTA format. (2) If the protein of interest does not belong to any protein family, its sequence should be compared with other protein sequences in the database by BLASTp software or others and all the sequences with more than 20% identity should be selected and saved as FASTA format.Bos Taurus (B. Taurus) and Homo sapiens (H. sapiens), and MsbAs, one of the largest superfamilies of proteins characterized by a highly conserved adenosine triphosphate (ATP) binding cassette (ABC), from Escherichia coli (E. coli), Vibro cholera (V. cholera), Gloeobacter violaceus (G. violaceus), Shewanella oneidensis (S. oneidensis), and Bradyrhizobium japonicum (B. japonicum)  Table  were anaE. coli BL21 (DE3) and expressed in the presence of 0.5 mM IPTG. After homogenization, the RNase3 was purified by His-Bind\u00ae metal chelating chromatography under denaturing conditions according to the manufacturer's protocol (Novagen). The generation of mAb was carried out as described [\u00ae Antibody Purification Kit (Millipore).The recombinant clones were transformed into escribed . HybridoAfter electrophoresis, the proteins were transferred onto a polyvinylidene fluoride membrane . After blocking, hybridizing with the first Ab, washing, and probing with the secondary Ab, the target proteins were visualized using the SuperSignal West Pico (Pierce) or 3,3'-Diaminobezidine tetrahydrochloride (Amersco).Each protein sequence was saved in PROTEIN format using EditSeq in DNAStar and executed by PROTEAN . The seqTo analyze the performance of RMA in large-scale, the input sequences were selected by the following steps. First, all information related to mAbs in website of Santa Cruz Biotechnology, Inc. was retrieved  and the GFP, green fluorescent protein; EGFR, epidermal growth factor receptor; mAb, monoclonal antibody; MBP, maltose binding protein, MMP, matrix metalloproteinase; MSA, multiple sequence alignment; RMA, reinforced merging algorithms; RNA, ribonucleic acid; RNase, ribonuclease; Smad, Sma-and-Mad related protein; TGF-\u03b2, transforming growth factor-\u03b2; UPM, unique peptide motifHTC carried out biological experiments, participated in the sequence analysis and drafted the whole manuscript. TWP participated in the design of the RMA program and its coordination. TF performed the statistic analysis. BHS participated in the design and modification of RMA program. PCW participated in the initial design of RMA program. CYT helped to modify the informatics' part of this manuscript. CTC helped to analyze the PROSITE pattern. SHL participated in the molecular cloning. MDTC conceived this study and helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "Nineteen of 24 tumours studied have shown a significant differential response. Three human colonic adenocarcinoma xenografts, in the short-term organ culture system, displayed the same response to 1-naphthol as primary tumours removed at surgery. 1-Naphthol, 1,2- and 1,4-naphthoquinone were also toxic to two human colonic adenocarcinoma cell lines, LoVo and COLO 206. The selective toxicity of 1-naphthol is mediated in part through an accumulation of 1-naphthol in the tumour tissue due to impaired conjugation by the tumour. The higher concentrations of 1-naphthol may then exert their toxicity either directly or by formation of naphthoquinones. Some indirect evidence was obtained for the possible involvement of 1,2- or 1,4-naphthoquinone in the cytotoxicity of 1-naphthol. Our studies suggest that further studies are warranted of the possible use of 1-naphthol or related compounds as antitumour agents."}
+{"text": "A novel, green chemistry, glycosylation strategy was developed based upon the use of ionic liquids. Research studies demonstrated that thiomethyl glycosides could readily be activated with methyl trifluoromethane sulfonate, using 1-butyl-3-methylimidazolium tetrafluoroborate as a solvent. This green chemistry glycosylation strategy provided disaccharides with typical yields averaging 75%. The ionic liquid solvent could be readily reused for five sequential glycosylation reactions with no impact on product yield. N-alkylpyridinium, 1,3-dialkylimidazolium, or trialkylsulfonium species, as shown in Owing to their unique chemical and physical properties, room temperature ionic liquids (ILs) have received significant attention as alternative solvents for a host of different applications. For example, it has been reported that ILs can be used in place of conventional organic solvents in synthesis, catalysis, electrochemistry, and liquid/liquid extractions. Commonlyal. reported that the glycosidations of glucopyranosyl fluorides with assorted alcohols employing an ionic liquid and a protic acid catalyst proceeded, under mild conditions, to afford the corresponding glycosides in 54\u201391% yields.BF4 per se was not quenched with low molar equivalents of water, and [bmim]BF4 acted not only as reaction media, but may also performed like \"molecular sieves\" at the same time.This interesting stability effect may be due to the reported ability of ionic liquids to act as liquid molecular sieves.\u201326 To ex4 for thioglycosylation reactions was accessed by repeating the synthesis of 3a, 4a and 3c. In brief, upon completion of the thioglycosylation reaction and extraction of the products, the ionic liquid was washed, filtered through a pad of Celite, and dried at 70\u00b0C, under reduced pressure. Following this procedure, the recovered ionic liquids were reused for the thioglycosylation reaction at least five times, without any loss in efficiency, to provide the same yields and selectivities as described in The recyclability of [bmim]BF4) provides a good yield and stereoselectivity with an environmentally benign protocol.In summary, we have demonstrated the application of ionic liquids for the thioglycosylation methodology employing methyl triflate as an activation agent. Moreover, the results indicate that the use of ionic liquid ([bmim]BFThe experimental details can be found in Supporting Information File 1File 1contains experimental details."}
+{"text": "Comparative genomics has revealed an unexpected level of conservation for gene products across the evolution of animal species. However, the molecular function of only a few proteins has been investigated experimentally, and the role of many animal proteins still remains unknown. Here we report the characterization of a novel family of evolutionary conserved proteins, which display specific features of cytoskeletal scaffolding proteins, referred to as LRCHs.LRCH gene in flies, dLRCH, we explored its function in cultured cells, and show that dLRCH act to stabilize the cell cortex during cell division. dLRCH depletion leads to ectopic cortical blebs and alters positioning of the mitotic spindle. We further examined the consequences of dLRCH deletion throughout development and adult life. Although dLRCH is not essential for cell division in vivo, flies lacking dLRCH display a reduced fertility and fitness, particularly when raised at extreme temperatures.Taking advantage of the existence of a single These results support the idea that some cytoskeletal regulators are important to buffer environmental variations and ensure the proper execution of basic cellular processes, such as the control of cell shape, under environmental variations. The division of animal cells relies on the choreographed reorganization of the mitotic spindle, which is responsible for chromosome segregation. To ensure that the two daughter cells receive identical genomic complements, microtubule dynamics must be coordinated with a stereotyped series of changes in cell shape, leading to cytokinesis. A failure to coordinate cell shape transformations with chromosome separation can lead to aneuploidy and contribute to cancer Drosophila member of the ERM family and we and other have shown a role for Moe in regulating cortical stability and rigidity during mitosis Drosophila cells destabilizes the cell cortex throughout mitosis, leading to cortical deformations and abnormal distribution of acto-myosin regulators It is well established that ERM proteins, named after the vertebrate members Ezrin, Radixin and Moesin, link actin filaments to membrane proteins dLRCH), as a potential physical interactor of Moe dLRCH in Drosophila S2 cells causes short-lived blebs that deform the cortex during mitosis, as well as alteration of spindle positioning. However, flies lacking dLRCH develop to adulthood, showing that dLRCH activity is not essential for cell division in vivo. Nonetheless, dLRCH deficient flies are female sterile, display shortened longevity and reduced resistance to extreme conditions. Consistently with the evolutionary conservation of LRCH proteins in animals, this first functional analysis therefore supports that dLRCH is required for proper development and physiology of Drosophila.To further explore the mechanisms regulating cortical organization during mitosis, we searched for putative partners of Moe. A two-hybrid screen identified the product of a candidate gene, CG6860 (hereafter referred to as Drosophila genome predicts that CG6860 encodes a novel protein of 809 amino acids (aa) that we named dLRCH, since it comprises Leucine-Rich-Repeats (LRR) and a Calponin Homology (CH) domain  domain . The N-tAlthough LRR and CH domains are widespread in eukaryotes, the combination of both LRR and CH within a same protein appears restricted to animal species, and specific to dLRCH in flies . In humaRecent work has highlighted the importance of the spatio-temporal localization and activity of cytoskeletal regulators during the successive steps of cell division Drosophila S2 cells were transfected with constructs encoding a GFP-tagged dLRCH protein, a tool that is also suitable for live imaging (see below). GFP-dLRCH is distributed at the cell cortex where it colocalized with the actin network, at the onset of mitosis . We then compared the consequences of dLRCH and Moe inactivation during cell division. The depletion of either dLRCH or Moe led to significant cortical blebbing in pro/metaphase and ana/telophase, albeit with a lower proportion of cells displayed defects in dLRCH-depleted cells compared to those lacking Moe  and enriched at the cleavage furrow in anaphase cells . The various defects observed during the division of dLRCH-depleted cells, i.e. cortical blebs and spindle mis-positioning, are however likely to involve Moe, since the simultaneous depletion of dLRCH and Moe did not lead to any aggravation of the defects exhibited in the absence of Moe dLRCH, removes \u223c30 kb that include most of dLRCH coding regions, as well as CG31804, a predicted gene showing very poor evolutionary conservation dLRCH represents a null dLRCH allele was confirmed by genomic PCR, with homozygous mutant embryos (Df(2L)dLRCH were viable and did not display gross developmental defects. Accordingly, it has been reported that a transposable element inserted in dLRCH exonic sequences, CG6860T1\u201336C, does not affect viability Df(2L)dLRCH mutants developed to adult stage, albeit with a slightly decreased viability when compared to wild-type dLRCH displayed decreased fertility when compared to their sibling controls (Df(2L)dLRCH homozygous females were sterile (Df(2L)dLRCH females appeared normal, we observed a weakly penetrant phenotype characterized by an abnormal number of germ cells dLRCH mutants opened the possibility that the absence of dLRCH impinges on development and physiology. We reasoned that if the activity of dLRCH is not essential under optimal culture conditions, its absence might be further deleterious under conditions of stress. We examined whether either low or high temperature, i.e., 17\u00b0C or 29\u00b0C that are close to the extreme allowing Drosophila development, impact on the survival of Df(2L)dLRCH mutants when compared to control. At these temperatures, we observed that Df(2L)dLRCH mutants displayed increased lethality dLRCH mutants exhibited a reduced longevity in both sexes, already detectable in heterozygous conditions (Df(2L)dLRCH females showed the most marked effect, even though reducing female fecundity is often thought to have a positive impact on lifespan in vivo results show that whereas dLRCH function is not essential in flies, its absence significantly affects the fitness of animals, especially under non-optimal conditions of development and during adult life.The slightly reduced viability of ethality , indicatnditions . InteresDrosophila cells implicate LRCH proteins in the organization of the cortex during cell division. dLRCH protein localizes to cortical regions undergoing contraction and depletion of dLRCH consistently provokes defects in both cell shape and positioning of the spindle during mitosis. However, the defects observed in cultured cells do not prevent a coherent development, since embryos lacking dLRCH reach adulthood. Nevertheless, our genetic analyses indicate that dLRCH activity is likely to be required for optimal resistance of individuals facing environmental constraints and aging.We identified here a novel family of putative cytoskeletal scaffold proteins, referred to as LRCH. Our studies in LRCH genes. Calponin Homology domains (often arranged in 2\u20134 tandem repetitions) are well established as protein domains mediating interaction with actin filaments LRCH proteins are characterized by a unique combination of protein domains that are otherwise common in eukaryotes, the LRR and CH domain. The distinctive domain organization of dLRCH defines a novel family of proteins, strongly conserved throughout the evolution of animals. Indeed, the human genome, as well as that of other mammals, contains four highly related While the LRCH family of proteins is well conserved in animals, little is known concerning their role in development or physiology. A few genetic studies have suggested that a variant in hLRCH1 is associated with knee osteoarthritis dLRCH-depleted cells as they passed through mitosis. dLRCH-depleted cells display abnormal transient cortical protrusions, or blebs, indicating that dLRCH contributes to cell shape control during mitosis. The formation of blebs results from a localized rupture of the interaction between actin filaments and the plasma membrane dLRCH-depleted cells form from early stages of mitosis and occur notably in the equatorial region at later stages. It has been shown that ERM proteins are normally recruited during the earliest steps of bleb retraction in normal cells, and Ezrin is required to retract short-lived blebs induced by the alteration of cortical tension in mammalian cells dLRCH-depleted cells show weak Moe-like defects during mitosis, LRCH proteins might contribute to proper organization of the mitotic cortex. We propose that the lack of LRCH activity causes transient alterations of the cell cortex, which are rapidly corrected in the presence of Moe, explaining why these abnormal blebs are only short-lived, as well as the absence of additive defects following the simultaneous depletion of both dLRCH and Moe.The sub-cellular localization of dLRCH during mitosis supports the idea that LRCH proteins function as cytoskeletal regulators during cell division. dLRCH localizes at the actin-rich cell cortex, where it partly overlaps with P-Moe throughout cell division Several lines of evidences suggest that, like in flies, LRCH and ERM proteins are also involved in the control of cell division in mammals. First, ERM and LRCH proteins show similar localization during the division of HeLa cells see . Human ELive imaging revealed the instability of spindle orientation in the absence of dLRCH during mitosis. Such a defect likely reflects an alteration of the cross-talk that normally occurs between the cortical actin network and microtubule tips during cell division e.g., EB1) or hetero-dimerisation  form a microtubule binding unit It remains yet possible that LRCH proteins contribute more directly to microtubule organization during cell division. It is worth noting that EB1, a protein binding microtubule plus-ends through a different subtype of CH domain, is required for proper positioning of the mitotic spindle dLRCH in vivo, we generated a loss of function dLRCH allele in flies, by inducing a targeted genomic deficiency that removes only dLRCH and a neighboring predicted gene (CG31804); CG31804 is poorly evolutionary conserved, even in closely related species, and both micro-array and deep sequencing experiments have not revealed expression, aside weak levels in the adult testis (Flybase website (accessed 2010) http://flybase.org/). If an influence of CG31804 cannot be formally ruled out, these results collectively suggest that the phenotype observed in Df(2L)dLRCH is primary due to the absence of dLRCH activity. In any case, embryos lacking dLRCH progress through an apparently normal development, showing that dLRCH is dispensable for cell division in vivo. Furthermore, it is well known that the division of spermatids in males is particularly sensitive to alterations in the molecular mechanisms controlling mitosis Df(2L)dLRCH males further argues that cytokinesis occurs normally in the absence of dLRCH in vivo.Functional assays in cultured cells have been proven as an efficient means of identifying the molecular pathways involved in the control of both cell shape Drosophila development and leads to fully fertile males, but sterile females in vivo, its function is indeed required for chromosome alignment and segregation during female meiosis. Interestingly, adult females lacking dLRCH are sterile and even Df(2L)dLRCH heterozygous display a phenotype of hypo-fertility. These data therefore open the possibility that dLRCH might also be involved in female meiosis, a speculation that must await future experimental investigation for confirmation.Similarly, it was recently shown that despite the dramatic defects observed in dividing S2 cells, the absence of Wac, a component of the Augmin complex, does not affect Drosophila raised under optimal culture conditions, but is required to maintain sensory organ fate under fluctuating temperature conditions dLRCH negatively impacts on resistance of flies to extreme temperature conditions, as well as longevity. In addition to apparently redundant transcriptional enhancers Phenotypic characterization is a difficult task that often limits the functional outcomes of reverse genetic approaches. If evolutionary conservation clearly represents a signature of molecules that might be involved in important developmental mechanisms, the selective pressure applied both on efficient reproduction and adult fitness, two parameters rarely examined in cellular and developmental studies. For example, it has been shown that the conserved miRNA miR7 is not essential for the development of http://smart.embl.de/). To determine the evolutionary repartition of proteins containing LRR and/or CH domains, Ensembl database (Homo sapiens) and FlyBase (Drosophila) were screened via Ensembl or Smart for PFAM domains . Individual sequences were extracted from Ensembl database: Homo sapiens , Mus musculus , Fugu rubripes , and Ciona intestinalis (ENSCINP00000002827), NCBI: Gallus gallus , WormBase: Caenorhabditis elegans (C14F11.2), and Caenorhabditis briggsae (CBP28441), FlyBase: Drosophila melanogaster (FBpp0080521) and Vector Base: Anopheles gambiae (AGAP010012-PA). Amino-acid sequences were aligned using CLUSTALW2 and evolutionary relationships viewed on a phylogram tree.Putative structure of the dLRCH protein (FBpp0080521) was predicted by analyzing its sequence with the SMART package (Drosophila S2 cells were cultured in Schneider's medium (Invitrogen) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and Penicillin-Streptomycin (PS). dsRNA were synthesized and purified according to the T7 RiboMAX\u2122 Express Large Scale RNA Production System (Promega). For dLRCH extinction, we used two non-overlapping regions to avoid off-target effects. For Moe and Slik extinction, we used a previously reported dsRNA st, 3rd and 6th day of culture. Immuno-fluorescence assays or live imaging were performed at the 6th day.dLRCH sequences were subcloned into pEFGP-C1 that contains an actin promoter. Coding sequence of Ezrin, Radixin, Moesin on one hand, and hLRCH3 on the other hand were cloned into pEGFP-N1 or pEGFP-C1, respectively. Cells were transfected with GFP-dLRCH or GFP-hLRCH3-expressing plasmids using Fugen HD and immuno-fluorescence analyses were performed 60 hours after transfection. A stable cell line expressing GFP-dLRCH was established by co-transfection of GFP-dLRCH and Hygromycin resistance plasmids.For immuno-fluorescence analysis, cells were cultured for 6 hours on glass coverslips, fixed in 4% paraformaldehyde in Schneider's supplemented medium , during 30 min. Cells were blocked with 2% BSA and 0.02% Saponin in TBS during one hour and then incubated with primary antibodies. We used anti-\u03b1Tubulin (Sigma-Aldrich) at 1\u2236200, anti-\u03b3Tubulin Fixed cells were mounted in Vectashield containing DAPI (Vector Laboratories). Images were acquired using a Nikon Eclipse 90i microscope with a MicroMAX (Princeton Instruments) camera. Deconvolution was performed using Huygens software (Scientific Volume Imaging). Live cell imaging was performed using stable cell lines expressing Tubulin-GFP in situ hybridization experiments following standard procedures. Embryos were mounted in glycerol-containing medium and photographed with a Nikon Eclipse 90i microscope. A null allele for dLRCH was generated through a small and targeted deletion using FLP recombinase f01198 and d04045 obtained from Exelixis Df(2L)dLRCH stocks coming from independent chromosomal events for further analyses (Df(2L)dLRCH mutant flies were kept and selected using GFP-expressing CyO balancers. The 1118w strain, or the parental d04045 line, was used as control, and the PL106/FM0y,w,MoeMoe mutant.Dig-labeled sense and antisense probes were synthesized from a full length CG6860 cDNA, and used for analyses . The resanalyses  using trFor experiments aiming at estimating viability, eggs were collected from well-fed females kept on agar medium with drops of live yeast. After hatching, 50\u201360 larvae were transferred in polystyrene vials containing corn/yeast agar medium and allowed to develop in different incubators , where light was provided from 8am to 8pm. The longevity of adult flies was recorded using new born flies passed into fresh vials containing the same medium and a drop of live yeast, in an incubator at 25\u00b0C with an identical light/dark regime. The number of dead flies was recorded each day, up to death of the last fly. Vials were renewed twice a week.Figure S1Evolutionary conservation of Drosophila and human LRCH proteins. Alignments of protein sequences corresponding to the LRR motifs (A) and the CH domain (B) between dLRCH and its 4 human orthologs. The two respective regions (positions 79\u2013281 and 669\u2013772 within dLRCH) were aligned using ClustalW. Identical residues are in red; those in green and blue are highly or weakly similar across the five sequences, respectively. C. Schematic representation of conservation levels between Drosophila and human LRCH proteins. Each bar (arrow) represents a 10 aa window, displaying from 5\u201310 (black) or 3\u20134 (grey) identical residues in the five LRCH proteins. White bars indicate a poor conservation, with 0\u20132 invariant residues. Positions of the two functional domains are underlined.(8.00 MB TIF)Click here for additional data file.Figure S2Inactivation of dLRCH in cultured cells and whole animals. A. Living S2 cells stably expressing GFP-dLRCH in control conditions (no dsRNA or control dsRNA targeting Sip1) or treated with dsRNA targeting the N-term or C-term region of dLRCH ORF . Pictures were taken using the same exposure conditions. GFP signal is strongly reduced in cells treated with dLRCH-N & -C dsRNA but not in cells treated with control dsRNA. B. Genomic DNA extracted from single homozygous flies corresponding to independent recombination events was used as a template for PCR amplification, using primer specific for Sip1 (control) or dLRCH coding regions. No dLRCH amplification was observed in flies homozygous for the Df(2L)dLRCH, confirming that this represents a molecular null allele.(2.88 MB TIF)Click here for additional data file.Figure S3Reciprocal independence of dLRCH and the Moe pathway. A. Depletion of dLRCH does not affect P-Moe (red) distribution in dividing S2 cells, as shown by a normal accumulation of P-Moe at the equatorial cortex (arrows) during anaphase . DNA is in blue B. Time-lapse frames of GFP-dLRCH S2 cells in control (left), and after treatment with Moe dsRNA (right) show that, reciprocally, Moe depletion does not prevent the proper distribution of dLRCH, as shown by accumulation at the cleavage furrow (arrow) in 98.0% of control cells and 97.7% of Moe-depleted cells, n\u200a=\u200a300. C. Time-lapse frames of GFP-dLRCH S2 cells show that Slik depletion does not prevent dLRCH localization at the cleavage furrow (arrow), with GFP-dLRCH accumulating at furrow in 93.3% of control cells and 92.0% of Slik-depleted cells, n\u200a=\u200a150. D. The graph shows the proportion of the different genotypes, observed in the progeny from MoePL106/FMO females crossed with control or Df(2L)dLRCH homozygous males. The absence of a dLRCH allele does not modify the proportion of the different classes, including that of MoePL106 male escapers, when compared to control. The number of individuals counted was MoePL106/+: n\u200a=\u200a641; MoePL106/+; Df(2L)dLRCH/+: n\u200a=\u200a680; FM0/+: n\u200a=\u200a543; FM0/+; D(2L)dLRCH/+: n\u200a=\u200a541; MoePL106/Y: n\u200a=\u200a12; MoePL106/Y; Df(2L)dLRCH/+: n\u200a=\u200a8; FM0/Y: n\u200a=\u200a397; FM0/Y; D(2L)dLRCH/+: n\u200a=\u200a421.(5.57 MB TIF)Click here for additional data file.Figure S4Evolution of the putative CG31804 gene in insect species. CG31804 putatively encodes a 212 amino-acid long peptide, being evolutionary conserved only in species of the melanogaster subgroup (grey box). Identities with the CG31804 encoded peptide decrease from 77% (D. simulans) to 57% (D. erecta). In the D. Ananassae genome, a genomic region at approximately the same location was identified by DNA similarity, but the presumptive ORF is interrupted by several stop codons and peptides do not display homology with the CG31804 product deduced from species of the melanogaster subgroup. Corresponding genome regions in other related drosophila were analysed with alignment tools , which detect no significant DNA nor protein similarities.(6.46 MB TIF)Click here for additional data file.Figure S5Germline defects observed in Df(2L)dLRCH females. A. Quantification of the number of nurse cells in the egg chambers of control or Df(2L)dLRCH homozygous individuals, in 7 independent experiments. Control (w1118) flies showed a weak proportion of abnormal numbers of nurse cells , in all experiments. In contrast we observed high variability in the proportion of abnormal egg chambers in Df(2L)dLRCH homozygous females. In two separate experiments, these defects were seen in 60% of examined samples. B. Pictures of egg chambers dissected from control (left) and Df(2L)dLRCH (right) females illustrating the observed defects in the number of nurse cells. Nuclei (blue) were stained by Dapi and F-actin (red) by Phalloidin-Texas-Red.(3.36 MB TIF)Click here for additional data file.Figure S6Expression and localization of human ERMs and LRCH3 in Hela cells. A. RT-PCR experiments for each of the 3 ERM and the 4 hLRCH mRNA show that all seven genes are expressed in HeLa cells. B. Sub-cellular localization of Ezrin-GFP, Radixin-GFP or Moe-GFP (green) and F-actin (red) during telophase indicates that all three human ERM proteins are located at the cleavage furrow (arrows). C. At the end of mitosis, GFP-hLRCH3 (green) is detected at the cleavage furrow as shown in co-labeling with \u03b1-Tubulin or P-ERM (red). DNA is in blue in merged images.(2.02 MB TIF)Click here for additional data file."}
+{"text": "Recent studies have demonstrated that it is common for women to report inconsistent fertility motivations and family planning behaviors. This study examines these inconsistencies among urban Honduran women interviewed at two points in time and presents reasons for inconsistent fertility motivations and contraceptive behaviors at follow-up.Data come from a one-year panel study conducted in Honduras from October 2006 to December 2007. A total of 633 women aged 15-44 years were interviewed at baseline and follow-up and have non-missing information on the key variables of interest. At baseline and follow-up, women were asked how much of a problem it would be  if they got pregnant in the next couple of weeks. At follow-up, women were asked an open-ended question on reasons it would be no problem, a small problem, or a big problem. The open-ended question was recoded into a smaller set of response categories. Univariate and bivariate analyses are presented to examine inconsistencies and reasons for stated inconsistencies.At follow-up, over half the women using a contraceptive method said that it would be no problem if they got pregnant. Nearly half of the women changed their perceptions between baseline and follow-up. Common reasons for reporting no problem among contraceptive users were that they accepted a child as God's will or that children are a blessing, their last child was old enough and they wanted another child. Common reasons for reporting a big/small problem among non-users of family planning (who have an unmet need for family planning) were that they were not in a stable relationship, the husband was not present, and they would expect a negative response from their family.Inconsistent fertility motivations and contraceptive behaviors are common among effective contraceptive users. Women who are using contraception and become pregnant will not necessarily report the pregnancy as unintended, given the widespread acceptance of unintended pregnancies in Honduras. Family planning providers need to recognize that fertility motivations vary over time and that women may not have firm motivations to avoid a pregnancy. Information on whether a woman wants to get pregnant soon, delay a pregnancy, or not have any (more) children is used to measure pregnancy intentions. Hence, an unintended pregnancy is defined as a pregnancy that is reported to have been unwanted  or mistimed  . WorldwiMany women who are not using contraception are at risk of an unintended pregnancy. A study from the U.S. demonstrated that among women who reported a pregnancy as unintended, about half were not using contraception at the time of pregnancy . A numbeStudies from developing countries have also demonstrated that ambivalence towards pregnancy, and inconsistent fertility motivations and contraceptive use are common among contraceptive users and non-users. One study found that in Burkina Faso and Ghana, around 13% of women who wanted to delay or limit childbearing said that it would not be a problem if they became pregnant soon . These fWhile quantitative data suggest that inconsistencies between fertility motivations and contraceptive use are common, few studies have attempted to determine the reasons for this ambivalence; the studies that have examined the reasons for these inconsistencies generally come from the U.S. ,15,19,20This study fills these gaps by examining reasons given for fertility motivations among women who had inconsistent fertility motivations and contraceptive use behaviors using data from women in four cities of Honduras. Because the study collected two rounds of data (at baseline and one-year follow-up), it is also possible to examine the extent that fertility motivations change over the one-year follow-up period among women who were all using contraception at baseline and were predominately using their method to space their next birth.The data come from a panel study that examined contraceptive continuation among users of reversible female contraceptive methods in four major urban areas of Honduras: Tegucigalpa, San Pedro Sula, Santa Rosa de Cop\u00e1n/La Entrada, and Gracias. Data were collected at baseline and a follow-up interview took place one year later. Baseline data, collected between October-November 2006, were comprised of exit interviews with eligible women attending a family planning clinic to receive the oral contraceptive pill, injection, or intrauterine device (IUD). The selected clinics included seven Secretary of Health clinics , one Secretary of Health hospital, and five Honduran Family Planning Association (Asociaci\u00f3n Hondure\u00f1a de Planificaci\u00f3n de Familia or ASHONPLAFA) clinics. Eligible women were aged 15-44 years old, and were either new or continuing users of one of the above mentioned methods. All women who were eligible and visiting the target clinics during the study period were eligible for interview; no women refused to participate. At baseline, a total of 800 women participated in the study. Contact information, including addresses, maps, and directions to the women's homes, provided at baseline were used to locate the women and arrange for follow-up interviews. Follow-up interviews were conducted in October-December 2007. A total of 671 women (84%) from the baseline sample were found and interviewed at follow-up. Among those women who were not interviewed, 15% were not found and a small number refused (7 women) or had died (2). The comparison of those women who were successfully interviewed and those lost to follow-up revealed few differences between the groups .At baseline, an interviewer asked women to respond to questions on their demographic characteristics, birth histories, previous use of contraception, perceptions of service quality, motivation to avoid pregnancies, and the family planning decision-making environment. At follow-up, women were asked about use of contraception during each month since the baseline interview, experience of and reactions to side effects, and updates on demographics, fertility motivations, and the decision-making environment. Ethical clearance was granted by the Institutional Review Board (IRB) of the University of North Carolina at Chapel Hill, the Honduran Secretary of Health, and ASHONPLAFA. Informed consent was obtained from each participant at the start of each interview.At baseline and follow-up, women were asked to rate how much of a problem a pregnancy in the next few weeks would be for them. The close-ended responses were big problem, small problem, or no problem. At baseline and follow-up, women who were using effective contraception were considered to have inconsistent fertility motivations and contraceptive use behaviors if they responded that getting pregnant in the next few weeks would be no problem. Users of contraception gave consistent responses if they said that getting pregnant in the next few weeks would be a small/big problem. Women who were not using an effective contraceptive method at follow-up were considered to be inconsistent and have an unmet need for contraception if they responded that getting pregnant in the next couple of weeks would be a small/big problem. At follow-up, non-users of contraception were consistent and had no need for family planning if they said that getting pregnant in the next couple of weeks would be no problem. As a test, all analyses were run with the small-problem group included in the no-problem group. No changes were noted in the analyses, most probably due to the small sample size of the small-problem category. Responses at baseline were compared to follow-up.As part of the follow-up questionnaire, women were also asked an open-ended question to provide, in their own words, a reason for why they reported that a pregnancy in the next few weeks would be no problem, a small problem, or a big problem. This question is used to provide a perspective on some of the factors that influence women's motivations to become or to avoid a pregnancy. Data on the open-ended question on why it would be no problem or a small/big problem if the woman becomes pregnant were recoded into a smaller number of categories. Categories were developed based on a cursory review of the data and additional categories were developed as needed. Two individuals (the first and second authors) recoded the data independently and their coding schemes were then compared (with the third author included) and when there were disagreements, the response was discussed and a consensus was achieved by the three-author team. Some women gave multiple reason responses; this happened among 112 of the women who responded to the open-ended question on their reason why getting pregnant would be no problem, a small problem, or a big problem.The analysis sample was reduced from the full sample of 671 women included at follow-up because of missing data on the key variables of interest (the problem question and the open-ended question). In particular, 35 women were pregnant at the time of the follow-up interview and were not asked if becoming pregnant soon would be a problem. Of the remaining 636 women, three had missing data on reasons for why getting pregnant would be a problem. Therefore, 633 women gave reasons why getting pregnant soon would be no problem or a small/big problem; this is the analysis sample. Including the 112 women who gave two reasons, there are a total of 745 reasons included in the analyses. Descriptive analyses are presented including univariate and bivariate associations of the problem question and recoded open-ended question.Table About one-fifth of the women interviewed were less than 20 years old Table  and 60% Not surprisingly, many women changed their method use during the one-year follow-up period (not shown). Overall, at the end of the one-year period, 17% of women were no longer using a method (not shown). Women also switched between methods over the follow-up period with the most women switching away from injections and adopting another method (not shown). At follow-up, 47% of the analysis sample was using an injectable method, 18% had an IUD, and 11% were taking the pill. Table In Table In Tables Table Of the 303 women who said that getting pregnant soon would be a big/small problem, 82% were using a contraceptive method at the time of the interview; this is a consistent response (consistent/met need). On the other hand, 19% of the women were not using a contraceptive method, even though they stated that getting pregnant soon would be a big/small problem. These women have an unmet need for contraception, and they are inconsistent in their fertility desires and contraceptive behaviors. The most common reasons cited for why a pregnancy soon would be a small/big problem among the women with an unmet need for contraception Table  were thaAmong women with consistent contraceptive behavior and fertility motivations, the main reasons cited for why getting pregnant would be a big/small problem were that the last child was still young (30%), that the family's economic situation was not adequate to have another child (16%), or that the woman was at that time studying/working (13%). Other reasons cited for why getting pregnant would be a big/small problem were that the woman had finished childbearing (10%) or that she was unable to have another child due to past/current medical complications (7%).This study demonstrates that 27% of the sample of urban Honduran women who were using the IUD, injection, or the pill at baseline reported that it would be no problem if they became pregnant in the next few weeks. These women have inconsistent fertility motivations and contraceptive use behaviors at baseline. At one-year follow-up, 17% of the sample was no longer using an effective method of contraception and only 59% were using their baseline method. The extent of changes in contraceptive method use between baseline and one-year follow-up is indicative of potential problems with the methods  as well as changing pregnancy desires in the period. Notably, about half of the women who were not using a contraceptive method at follow-up have an unmet need for family planning; that is, they reported that it would be a big/small problem if they got pregnant in the next few weeks. At follow-up, we also found that more than four-fifths of women who report that getting pregnant in the next few weeks would be no problem were effective method users; these women are considered to have inconsistent fertility motivations and behaviors. The most common reasons for reporting no problem among these women were that they would accept the pregnancy, children are God's will, and that children are a blessing and are always welcome. Conversely, the women who reported that a pregnancy in the next few weeks would be no problem and were not using contraception (a consistent response) tended to give reasons related to motivations to get pregnant.This study further demonstrates that 19% of the women interviewed who are not using any contraceptive method report that it would be a big/small problem if they became pregnant in the next few weeks. These women are considered to have an unmet need for contraception. Among these women, the most common reasons for why a pregnancy would be a big/small problem are related to partner and family issues. On the other hand, more than four-fifths of the women who were using a contraceptive method at the time of the follow-up interview gave a consistent response that it would be a big/small problem if they became pregnant in the next few weeks. These women gave reasons related to their economic and educational situations as well as their plans to delay a pregnancy. These results illustrate that women who are consistent in their contraceptive behaviors and fertility desires appear to plan their pregnancies, whereas the women with an unmet need for contraception demonstrate partner and family support concerns that influence their motivations for a future pregnancy.Most studies on fertility desires focus on retrospectively reported pregnancy intentions or examine which women have an unmet need for contraception based on their stated fertility desires ,22,23. TA limitation of this study is that we cannot compare over time the reasons women gave as to why getting pregnant would be a big, small, or no problem as this information was only collected at follow-up. Also, while we report on inconsistencies between fertility motivations and contraceptive use behaviors, we may in part be measuring ambivalence toward contraceptive use rather than problems with the meaning or measurement of fertility motivations . ContracOur study demonstrates important findings for family planning programs. First, just examining standard fertility intentions - wants now, wants to delay, wants no more - will not provide an accurate prediction of who needs or will use long-term effective methods. Second, we note that fertility intentions can change over time. Third, women who are using contraception and have inconsistent fertility motivations with their current use behaviors are more likely to accept a future pregnancy as the will of God and raise the child as a blessing they need to nurture. Hence, the outcome of an unintended pregnancy may be an intended birth. Programs may need to focus on giving women more autonomy and confidence when planning a family to ensure they are using a method that meets their current fertility intentions. Furthermore, women who have an unmet need for family planning and report that a pregnancy in the next few weeks would be a big problem may need to be counseled about the advantages of consistent and effective method use to avoid an unintended pregnancy.This study demonstrates that many effective family planning users have inconsistent fertility motivations. Future studies are needed to examine whether less motivated women are more likely to discontinue use when they experience partner or family opposition; side effects; or changes to their economic or educational situation. Qualitative studies are also needed to determine whether the most motivated women are receiving the methods that are best suited to their fertility desires. Among women who use temporary methods of contraception, even though they might state no intention of a future pregnancy (or a desire to delay a pregnancy), they may not have strong desires to avoid pregnancy and this might relate to their method choice or the effectiveness of method use. Family planning program officers need to be aware that women's fertility intentions can change within a short period of time, even though they might continue to use a contraceptive method. Moreover, if a pregnancy happens, it will not necessarily be reported as unintended, given that most births would be accepted among women in Honduras. Family planning program managers should consider strategies to ensure that motivated users have access to follow-up care, if needed, to address method concerns such as side effects. Finally, a greater understanding of fertility motivations and how they influence the effectiveness of contraceptive use is needed to help family planning providers ensure that they are counseling women appropriately and could help reduce the prevalence of unintended pregnancies in Honduras and other countries where unintended pregnancies are common.The authors declare that they have no competing interests.ISS conceived of the idea, participated in data coding, analysis, and writing. LI led data coding and analysis and revised the manuscript. JB participated in data coding, analysis, and writing. JL participated in data collection and contributed to paper development. All authors read and approved the final manuscript."}
+{"text": "Since the beginning of the 90s lots of cationic plant, cysteine-rich antimicrobial peptides (AMP) have been studied. However,  Plants are constantly exposed to several pests and pathogens in nature. They have developed complex defense mechanisms to protect themselves against the attack of pathogens , 2010. TDefensins are small cationic peptides of 45\u201354 amino acid residues with a conserved signature of cysteines, which can form three to four disulfide bridges. Plant defensins exhibit a conserved tertiary structure that consists of a triple-stranded antiparallel \u03b2-sheet and one \u03b1-helix that are stabilized into a compact shape by the disulfide bridges. These bridges form a cysteine-stabilized \u03b1-helix \u03b2-sheet motif (CS\u03b1/\u03b2) . In addiTriticum turgidum) and barley (Hordeum vulgare)  have been studied. Plant defensins were first described in the seeds of wheat (vulgare) . They weThe role of defensins in the preformed defense of plants is well reported. Several reports show that defensins are an integral part of the plant innate immune system. Most plant defensins already characterized show a constitutive pattern of expression with up regulation in response to pathogen attack, injury and some abiotic stresses .Several features make clear that defensin peptides are involved in plant defense . Their din vitro antimicrobial activity and, currently, there are several reports describing the production of transgenic plants constitutively expressing foreigner defensins. Hence, they possess an enormous multiplicity of biological activities, such as antimicrobial, insecticidal, inhibiting protein synthesis, mediating abiotic stress, and Zn tolerance, and as inhibitors of digestive enzymes , whose cysteine residues interact in the following order: Cys1-Cys10, Cys2-Cys5, Cys3-Cys7, Cys4-Cys8, and Cys6-Cys9  motif, which forms one \u03b1-helix followed by three anti-parallel \u03b2-sheets. The amino acid sequence is also quite conserved, especially due to the presence of six to eight cysteine residues, which form three to four disulfide bridges in the sequence of Cys1-Cys8, Cys2-Cys5, Cys3-Cys6, and Cys4-Cys7 . Neverthys6-Cys9 . The addys6-Cys9 .Nicotiana alata (NaD1), Petunia hybrida (PhD1 and PhD2), and ZmESR6 isolated from developing maize kernels. These defensins contain an extra acidic C-terminal prodomain whose function is still unknown, although it has been suggested that it is involved in vacuolar targeting or in eliminating potential detrimental effects caused by the basic nature of the defensin , Pachyrrhizus erosus (SPE10), Petunia hybrida (PhD1), Pisum sativum (Psd1), Raphanus sativus (Rs-AFP1), and Saccharum officinarum (Sd5) . An amino acid sequence alignment of antifungal defensins from plants shows that they do not present conservative amino acid sequences, except the cysteine residues and a glycine residue positioned in the second \u03b2-sheet . This preservation in the primary sequence gives them a three-dimensional conformation denominated \u03b3-core motif, consisting of two antiparallel \u03b2-sheets, with an interpolated turn region. Earlier studies classified plant defensins as belonging to the \u03b2-\u03b3-\u03b1 Group, according to their relative structural \u03b3-core (R. sativus defensin 1 (Rs-AFP1) was determined by 1HNMR, and mutation analyzes was also performed using the peptide isoform Rs-AFP2  have been widely extended during the last few years. Among the peptides with antifungal activity, whose structures have been elucidated, are included the defensins from  \u03b2-sheet . Accordil \u03b3-core . It has l \u03b3-core , not onll \u03b3-core . This ch Rs-AFP2 . In bothThere are two major hypothesis that tries to explain the mechanism of action of antimicrobial defensins: (i) the carpet model and (ii) the pore model. In both models, defensins are described to interact with the negatively charged molecules present at the cell membrane of pathogens, causing an increase of its permeabilization, leading to cell leakage and death by necrosis. While the carpet model emphasizes the pore formation of several peptides into the membrane, the pore model shows that peptides form oligomers that, then, form multiple pores into the cell membrane. However, there is an alternative hypothesis, where defensins do not damage the cell membrane, but interact with the phospholipids, leading to an increase of ion permeability, or even to the transportation of these peptides to the intracellular environment . Hence, in vitro assays using only the \u03b3-core sequence of Mtdef4 and MsDef1  showed that the high content of positively charged residues with the core of MtDef4 could, alone, provide antifungal activity, in contrary to the core of alfAFP, which was inactive against filamentous fungi  at a dependent concentration, indicating physical damage of cell membranes. In comparison, alfAFP induced a less effective membrane permeabilization, and did not induce a concentration dependent SG uptake , in which the site related to the second loop of the defensins that contains the \u03b3-core region is described as the most important site for their antifungal activity. This is highly positively-charged in the cartoons where electrostatic surfaces were designed in vacuum. Therefore, it corroborates with the antifungal assays and the in silico studies performed by many researchers over the last 20 years.Further reports displayed a comparison between the electrostatic potential surfaces of different defensins with their potential antimicrobial activities . HoweverF. solani . In this way, analyses of the defensin from Pachyrrihizus erosus, SPE10, provided the selection of the binding pattern Arg36-Trp42-Arg40 as essential for dimer formation. Moreover, it was demonstrated that Trp42 is fundamental for antifungal activity of plant defensins, as it is absent in non-antifungal peptides Therefore, dimers of SPE10 are arranged in a side-by-side manner with the \u03b1-helix of one monomer interacting with the \u03b2-sheet of the second monomer, leading to a stretched and twisted molecular surface. Conformational changes on Arg36 and Trp42 would alter the dimeric interface of SPE10, destabilizing the dimer . Hence, plant defensins that form dimers coupled with their positively charged surface area become highly efficient molecules against pathogenic fungi, as they can strongly interact with the negatively charged glycoproteins located at the fungal cell walls  was obtained by Rs-AFP2, when assayed against Pyricularia oryzae. Its IC50 ranged from 0.08 to 5 \u03bcM. Since that lots of defensins were reporter to show high biological activity in the range of micromolar to nanomolar as will soon be shown. One of the first studies that attempted to highlight this class of plant antimicrobial defensins was carried out with two peptides isolated from Radish seeds, Rs-AFP1 and Rs-AFP2. Both peptides were assayed against 20 different plant pathogenic fungi and the lower protein concentration required for 50% inhibition of fungal growth (ICAesculus hippocastanum (Ah-AMP1), Clitoria ternatea (Ct-AMP1), Dahlia merckii (Dm-AMP1), Heuchera sanguinea (Hs-AFP1) against eight different fungi in the presence, or absence, of inorganic ions. The lower IC50, around 0.1 \u03bcM, was acquired when Ah-AMP1 was tested towards Cladosporium sphaerospermum, Leptosphaeria maculans, and Septoria tritici. Hs-AFP1 presents the same antifungal activity when assayed against Septoria tritici. In all the studies, inorganic ions decreased IC50. When visualized under a microscope, it was possible to see that such antifungal peptides caused distinct morphological changes during germ tube elongation and hyphae development, like multiple hyphae buds or the diminished of the rate of germ tube elongation (Table 1).Few years after Terras report, ongation ; Table 1Phaseolus vulgaris, permeabilizes Mycosphaerella arachidicola membrane, among other fungi  in a eukaryotic expression, system based on the methilotrophic yeast Pichia pastoris. The high amount of Psd1 produced by P. pastoris expression system (13.8 mg/L), allowed investigations about the conformational features between wide type and recombinant form of Psd1 (rPsd1). Besides being active towards filamentous fungi, such as Neurospora crassa, Psd1 did not demonstrate any activity against yeasts, even at high (20 \u03bcM) concentrations  active against F. oxysporum . A comparison between the expression of Pdc1, a corn defensin, in yeast and E. coli was done and in both cases the peptide kept its antimicrobial activity, however, Pdc1 expressed in yeast (IC50 7.5 \u03bcM) was more efficient than when expressed in E. coli (IC50 30 \u03bcM) in arrest F. graminearum growth. The presence or absence of a His-tag also influences its activity, suggesting that defensins are sensible to covalent modifications on its terminal ends  resides in the carboxy-terminal region. They evaluated six different defensin chimeras obtained from molecular combinations of MsDef1, active towards F. graminearum. They also analyzed MtDef2, a defensin from Medicago truncatula, which did not have any activity towards F. graminearum. Among the six chimeras, only the ones harboring the MsDef1 portion on the C-terminal displayed some activity against F. graminearum . Therefore, plant defensins with antifungal activity have become the first molecule for the development of transgenic crops resistant to phytopathogens.Although there are many transformed plants in the market with additional genes coding to proteins that confer resistance towards herbicides and insect-pests, there is still no transgenic plant available against phytopathogenic fungi, nor even containing plant defensins as the resistant factor. Nevertheless, several studies describe the efficient activity of antifungal defensins when transformed into different host plants . The transgenic plants were tested in vitro and in vitro against Magnaporthe oryzae and Rhizoctonia solani, the main causes of rice losses in agriculture, revealing that overexpression of Rs-AFP2 can control the rice blast and sheath blight diseases  was more resistant to the attack of Verticillium dahliae, when compared to non-transformed plants  as well as the defensin PDF1.2, providing an enhanced resistance to Verticillium dahliae (Similar results were obtained when an ionotropic glutamate receptor (RsGluR) was transformed into  cinerea . Microar dahliae .Plant defensins correspond to a world of possibilities for defense mechanisms, and new peptides with different activities are still to be discovered, as well, studies with thousands of plant species need to be performed. Nowadays, several peptides already show satisfactory efficacy against such pathogens with strong potential to be applied for the production of a commercial fungicide or application into transgenic plants. But, the question remains. Why is there still no product containing antifungal plant defensins \u2013 in its natural form or in nanocapsules \u2013 already available in the market?It is interesting that plant defensins with antifungal peptides are mostly studied for pathogens located in tropical areas, including Latin American, African, and some Asian countries. Moreover, the loss of commercially important crops due to the attack of phytopathogenic fungi is considered worldwide, until now, less detrimental than the losses caused by drought stress and insect-pests. Therefore, the efforts focused on the release of novel plant varieties resistant to drought stress and insect-pests are more significant, as there is mounting pressure to control these adversities in order to provide an increase in crop production. However, the development of transgenic plants expressing antifungal defensins or the production of defensin-based biofungicide depends, mainly, on the determination of regional research teams focusing on specific fungal targets, so these products can reach the market.Furthermore, there is a long process required for analyzing the efficiency, environmental risks, safety towards animal and human consumption, and reproducibility of transformed plants expressing certain molecules, as well as the need for having an extremely stable, effective, and easy-to-produce peptide to be used in the fabrication of a biofungicide. Therefore, it is possible that there are already plant defensin-based products on the horizon that will soon be released on to the market.Also, it is expected that, in the near future, antifungal defensin-based commercial agro-products be targeted as essential for the increase of crop production. This will stimulate and accelerate the transition between biotechnological research and field application of bioproducts.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 aim of this study was to evaluate the effects of ginger on pain relief in primary dysmenorrhea.ZThis was a randomized, controlled trial. The study was based on a sample of one hundred and twenty students with moderate or severe primary dysmenorrhea. The students were all residents of the dormitories of Shahed University. They were randomly assigned into two equal groups, one for ginger and the other for placebo in two different treatment protocols with monthly intervals. The ginger and placebo groups in both protocols received 500\u2009mg capsules of ginger root powder or placebo three times a day. In the first protocol ginger and placebo were given two days before the onset of the menstrual period and continued through the first three days of the menstrual period. In the second protocol ginger and placebo were given only for the first three days of the menstrual period. Severity of pain was determined by a verbal multidimensional scoring system and a visual analogue scale.There was no difference in the baseline characteristics of the two groups . The results of this study showed that there were significant differences in the severity of pain between ginger and placebo groups for protocol one (P\u2009=\u20090.015) and protocol two (P\u2009=\u20090.029). There was also significant difference in duration of pain between the two groups for protocol one (P\u2009=\u20090.017) but not for protocol two (P\u2009=\u20090.210).Treatment of primary dysmenorrhea in students with ginger for 5\u2009days had a statistically significant effect on relieving intensity and duration of pain.IRCT201105266206N3 Primary dysmenorrhea is the term for painful menstrual cramping, there is no pathological evidence for the condition and it occurs in up to 50% of menstruating females . It can One reason that has been suggested as an explanation for primary dysmenorrhea is an increased production of uterine prostaglandins derived from cyclooxygenase (COX)-2 activities ,4. StudiGinger has a long history of traditional use. It contains several constituents such as gingerol, gingerdiol, and gingerdione, beta-carotene, capsaicin, caffeic acid and curcumin ,10.Several studies have demonstrated that ginger has beneficial effects to cancer prevention , pregnanThe study was a double blind, placebo-controlled and parallel-group study with balanced randomization [1:1] for the two groups. The study was conducted in Tehran, Iran from June 2008 to December 2008.2 or greater than 25\u2009kg/m2, and mild dysmenorrhea.A sample of female students aged 18 and over was selected for the trial. Inclusion criteria consisted of: being single, having a menstrual cycle that lasts from 21 to 35\u2009days with 2 to 6\u2009days of flow and average blood loss of 20 to 60\u2009ml [The study was conducted at Shahed University dormitories from June 2008 to December 2008 in Tehran, Iran.Zingiber officinale R. rhizomes were collected in April 2008 from the Iranian Institute of Medicinal Plants  field. A voucher specimen of the plant (number 1483) has been deposited in the Central Herbarium of the institute. The rhizomes were dried in a dark condition at room temperature and ground to a powder that was encapsulated.The placebo capsules contained toast powder. The capsules were similar in shape, taste and color but one set contained 500\u2009mg ginger powder per capsule and the others were placebo capsules. The ginger capsules did not have distinguishable smell. The capsules were prepared in the Institute of Medicinal Plants and put into coded packages. Capsules and their packages were identical in appearance.After confirmation of a patient\u2019s eligibility and after written informed consent was given, the students were randomly divided into two equal groups to be administered with ginger or placebo. The ginger and placebo groups received 500\u2009mg capsules of ginger powder and placebo respectively three times a day in two different treatment protocols. Both treatment protocols were given at monthly intervals as follows:Treatment protocol 1: In this protocol ginger and placebo were given two days before the onset of the menstrual period and continued through the first three days of the menstrual period.Treatment protocol 2: In this protocol ginger and placebo were given only for the first three days of the menstrual period  and duration of pain between two groups, P value\u2009\u2264\u20090.05 was considered as significant.The ethics committee of Shahed University approved the protocol. We obtained written informed consent from participants after comprehensive explanation of procedure involved.The study involved a total of 105 students. Thirteen students who had received placebo discontinued the trial before completing the evaluation due to the fact that they indicated did not like to be involved in this research project any longer. More information on reasons for leaving was not captured. However, there were no significant differences between characteristics of 13 patients who left the placebo group and those who remained in the study  and menstruation characteristics Table\u2009.The results of this study showed that there were significant differences in severity of pain between ginger and placebo groups for protocol one  and 4 students in the placebo group (8.7%) reported adverse effects. Students in the ginger group reported heartburn while students in the placebo group reported nausea.The results indicate that administration of 1500\u2009mg ginger powder daily for three days is a safe and effective way to produce analgesia in students with primary dysmenorrhea.Although the 1.4 and 2.0 point (cm) reduction in pain severity seen under protocol 1 and protocol 2 were statistically significant, as suggested if we consider a reduction of 3 point (cm) pain severity in the VAS as clinically significant , then onStudies have shown that the NSAIDs are effective in treating dysmenorrhea comparing to placebo -26, but The results of present study showed that taking ginger 2\u2009days before the onset of the menstrual cycle was significantly better at decreasing the duration of pain. However, there was some indication that the magnitude of decrease in pain duration for protocol 2 was better than protocol one. The explanation for such an observation needs more investigation, although one might argue that this apparent superiority was due to under reporting by students using protocol 2.Several factors including increased production of prostaglandins may be involved in menstrual pain . To explThe findings of this research are in accordance with a previous observation that compared efficacy of ginger to some NSAID drugs . SimilarThis study is somewhat limited by a lack of observation regarding the effects of ginger on the other symptoms associated with dysmenorrhea. Furthermore, 13 patients from placebo group left the study. Unfortunately we did not collect data on reasons for leaving and this is a limitation. Moreover we did not analyze the gingerol or shogaol content of our ginger powder nor was it made in a standardized fashion. Consequently we do not know how much of key constituents were in the capsules making it hard to know what dose of other ginger capsule to give in the future and to determine if all the capsules were uniform in there gingerols/shogaol content. Lastly, it is worth noting that no measure of adherence or blinding was assessed. Consequently, effect sizes could be exaggerated if blinding was not achieved. Conversely if some of the participants did not take enough capsules or at the right times it could biases our results to the null. However, it was found that the effects of ginger are large enough to be clinically significant and to alleviate primary dysmenorrhea.The results suggest that ginger may be an effective and safe therapy for relieving pain in women with primary dysmenorrhea if administered at the onset or during the 3\u2009days prior to menstruation. Furthermore studies with larger number of patients concerning the efficacy and safety of different doses of ginger as well as its effects on the other dysmenorrhea symptoms are suggested to supplement this research.The authors declare that they have no competing interests.PR was the main investigator, designed the study, collected the data, and performed analysis. AM contributed to the analysis, critically evaluated the paper, and provided the final draft. HF prepared the materials for this study. SK helped the main investigator to writing the manuscript. MN helped the main investigator to design the study. All authors read and approved the final revision of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/92/prepub"}
+{"text": "In this study, we investigated the sensitivity of a DIC system in determining surface strains in a mouse tibia while loaded in compression through the knee joint. Specifically, we examined the effect of speckle distribution, facet size and overlap, initial vertical alignment of the bone into the loading cups, rotation with respect to cameras, and ex vivo loading configurations with the tibia attached to the entire mouse, or to the femur and foot, or only to the foot, showed different strain contour maps.We loaded tibiae of C57BL/6 mice  up to 12\u00a0N at 8\u00a0N/min. Images of speckles on the bone surface were recorded at 1\u00a0N intervals and DIC was used to compute strains. Results showed that speckles must have the correct size and density with respect to the facet size of choice for the strain distribution to be computed and reproducible. Initial alignment of the bone within the loading cups does not influence the strain distribution measured during peak loading, but bones must be placed in front of the camera with the same orientation in order for strains to be comparable. Finally, the ex vivo murine tibial loading tests.This work provides a better understanding of parameters affecting full field strain measurements from DIC in  These studies have thus often related mechano-adaptation of bone to the strain distributions that estimated either experimentally on the basis of measurements made only at single locations of the bone surface using strain gauges in vivo, or in representative ex vivo loading. Other times, strains have also been estimated computationally with finite element analysis, which requires that assumptions are made regarding the material properties, geometry, and loading conditions. These methods are clearly limited in their scope to provide direct measurements of strain across an entire bone surface and so more recently, digital image correlation (DIC) has been used to overcome these limitations offering a viable method for measuring high resolution full-field strains on the whole bone surface  cameras while loaded. The field of view is then divided into subsets called \u2018facets\u2019, which are unique correlation areas containing a specific number of pixels, and the displacement is computed by tracking the relative movements between the facets. The facets track features of speckles during loading and measure their deformation. A large facet size improves the accuracy of measurements, but a small facet reduces the computation time and captures local effects. The facets overlap each other ensuring continuity of strain across facets. A large overlap increases the computational time for calculating strains and can result in redundant measurements. The final result is the measurement of the axial, transverse, and shear surface strain.ex vivo testing conditions.We have previously used DIC to determine surface strains in mouse tibiae while loaded in compression through the knee joint . We showex vivo bone loading configurations  on the mouse tibial surface strains.Here we test how sensitive DIC strain measurements are to the test set-up. In this study we generate 3D contour maps of the strain fields on the bone surface during mouse tibia loading  and dete2Fresh frozen male C57BL6 mice  at either 12 or 18 weeks of age (w.o.) were used in this study. Mice were defrosted and soft tissue was removed from both their tibiae. Each tibia was covered with a thin layer of matt, water-based, white paint , and then speckled with matt, acrylic, black ink  using a high precision air brush . We determined the optimal size of speckles (see below).\u2113, Right and left tibiae of each mouse were speckled together to ensure similar speckle distributions. To measure strain during the application of load, limbs were loaded at a rate of 8\u00a0N/min up to 12\u00a0N with 50\u00a0N load cell  using custom built loading cups, which applied an axial load on the tibia across the knee and ankle joints . 12\u00a0N isPreceding the actual measurement of load-induced strains on the bone surface, three images of the non-loaded tibia surface were captured in order to evaluate the amount of experimental noise (accuracy) generated by the camera system during image acquisition. Load-induced strains were measured twice on each tibia to ensure reproducibility of measurements. Maximum and mean strains on the surface were calculated using the linear strain computation algorithm of ARAMIS. This uses the displacement of the data points (centres of the facets) to calculate the strain. All strains were computed with a computation size of 5, i.e. a 5\u00d75 field of 3D points was used to calculate strain value at the facet centre, and a validity quote of 65%, i.e. at least 16 of the 25 data points (for a field of 5\u00d75) around the facet centre must exist for the strain to be computed at this location.All bones were kept wet before and between measurements by wrapping them in saline moistened gauze.2.1Speckle 1 had dots of ~4 pixels in diameter with 40% black/white density, Speckle 2 had dots of ~8 pixels in diameter with 45% black/white density and Speckle 3 had dots of ~12 pixels in diameter with 55% black/white density. These three speckle types were produced by the same ink and high precision air brush, by varying the aperture of the air brush nozzle. The images were analysed by using the facet size (19\u00d719) and overlap (21%) proposed as default by the DIC system. To define which speckle pattern gives the most accurate results with our set-up, we compared the noise generated by speckles at zero loading. As a result from this test Speckle 2 was applied to all the following tests.The dependence of the strain map on speckle distribution and size was tested using three different speckle patterns consecutively on the left and right tibia of three 18 w.o. mice. Specifically, 2.2During processing of images, we compared strain contour maps of six tibiae (12 w.o.) calculated using three quadrangular facet sizes of 15\u00d715, 19\u00d719 and 20\u00d720 pixels with two overlaps of 5\u20137% and 20\u201321%. Noise at zero load, and maximum and average strains for four different load cases  were calculated. As a result from this test facet size 19\u00d719 with overlap 20% was used to process images of all the following tests.2.3Because cups were originally designed to allow loading of the right leg, we compared DIC results captured from loaded right and left legs of six 18 w.o. mice in order to test whether any difference in strains engendered could be detected.2.4A series of 10 consecutive tests were carried out on the right tibia of one 18 w.o. mouse and the strain distribution for each test was recorded, maintaining all parameters constant, to determine repeatability in measuring strains on a single mouse bone. Between each test, the specimen was not removed from the loading cups, but the load was completely removed. The time between each load was 5\u00a0min, and the bone was kept moist throughout the testing.2.5Position 1, Position 2, Position 2 was achieved by locating the knee joint at the edge of the notch within the cup and measured by the angle of the fibula in the first picture of the bone under static condition.The loading cups used here were specifically designed to load the tibia axially . Tests c2.6Rotation 1, Rotations 2 and 3, Sensitivity of the strain map to the rotation of the surface of interest in front of cameras was also measured. Strains from both the right and left tibiae of two 18 w.o. mice were computed when the bone was loaded with its medial surface parallel to cameras (2.7ex vivo loading configurations: (1) with the entire mouse body attached, (2) with only the foot and the femur and its muscles attached, and finally (3) with the tibia and foot alone.Right and left tibiae from three 12 w.o. mice were loaded in three different 2.8Position 1 and Position 2, were analysed with a t-test. Difference in strain values between three speckles and three rotations the three loading configurations were compared using analysis of variance (one-way independent ANOVA) and Post Hoc procedures for multiple comparisons. All tests were two tailed and p-values smaller than 0.05 were considered to be significant.Normal distribution of the maximum and average strains and their homogeneity of variance were analysed by the Shapiro\u2013Wilk test and Levene\u05f3s test, respectively . Difference in maximum and average strains between left and right legs, and between Precision error (PE) of measurement on a single bone was estimated in terms of coefficient of variation (CV) of repeated measurements given in percentage .3The spatial strain distribution over the medial side of the tibia was calculated, based on the three components of displacement measured by the DIC system. The results reported here show the strain in the axial (loading) direction as transverse strain and shear strain had relatively low magnitude compared to the axial strain.Tibial axial compression of 12\u00a0N results in tension on the antero-medial side of the tibia  with a p3.1Speckle 3 the tensile strain peak was significantly higher (p<0.005) than in Speckle 1 (0.472\u00b10.038% in Speckle 3 vs. 0.412\u00b10.017% in Speckle 1) probably due to an increase in the speckle density in the specific location  generated statistically significant smaller (p<0.05) noise than Speckle 1 (0.040\u00b10.005), but was similar noise to Speckle 3 (0.045\u00b10.008).Results from the speckle dependence test showed that the average strain distributions obtained over the medial tibial surface from three different speckles were similar . Howeverlocation . Speckle3.2Overlay plots displaying strains at four different stages of the loading  show tha3.3p=0.481 and 0.387, for maximum and average strain, respectively).When comparing left and right leg, we observed a very similar strain distributions, peak and average strain magnitude on the medial surface of two tibiae with a maximum and average surface strain respectively of 0.454\u00b10.054% and 0.261\u00b10.038% for the left leg, and 0.465\u00b10.044% and 0.255\u00b10.033% for the right leg . The PE_CV was equal to 6.15% for the maximum strain and to 5.38% for the average strain.3.5Position 1 and Position 2 within cups were similar at 12\u00a0N, with a maximum and average surface strain respectively of 0.460\u00b10.031% and 0.262\u00b10.026% when the tibia was loaded in Position 1, and 0.456\u00b10.028% and 0.283\u00b10.021% when loaded in Position 2 . The tibia initially loaded in Position 2 almost completely self-aligned to Position 1 while loaded up to 12\u00a0N.Strain distributions obtained for tibiae loaded in 3.6Rotation 1, maximum and average strains were 0.410\u00b10.052% and 0.237\u00b10.028%, respectively. Higher peak strain magnitudes (p<0.05) were obtained when the bone surface was rotated at 45\u00b0 to the camera axis (Rotation 2), with a maximum surface strain of 0.495\u00b10.057% (average strain was 0.257\u00b10.019%)  than in tibiae loaded with cameras in Rotation 1 and 2 .Strain distributions differed when the tibiae were rotated with respect to the camera plane. When bone was positioned and loaded in \u00b10.019%) . When bo3.7p<0.001) decreased when only the femur and its soft tissues were still attached (0.340\u00b10.022%) and decreased (p<0.001) even more markedly with the tibia and foot alone (0.262\u00b10.029%); peak strains in the latter two configurations also differed significantly (p<0.001) from each other. Furthermore, the average strain level across the entire surface was significantly higher (p<0.001) when the tibia was loaded in the full mouse configuration (0.319\u00b10.021%) compared to either of the other two  configurations, which did not differ from each other. The load-deformation curve was similar for three loading conditions, indicating that there was no failure during the load.Local regions of high strain (0.470\u00b10.017%) were seen on the medial side of all tibiae loaded with the full mouse body, but these levels of strain were only very rarely observed when tibiae were loaded in either of the two other loading configurations . Across 4ex vivo murine tibial loading tests. A summary of tests conducted with respective results is shown in In this study we demonstrate the sensitivity of DIC strain measurements on the murine medial tibial surface to different testing parameters during loading. Although only 4\u20136 bones were tested for each sample group, we were able to detect statistical significant differences in variables investigated, offering an overview of parameters affecting full field strain measurements in Speckle 1) as well as ~12 pixels in diameter with a higher density (Speckle 3), it was not possible to calculate the strain in all facets on the bone surface. This is in agreement with previous studies, which have indicated that a speckle with less than 4 pixels in diameter is not recognisable by the DIC system  and noise increased. This means that large speckles require larger facets in order to be computed because otherwise it would not be possible to detect contrast in the specific areas. The optimal facet size and overlap will depend on speckle size and density and vice versa. Therefore the best post-processing settings should be identified for each sample tested. Our results suggest that for a mouse tibia bone a ratio of 1:2 should be kept between speckles diameter and facets size (i.e. speckles of ~8 pixels diameter and facet size of 19\u00d719 pixels) for the strain to be computed.Position 1). Furthermore, when the full mouse was attached to the tibia, it was not possible to load the leg within cups in any other position than the two explored. This indicates that this load set-up together with the shape of cups make this particular system relatively insensitive to how the knee is originally placed in cups, as the tibia will align itself during compression.Initial alignment of the tibia into the loading cups did not affect the surface strain at 12\u00a0N as the compressive force acting on the knee and ankle joints causes an automatic adjustment during loading of the knee to the recommended axial loading position . When rotated with respect to the camera plane, speckles acquire a different shape and size as the bone surface was closer or farther from cameras. If the surface is curved, rotation with respect to cameras may also make it impossible for the cameras to see the whole surface and data will be lost (as shown in the +45\u00b0 rotation). For reproducible and accurate results, the imaged surface should be parallel to the camera plane.Ex vivo testing is often performed to estimate in vivo conditions. Here we show that disarticulation of the limb significantly affects results ( results . If accuin vivo loading of mouse right tibia knee joint, but proved to work similarly for both left and right legs. The same cups were previously used for investigating strains on the tibia of 8 w.o., female, B6 mice (The loading cups we used for our study were initially developed for  B6 mice . When co B6 mice  were reh4.1ex vivo murine tibial loading tests. The heterogeneity of the bone surface determines local deformations and strains, which are captured by tracking the displacements of unique material points on the surface at high resolution but cannot be captured using strain gauges or approximate finite element models. Revealing local microstructural strains in cortical bone is not only essential for enhancing our understanding of bone adaptation, but can also provide insights in other fields, such as bone damage development and fracture.This work provides a better understanding of parameters affecting full field strain measurements from DIC in The authors have no conflict of interest."}
+{"text": "SHF) and Patient\u2010Centeredness in Health Care\u2019 was launched to establish and implement quality criteria related to collaboration with patient groups.The importance of patient participation and involvement is now widely acknowledged; in the past, few systematic health\u2010care institution policies existed to establish sustainable co\u2010operation. In 2004, in Germany, the initiative \u2018Self\u2010Help Friendliness (SHF by summarizing a number of studies and (ii) a new survey on the importance and feasibility of SHF.The objective of this study was to describe (i) how patients were involved in the development of SHF was shaped, tested and implemented in 40 health\u2010care institutions in Germany. Representatives from 157 self\u2010help groups (SHGs), 50 self\u2010help organizations and 17 self\u2010help clearing houses were actively involved. The second objective was reached through a survey of 74 of the 115 member associations of the biggest self\u2010help umbrella organization at federal level (response rate: 64 %).In a series of participative studies, SHGs regarding co\u2010operation, their involvement in the definition of quality criteria of co\u2010operation, having a crucial role during the implementation of SHF and accrediting health\u2010care institutions as self\u2010help friendly. The ten criteria in total were positively valued and perceived as moderately practicable.Patient involvement included the following: identification of the needs and wishes of SHF as a systematic approach to closer collaboration of professionals and SHGs. Some challenges have to be taken into account involving patients and the limitations of our empirical study.Through the intensive involvement of self\u2010help representatives, it was feasible to develop  More than three decades ago, Hatch & Kickbusch stated that \u2018potential benefits from self\u2010help are not confined to participants\u2019, but may lie in \u2018improvements in the quality and the structure of institutional services and in the behavior of professionals\u2019 p.195).95.1 HencSince then, many efforts have been made to generally acknowledge self\u2010help groups (SHG) or \u2018voluntary and self\u2010help organizations\u2019 as mentioned in the WHO's strategy \u2018Health 2020\u2019.,,A nationwide self\u2010help\u2010support\u2010system, inclu\u2010ding around 300 clearing houses for self\u2010help support, was developed and established over the last 30 years in Germany, predominantly to promote regional SHGs, but also self\u2010help organizations.individual patient participation \u2013 specifically in the context of shared decision making \u2013 is not uncommon, there is a lack of systematic approaches at the collective level, that is, the involvement of patient or self\u2010help groups in health\u2010care organizations.,,At the meso\u2010level of single health\u2010care institutions, however, general provisions rarely exist for the participation and systematic involvement of patients in \u2018their\u2019 services; the few measures described in the literature usually deal with provisions for the financial funding of self\u2010help support.groups\u2019  and \u2018self\u2010help organizations\u2019 for the more formally organized non\u2010profit associations at national or federal state level. Particularly, the latter ones include what in other contexts or countries is called patient advocacy, consumer or user associations, or \u2018HCPO\u2019 .The notion of SHF implies a partnership approach (as opposed to the above\u2010mentioned \u2018superordinate\u2013subordinate relationships\u2019) for sustainable collaboration with patients. Our understanding of partnerships is close to Baggott's working definition as \u2018a range of collaborative working arrangements, institutions and processes, involving organizations and individuals, that seek to improve the health and wellbeing\u2019 (p.11).In the previous studies, we have reported on the approach in general,the challenges and possibilities of involving patients at the collective level in the SHF approach, that is, representatives of self\u2010help groups and organizations, by summarizing a number of pilot studies and projects.a recent major study, specifically dedicated to the assessment of the importance and feasibility of quality criteria of good collaboration.The advantages and shortcomings of both the involvement of self\u2010help representatives (SHRs) and the empirical study on collaboration criteria will be discussed at the end.www.selbsthilfefreundlichkeit.de).Our account of the involvement of self\u2010help associations looks at a series of empirical studies and practice\u2010oriented development projects with regard to SHF between 2004 and 2013 (see Table The concept of SHF stems from two sources. The first is a former survey of 345 contact persons of 658 SHGs in Hamburg (response rate: 52.4%).The second source is the annual meetings of SHG members and medical doctors, jointly organized by the Medical Chamber of Hamburg and the local clearing house for SHGs. In one of these meetings, the notion of \u2018self\u2010help friendly hospitals\u2019 arose; this term was used in an intensive discussion between representatives of Hamburgian hospitals and the clearing house about a formal co\u2010operation statement between hospitals and SHGs in 2003, and to initiate a model project on SHF in hospitals. In autumn 2004, funds for an explorative research project were granted from the Federal Association of Company Health Insurance Funds (BKK BV).In 2005, representatives of 30 self\u2010help organizations with experience of co\u2010operation, and 20 self\u2010help clearing houses, answered a questionnaire concerning their wishes, expectations and assessments of different quality criteria and recommendations. Their assessments and recommendations provided a significant basis for the identification of relevant quality criteria for SHF.The steering group of the model project consisted of the project manager (an experienced former self\u2010help supporter and consultant for change management), three hospital quality assurance managers, two staff members of the local clearing house and four representatives of SHGs. Scientists of the Hamburg Department of Medical Sociology worked as consultants and moderators. Eight criteria for good colla\u2010boration between hospitals and SHGs were developed and agreed on:The hospital offers rooms, infrastructure and possibilities for public relations.Patients of the hospital are personally informed about self\u2010help on a regular basis.The hospital supports public relations of the SHG.The hospital appoints a staff member as a contact person for self\u2010help.Staff and SHG members meet regularly for information exchange.SHGs are involved in further education/training of staff.SHGs are involved in quality circles and ethics committees.The collaboration is formally agreed on, and the activities will be documented.A quality circle as mentioned in #7 is a work group of employees or persons involved in a process who meet regularly to investigate and discuss their quality problems to develop solutions and actions for improvement. Most of the criteria refer to improving support for SHGs, while criteria 5\u20137 aim at permanent involvement of SHGs to improve service quality.In Hamburg, the implementation of the self\u2010help criteria was successfully tested in two hospitals. After a formal external audit, the two hospitals were awarded a \u2018Quality Seal for Self\u2010Help Friendliness\u2019 in mid\u20102006. Eight members of SHGs had been trained specifically for this task and participated in the on\u2010site visitation. These quality criteria were published as a brochure with guidelines on how to plan and to implement them.After the test phase in Hamburg, it took some time until the welfare organization \u2018Der PARIT\u00c4TISCHE North Rhine\u2010Westphalia\u2019 became the project holder of the next development project (2008\u20132010) and founded its own agency for SHF, with a social worker experienced in self\u2010help affairs. This project produced the following process pattern for becoming self\u2010help friendly:The agency for SHF (or a self\u2010help clearing house) contacts and informs the hospitals.First consultation of the agency takes place in the hospital.The agency contracts the hospital and mediates contacts with self\u2010help clearing houses.The staff of the self\u2010help clearing houses counsels hospital staff and mediates SHGs.The hospital and SHGs collaborate in a quality circle.Measures to fulfil the quality criteria are put into practice and are part of the internal quality management system.The hospital applies for a certificate .The quality report of the hospital is signed by representatives of SHGs.Certification is awarded .The agency conducted consultations with 31 hospitals in North Rhine\u2010Westphalia (NRW), 17 of which finished the process with a distinction. The requests for consultation overstrained the capacities of the agency .www.selbsthilfefreundlichkeit.de). However, they also showed that some aspects were either too demanding, or even resulted in barriers to further spreading the concept; for example, the agency concept was too expensive. Hence, the present approach is to give a small amount of funding to the existing clearing houses whenever they have an additional workload caused by their support for institutions adopting SHF. Also, the concept of a formal quality seal after an external audit was dropped as it was too time\u2010consuming, not only for the hospitals but also for the SHRs. Today, health\u2010care institutions can apply for a distinction of the network \u2018Self\u2010Help Friendliness and Patient Centeredness\u2019 if they are able to present a \u2018quality report\u2019 in which patient representatives have certified that at least one measure for each quality criterion has been put into practice and included in the internal quality management system. This has proved most feasible and, at the same time, guarantees that no advertising of SHF claims can be made of any institution without the consent of SHRs.Both projects in Hamburg and in NRW produced valuable insights and downloadable material that can be used by other hospitals as a blueprint for becoming self\u2010help friendly  participated in the process; they comprised general practice, gynaecology and obstetrics, internal medicine, urology, ophthalmology, orthopaedics, ENT medicine and paediatrics. The medical and lay persons met four times as a quality circle and jointly developed measures to put the quality criteria into practice. At the end of the process, the success of implementing the criteria was put on record in a quality report which was signed by both parties. The practices were awarded a distinction as being particularly self\u2010help friendly.The process in two pilot hospitals for rehabilitation was as participative as in the hospital sector and finished in 2013. The process started with a working group on quality improvement of an umbrella organization of rehabilitation institutions. The resulting preliminary set of criteria was discussed with 14 SHRs in a special workshop for this purpose in December 2011. They produced five quality criteria that were tested and finally confirmed in a model project with two rehabilitation hospitals. The participating SHGs in the model project were as follows: the Interest Group of Contergan Victims, the Federal Osteoporosis Association, the German Multiple Sclerosis Society and a local SHG of stroke patients. At the end, the successful implementation of self\u2010help friendliness was confirmed by the SHRs and led to a distinction for the two hospitals.active members, among them 39 local self\u2010help clearing houses, seven self\u2010help organizations, 27 hospitals and 15 rehabilitation hospitals. Eighteen hospitals and four rehabilitation hospitals currently hold distinctions as self\u2010help friendly health\u2010care institutions. In each case, the list contains an enumeration of the collaborating SHGs .The national network of institutions interested in becoming (or remaining) self\u2010help friendly has grown steadily since its start in 2009. In November 2015, the network  had 105 What do SHRs think of the importance and feasibility of the SHF approach as a whole? As we had only qualitative knowledge concerning this issue, we started a quantitative survey in 2012.individual level , together with items published in.collective level of self\u2010help friendliness and was called \u2018involving SHGs\u2019. This scale consists of ten statements describing the health\u2010care institution's quality of collaboration with the organized self\u2010help.The survey instrument was based on previous research on the evaluation of SHF in hospitals.Besides a few questions on demographics and the patient's self\u2010rated severity of own illness , and the tasks and functions of the organization (seven items), the questionnaire included one open\u2010ended question on the integration of SHF into quality management systems. All items measuring the importance and feasibility of SHF criteria had the same answer scale from 1 to 6 . Additionally, users could tick the box \u2018cannot assess\u2019.Procedure and respondents: We sent a postal questionnaire to 115 member organizations of the Federal Working Group for Self\u2010help (BAG SELBSTHILFE), a large umbrella organization of national\u2010level self\u2010help organizations for disabled and chronically ill persons. The member organizations could determine themselves who answered the questions. Questionnaires could be sent back either by mail, fax or e\u2010mail to the research institute; a reminder was posted 2\u20133 weeks after the first contact. We got 74 responses (64%).within the organization , 23 % described their function as inside and outside the organization and 8 % understood themselves as patient representatives in other contexts .Fifty\u2010eight percentage of the participating SHRs were male, and 50% were 60 years or older. Only 32 % of the respondents felt free of acute health problems at this point. Most of the SHRs (69 %) were primarily working in functions Descriptive analyses were performed as frequency distributions, cross tables and mean comparisons. Differences between function in the self\u2010help organization and other characteristics of the respondents  were statistically tested. The open\u2010ended question of the survey was analysed by quantitative content analysis.The focus of this paper is on the collective level of SHF, that is, on the involvement of patient or SHGs  in health\u2010care organizations. Therefore, we will only present our results concerning the subdimension \u2018involving SHGs\u2019 .Figure practical feasibility of the SHF criteria was rated lower than their importance. Considered to be easy to implement were those types of co\u2010operation that already exist in many places or can be expected to need little effort, that is, informing patients and/or their relatives about SHGs with relevant material on a regular basis, and supporting SHGs in their public relations , they were equal partners in the formulation of quality criteria and collaborated to develop a kind of \u2018quality seal\u2019 .Undertaking (applied) research \u2013 Self\u2010help representatives in pilot studies helped to assess the appropriateness and wording of research instruments; self\u2010help clearing houses and some self\u2010help umbrella organizations assisted with recruitment to studies and thus improved response rates; SHRs were responders in interviews and surveys.Analysis and write\u2010up \u2013 some SHRs gave feedback regarding preliminary versions to improve understanding and comprehensiveness, particularly in cases of open\u2010ended questions.Dissemination and implementation \u2013 there was little help with the dissemination, but a major impact on the implementation of SHF: SHRs were auditors in the accreditation process for the first self\u2010help friendly hospitals, advisors and/or participants in workshops and quality circles; and, most important for sustainability, they were and are witnesses and controllers for the granting and renewal of distinctions.Other than in the review quoted, we registered the most important beneficial impacts in the last phases \u2018implementation\u2019 and \u2018maintenance\u2019. Nevertheless, \u2018challenging impacts\u2019 found in the reviewDifficulties in recruiting a diverse range and representative sample of SHRs to a project.Sessions being overshadowed by personal experience stories, when the aim was to discuss research topics.Increased time and cost owing to the practical aspects of planning and discussing with the SHRs.Training and education for both SHRs and staff from self\u2010help clearing houses.Whereas these points can be regarded as challenges that are compensated for by the benefits of participation, we also have to mention some more severe limitations:Except for the first stimulus from SHGs in the annual meetings organized by the Medical Chamber of Hamburg, the whole process was initiated and driven by researchers and funders.Participation was not feasible in all steps of the programme.Although we tried to involve a broad range of SHRs, we had to accept that only a limited number of SHRs were prepared to participate.Roles were restricted to particular modes of contributing to the next step in the programme; there was no continuity in the representation of self\u2010help.These points imply that participation was incomplete in many respects. This problem is not unique, and some researchers conclude that a fully participatory project may be unrealistic.Two other aspects are unsatisfactory: firstly, there are general concerns about the representativeness and legitimacy of SHGs and \u2013 at higher organizational levels \u2013 about their independence, particularly from the drug industry.The second aspect is related to the poor evidence for measurable impacts of patient and public participation,Another challenging issue is the transferability of the approach to other countries. At least in terms of the mere quantity and nationwide coverage, there is probably no other country in the world providing such extensive professional support for patients and SHGs at all levels than Germany. These self\u2010help specific characteristics of the German health\u2010care system have certainly facilitated the present acknowledgement and appreciation of SHF. Transferability seems to be no problem in a system similar to that in Germany, such as in Austria, where about 40 hospitals have gone through the process to become self\u2010help friendly.Second objective of this paper was our recent major survey of SHRs\u2019 views. The high values for importance were expected, specifically because the dimensions and criteria were formulated in accordance with them. The values concerning practical feasibility can be regarded as cautious optimism, as they mostly lie in the upper half of the scale. Only two values are below the middle of the 6\u2010point scale (2.8 and 3.3), namely involvement in meetings and/or quality management, and regular information exchange. This indicates that SHRs are rather sceptical regarding to what degree \u2018true\u2019 participation might be possible. These two criteria certainly deserve particular attention in any future development.Although there was a wide variety of representatives from nationwide self\u2010help organizations included in the study, the sample cannot be regarded as being representative, neither for the whole landscape of the self\u2010help sector in Germany  nor the professional health service institutions and the involved staff. Unfortunately, we have no information on the diseases of the respondents. However, the surveyed member organizations of the BAG SELBSTHILFE cover a wide range of illnesses, disabilities and mental disorders. Although not completely comparable, we would like to mention that a similar survey with quality managers in hospitals about the criteria's importance and practicality showed similar results.collaboration as a major domain of patient\u2010centredness in health services. Consequently, other important aspects for the well\u2010being of patients might remain untackled .Another shortcoming is the restriction of the study to the self\u2010help and patient organizations\u2019 points of view on Collaboration with self\u2010help associations is voluntary; therefore, the preparedness of health\u2010care institutions is a necessary prerequisite for better integration of patient and consumer organizations into the daily routines of health services. However, whereas \u2018normal\u2019 institutions demand the adaption of patients to the institution, self\u2010help friendly institutions are prepared to act the other way round, allowing participation and adapting to patients\u2019 wishes and needs. Hypothetically, collaboration could lead not only to (legitimate) opposing points of view and new challenges, but might also provide a podium for \u2018gadflies\u2019 and destructive critics. During the course of our research, however, we did not hear any complaints of this kind from the participating institutions. The most plausible reason seems to be that the dependency of patients on \u2018their\u2019 professional institutions is so high that open conflict generally will be avoided.\u2018Self\u2010help friendliness\u2019 is a catchy label, quite suitable as a short title and attractive for the public relations of health\u2010care institutions. Nevertheless, taking a second and more intensive look at the concept, it has a somewhat paternalistic connotation . We should emphasize that SHF is intended as an example of Rabeharisoa's idea of a \u2018partnership model\u2019, which was meant to describe collaboration with patient organizations as equal partners in scientific research.et al. provided an impressive account of the various values attached to public involvement in research (p.7).The development of SHF was certainly value\u2010driven. Gradinger In spite of these genuinely positive values, one should keep in mind Baggott's warning of taking a too \u2018rose\u2010tinted\u2019 view of partnerships (p.169): partnerships working with the generally less powerful self\u2010help organizations can easily be misused to enhance the power of professionals and protect (economic) institutional interests, instead of establishing collaborations of equals at eye level.Most of the studies mentioned in part 3 were financially supported by the Federal Association of Company Health Insurance Funds (BKK Bundesverband), Germany. The development and testing of the instrument for the measurement of self\u2010help\u2010oriented patient\u2010centredness in hospitals (SelP\u2010K), mentioned in part 3, were conducted within a research project \u2018Quality concept \u2018Self\u2010Help Friendly Hospital\u2019 as an approach for patient centred participative care\u2019, funded by the Federal Ministry for Education and Research, 2008/01\u20102011/12. The study reported in part 4 was funded by the Bundesarbeitsgemeinschaft SELBSTHILFE , the German umbrella organization for self\u2010help associations on the federal level.One of the authors (AT) is member of the volunteer Steering Committee of the German Network \u2018Self\u2010Help Friendliness and Patient Centeredness\u2019."}
+{"text": "Many of the molecular and pathological features associated with human Alzheimer disease (AD) are mirrored in the naturally occurring age-associated neuropathology in the canine species. In aged dogs with declining learned behavior and memory the severity of cognitive dysfunction parallels the progressive build up and location of A\u03b2 in the brain. The main aim of this work was to study the biological behavior of soluble oligomers isolated from an aged dog with cognitive dysfunction through investigating their interaction with a human cell line and synthetic A\u03b2 peptides. We report that soluble oligomers were specifically detected in the dog's blood and cerebrospinal fluid (CSF) via anti-oligomer- and anti-A\u03b2 specific binders. Importantly, our results reveal the potent neurotoxic effects of the dog's CSF on cell viability and the seeding efficiency of the CSF-borne soluble oligomers on the thermodynamic activity and the aggregation kinetics of synthetic human A\u03b2. The value of further characterizing the naturally occurring Alzheimer-like neuropathology in dogs using genetic and molecular tools is discussed. Aging dogs spontaneously deposit human-type amyloid \u03b2 (A\u03b2) peptide . Upon neurological examination, this subject displayed signs of cognitive impairment and magnetic resonance imaging (MRI) showed diffuse cortical atrophy. A\u03b2 immunostaining demonstrated extensive diffuse plaques in the neocortex and hippocampal regions; but no tau staining. Of importance, CSF and serum from this subject exhibited neurotoxic effects following treatment of a human neuroblastoma cell line and led to efficient aggregation of synthetic human A\u03b2 peptides.This project was reviewed by the University of Surrey Ethics Committee and verified that the aspects of the study pertaining to the Samoyed dog including use of body fluids excess and post mortem material did not come under the auspices of Animals (Scientific Procedures) Act 1986 (ASPA). The subject's owner gave informed consent to participation in the study. Full clinical and neurological examination and presentation, including MR imaging assessment are found in Supplementary Materials. Negative control CSF was obtained from a 10-year-old male Rottweiler suffering from nodular granulomatous episclerokeratitis following submission for routine teaching post-mortem and not subject to animal ethics guidelines. CSF derived from a 79-year-old patient with advanced sporadic AD (pos1-CSF) and from a 65 year old patient with advanced sporadic AD (pos2-CSF) were provided by The UK Multiple Sclerosis Tissue Bank.A 12-year-old neutered male Samoyed dog was presented for pain management and evaluation of difficulty in rising. Neurological examination revealed tetraparesis and reduced spinal reflexes and muscle tone consistent with a polyneuropathy. The difficulty rising was attributed to this, complicated by the sedative polypharmacy. The historical and consulting room behavior suggested a cognitive function deficit possibly complicated by a urinary tract infection. The brain MRI scan revealed a diffuse cortical atrophy and a hyperintensity in the white matter on T2W, particularly in the corona radiata Figure . Signs wNeuronal loss and degeneration was marked in the cortical region of the subject Figures  while inThe ability of PRIOC10 mAb to bind to A\u03b2 soluble oligomers in sub-serum and sub-CSF was assessed by Western blotting and Sandwich ELISA. Western blotting results indicate that PRIOC10 mAb bound to soluble oligomers in sub-CSF but not in sub-serum or negative control CSF (neg-CSF) derived from the Rottweiler Figure . Further1\u201340), or PrioAD13 (A\u03b21\u201342)  and almost matched CSF levels detected with the PRIOC10 vs. biot-PRIOC10 combination. This was followed by the PRIOAD13 vs. biot-PRIOC10 combination that displayed higher CSF levels of detection compared with the PRIOAD12 vs. biot-PRIOC10 combination (p = 0.0279). Of note, sELISA lead to detection of A\u03b2 in the sub-serum , and PSEN2 (p.P436P).Genome assembly CanFam3.1 and transcripts ENSCAFT00000013599.4 for 1\u201340, monoA\u03b21\u201342, scramA\u03b225\u201335, oligoA\u03b21\u201340, oligoA\u03b21\u201342, fibA\u03b21\u201340, fibA\u03b21\u201342, sub-serum, and sub-CSF on differentiated human neuroblastoma cell line RA-SH-SY5Y viability was investigated using the MTT assay. To achieve similar concentrations of synthetic A\u03b2 and CSF/serum-borne A\u03b2, standard curves of all synthetic A\u03b2 was generated and the subject's CSF and serum A\u03b2 oligomers values were determined by comparison to the appropriate standard curve. MonoA\u03b21\u201340, monoA\u03b21\u201342, scramA\u03b225\u201335, and sub-serum displayed no toxicity toward RA-SH-SY5Y neuroblastoma cells as compared to untreated cells (p \u2264 0.05)  and \u226444% cell viability for treatment with oligoA\u03b21\u201342, fibA\u03b21\u201342, and sub-CSF (p \u2264 0.05) , while treatment with sub-CSF lead to 44% cell death. These results show that the subject's CSF induced RA-SH-SY5Y cell death and confirmed the potent toxic effects of A\u03b2 soluble oligomers previously shown to affect neurons  and 72 h (t72), as described above, was used in the seeding reaction; as t12 represents maximal optical density (OD) expression of A\u03b2 soluble oligomers immunodetected with PRIOC10 , fibA\u03b21\u201340/fibA\u03b21\u201342 (t72), or scramA\u03b225\u201335 (t12 and t72) prepared by conversion during t12 and t72 to 2 mM monoA\u03b21\u201340, or monoA\u03b21\u201342 in order to assess their effects on the \u201clag-phase\u201d kinetic as measured by ThT fluorescence. We report that t12 oligoA\u03b21\u201340, oligoA\u03b21\u201342 but not post-conversion scramA\u03b225\u201335 led to substantial reduction of the \u201clag-phase\u201d (p \u2264 0.05) compared to t0 A\u03b2 . Both t72 fibA\u03b21\u201340 and fibA\u03b21\u201342 but not post-conversion scramA\u03b225\u201335 affected the A\u03b2 aggregation kinetic by shortening the reaction's \u201clag-phase,\u201d albeit the effect was limited when compared to the addition of the oligomers  and acceleration of human synthetic monomeric A\u03b21\u201340 and A\u03b21\u201342 aggregation as compared to a negative control CSF derived from the Rottweiler or scramA\u03b225\u201335 , or anti-A\u03b21\u201342 (PrioAD13) for immunodetection, and further confirms the A\u03b2 specificity of the soluble oligomers observed on western blotting. Of importance, the sELISA detected significantly higher levels of total A\u03b2 oligomers (PRIOC10 vs. 4G8), followed by A\u03b21\u201342 soluble oligomers (PRIOC10 vs. PRIOAD13 combination) then A\u03b21\u201342 soluble oligomers (PRIOC10 vs. PRIOAD12). In contrast with the result observed on Western blotting, we were able to detect soluble oligomers in the serum of our aged dog, albeit the levels were much lower than those observed in the CSF. Taken together, these results demonstrate the presence of different species of A\u03b2 soluble oligomers in the subject's CSF in agreement with the results reported by Head et al. . Following addition of CSF derived from our behaviorally impaired dog, the A\u03b2 aggregation kinetic was substantially altered and led to reducing the lag phase, confirming both the presence but also the potent effect of soluble oligomers. In contrast, serum, negative control CSF and scrambled A\u03b225\u201335 peptide did not affect the kinetic of the reaction. The ability of CSF-borne soluble oligomer to affect synthetic peptides derived from human \u03b2 sequences was perhaps expected as the canine APP shares about 98% homology with human APP. Nevertheless, this result is important as it demonstrates inter-species molecular interaction but does not suggest nor does it demonstrate any interspecies transmission between humans and dogs. Finally, studies are underway to determine whether the above findings are a more universal phenomenon recognized in different breeds of aged and behaviorally impaired dogs.A central feature in AD is fibril biogenesis leading to senile plaques (Powers and Powers, We have comprehensively demonstrated that this behaviorally impaired dog exhibited A\u03b2 and A\u03b2 soluble oligomers in its blood, CSF, and brain. We also show that the dog's A\u03b2 affects the survival of human-derived neuron-like cell line and has a direct effect on the aggregation kinetics of human synthetic A\u03b2 peptides. The study failed to demonstrate the involvement of phospho-tau and more genetic and molecular studies are needed to decipher its role in the neuropathology underlying cognitive dysfunction, yet we would advocate that dogs with behavioral impairments should be studied and the disease mechanisms investigated in a similar fashion as is the case with AD.CR: Performed Clinical assessment of the dog, performed MRI and revised manuscript; FS: Performed experiments; MD: Performed experiments; KF: Performed experiments; JB: Performed experiments; RG: Performed experiments; AR-L: Performed experiments; DG: Performed experiments; EH: Conceived experiments and revised manuscript; SB: Conceived experiments and revised manuscript; BS: Conceived experiments and revised manuscript; JH: Conceived experiments and revised manuscript; MT: Designed experiments, performed experiments and wrote 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": "Cytosolic recognition of DNA has emerged as a critical cellular mechanism of host immune activation upon pathogen invasion. The central cytosolic DNA sensor cGAS activates STING, which is phosphorylated, dimerizes and translocates from the endoplasmic reticulum (ER) to a perinuclear region to mediate IRF-3 activation. Poxviruses are double-stranded DNA viruses replicating in the cytosol and hence likely to trigger cytosolic DNA sensing. Here, we investigated the activation of innate immune signaling by 4 different strains of the prototypic poxvirus vaccinia virus (VACV) in a cell line proficient in DNA sensing. Infection with the attenuated VACV strain MVA activated IRF-3 via cGAS and STING, and accordingly STING dimerized and was phosphorylated during MVA infection. Conversely, VACV strains Copenhagen and Western Reserve inhibited STING dimerization and phosphorylation during infection and in response to transfected DNA and cyclic GMP-AMP, thus efficiently suppressing DNA sensing and IRF-3 activation. A VACV deletion mutant lacking protein C16, thought to be the only viral DNA sensing inhibitor acting upstream of STING, retained the ability to block STING activation. Similar inhibition of DNA-induced STING activation was also observed for cowpox and ectromelia viruses. Our data demonstrate that virulent poxviruses possess mechanisms for targeting DNA sensing at the level of the cGAS-STING axis and that these mechanisms do not operate in replication-defective strains such as MVA. These findings shed light on the role of cellular DNA sensing in poxvirus-host interactions and will open new avenues to determine its impact on VACV immunogenicity and virulence.IMPORTANCE Poxviruses are double-stranded DNA viruses infecting a wide range of vertebrates and include the causative agent of smallpox (variola virus) and its vaccine vaccinia virus (VACV). Despite smallpox eradication VACV remains of interest as a therapeutic. Attenuated strains are popular vaccine candidates, whereas replication-competent strains are emerging as efficient oncolytics in virotherapy. The successful therapeutic use of VACV depends on a detailed understanding of its ability to modulate host innate immune responses. DNA sensing is a critical cellular mechanism for pathogen detection and activation of innate immunity that is centrally coordinated by the endoplasmic reticulum-resident protein STING. Here, STING is shown to mediate immune activation in response to MVA, but not in response to virulent VACV strains or other virulent poxviruses, which prevent STING activation and DNA sensing during infection and after DNA transfection. These results provide new insights into poxvirus immune evasion and have implications in the rational design of VACV-based therapeutics. Cells sense the presence of invading pathogens by the use of pattern-recognition receptors (PRRs), a set of germ line encoded molecules recognizing pathogen-associated molecular patterns (PAMPs). Nucleic acids derived from viral infection are potent PAMPs that can be recognized in the cell cytosol and the endolysosomal compartment by dedicated PRRs. Recognition of RNA or DNA leads to the secretion of type I interferon (IFN) and other inflammatory cytokines and the expression of IFN-stimulated genes (ISGs), which restrict viral replication. Cytosolic double-stranded RNA is sensed by retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5). Both RIG-I and MDA-5 associate with the IFN-\u03b2 promoter stimulator 1 , a protein residing in the mitochondrion that mediates the activation of IFN responsive factors (IRFs) and nuclear factor \u03ba-light-chain-enhancer of activated B cells (NF-\u03baB) \u20134. cGAMP\u20135\u2013Poxviruses are a highly successful family of viruses infecting a broad range of species. Besides vaccinia virus (VACV), the prototypic member of the family and the virus that was used to eradicate smallpox, there are other poxviruses that can cause disease in humans  or in animals . In addition to virulent strains, nonvirulent strains also exist and are popular candidates as recombinant vaccine vectors. Modified vaccinia virus Ankara (MVA) is an attenuated VACV strain generated through more than 500 serial passages in chicken cells. Through that serial passaging MVA lost its ability to replicate in human cells due to severe deletions and truncations affecting expression of multiple genes compared to the reference strains Copenhagen (COP) or Western Reserve (WR) , 19. AccPoxviruses contain large dsDNA genomes of about 200 kbp with a coding capacity for more than 200 proteins. Poxviruses are unique in being the only dsDNA viruses to replicate exclusively in the cell cytoplasm. Replication in this compartment, however, makes this family of viruses particularly susceptible to detection by cytosolic DNA sensors. The success of poxviruses therefore implies the evolution of countermeasures to either avoid recognition or to dampen DNA sensing-induced innate immune activation. An example of such countermeasures is protein C16, which targets DNA-PK and inhibits IRF-3 activation in response to DNA . BesidesGaussia luciferase (GLuc) under the control of the promoter of the IRF-3-dependent gene IFIT-1  responds to PAMPs, (ii) provides consistent and high-throughput quantitative measurements, and (iii) is amenable to genetic manipulation. THP-1 monocytes expressing e IFIT-1  providede IFIT-1 . Infectie IFIT-1 , 28. Cone IFIT-1 . We then did not . The pro did not . Similarnfection . The prenfection . From thVACV is a dsDNA virus that replicates in the cell cytoplasm and therefore has the potential to be detected by cytosolic DNA sensing mechanisms, in which STING has a pivotal role. To determine whether STING had an impact on the IRF-3 response induced by VACV in THP-1 cells, we transduced THP-1-IFIT-1-GLuc cells with a lentivirus expressing shRNA against cellular STING, or a control shRNA, and selected them with puromycin. Control and shSTING cells were PMA-differentiated and STING levels assessed by immunoblotting. shSTING cells showed a reduction in STING expression compared to control cells . FurtherTo further confirm that MVA-induced activation of IRF-3 in THP-1 cells derived from DNA sensing, we generated a THP-1 cell line expressing shRNA against cGAS, the main cellular DNA sensor. Immunoblotting for cGAS confirmed cGAS depletion in shcGAS cells compared to control cells . AccordiCXCL10 and IFN-\u03b2 mRNA expression 6 h after HT-DNA transfection. DNA challenge triggered expression of CXCL10 (IFN-\u03b2 (The absence of IRF-3 activation upon COP or WR infection could be explained by (i) an ability of these viruses to mask their dsDNA genome in a manner that is lost in MVA, or (ii) the production of viral factors that prevent IRF-3 activation. We addressed the presence of viral factors by determining the capacity of all three VACV strains to inhibit IRF-3 activation mediated by exogenous DNA. First, PMA-differentiated THP-1 cells were infected with 2 PFU/cell of MVA, COP, and WR for 6 h and subsequently transfected with HT-DNA. After 16 h, HT-DNA transfection induced an \u223c30-fold increase in IFIT-1-driven GLuc activity in mock-infected and MVA-infected cells, and this was completely suppressed in COP-infected and WR-infected cells . We thenf CXCL10  and IFN-0  must be present in CPXV and ECTV, and presumably in other virulent poxviruses, because both ECTV and CPXV inhibited STING activation to the same extent as VACV .Whatever the exact mechanism is, it will add to the inhibitory capacity provided by other VACV factors targeting the IRF-3 signaling cascade downstream of cGAS/STING such as proteins C6 and K7, which act at the level of TBK-1 , 24, or 38\u2013Multiple human viruses target STING function. For instance, the human cytomegalovirus protein UL82 binds to STING and prevents its translocation and activation , the Kap48\u2013IFIT-1 promoter  supplemented with 10% heat-inactivated fetal calf serum  and 100 U/ml penicillin plus 100 \u03bcg/ml streptomycin . THP-1-IFIT-1-GLuc cells were a gift from Veit Hornung . These cells had been modified to express GLuc under the control of the promoter . THP-1-I4 cells per well in the presence of PMA (50 ng/ml). After 48 h, the cells were infected in quadruplicate with the indicated viruses in RPMI 1640 supplemented with 2% FCS at the indicated PFU per cell, and the medium was collected at the indicated times postinfection. When DNA stimulation was performed, PMA-differentiated cells were infected with the indicated viruses at 2 PFU/cell for 6 h and then transfected with HT-DNA at 0.5 \u03bcg/ml for a further 16 h. Transfections were performed in Opti-MEM (Life Technologies) in the presence of the transfection reagent TransIT-LT-1 (Mirus Bio) according to the manufacturer's recommendations. When cGAMP stimulation was performed, cells were infected as described above and then exposed to cGAMP at 10 \u03bcg/ml in the medium for a further 16 h. The luciferase activity was measured in a Clariostar plate reader (BMG Biotech) in the presence of coelenterazine (NanoLigh Technology) at 2 \u03bcg/ml. Data were normalized to mock-infected samples and are presented as a fold increase.THP-1-IFIT-1-GLuc cells were seeded in 96-well plates at a density of 5 \u00d7 105 cells per well in the presence of PMA (50 ng/ml). After 48 h, the cells were infected in triplicate with the indicated viruses in RPMI 1640 supplemented with 2% FCS at the indicated PFU/cell for 16 h, or for 6 h and subsequently transfected with HT-DNA for a further 6 h as described above. RNA was extracted using a Total RNA purification kit (Norgen Biotech) and transcribed into cDNA using Superscript III reverse transcriptase (Invitrogen) according to the manufacturer's protocol. cDNA was diluted 1:5 in water and used as a template for real-time PCR using SYBR green PCR master mix (Applied Biosystems) in a LightCycler 96 (Roche). Expression of each gene was normalized to an internal control (18S), and these values were then normalized to the nonstimulated mock-infected control cells to yield a fold induction. The primers used for CXCL10 detection were as previously described  and hIFN-\u03b2 detection  were as indicated here.THP-1-IFIT-1-GLuc cells were seeded in 24-well plates at a density of 2 \u00d7 10escribed . The priCell culture supernatants from virus-infected THP-1 cells grown in 24-well plates were assayed for CXCL10 using Duoset ELISA reagents (R&D Biosystems) according to the manufacturer's instructions.4 cells per well in the presence of PMA (50 ng/ml). After 48 h, the cells were infected in quadruplicate with the indicated viruses in RPMI 1640 supplemented with 2% FCS at the indicated PFU/cell. The medium was collected 24 h later and transferred to 96-well plates containing HEK293T cells previously transfected for 24 h with 70 ng/well of a reporter plasmid expressing firefly luciferase under the control of ISRE (Promega) and 10 ng/well of a control plasmid expressing Renilla luciferase  using polyethylenimine  at a ratio of 1:2 (\u03bcg of DNA:\u03bcl of PEI). Activation with recombinant hIFN-\u03b2 at 25 ng/ml was also included as a control. At 10 h after medium transfer or hIFN-\u03b2 activation, the cells were lysed in passive lysis buffer (Promega). The FLuc and RLuc activities were measured in a Clariostar plate reader (BMG Biotech), and FLuc/RLuc ratios were calculated for each well. Data were normalized to mock-infected THP-1 samples and are presented as fold increases.THP-1-IFIT1-GLuc cells were seeded in 96-well plates at a density of 5 \u00d7 10THP-1-IFIT-1-GLuc cells were first depleted for SAMHD1 using specific short hairpin sequences expressed from the HIV-1-based shRNA expression vector HIVSiren  and sele366 , STING , cGAS , IRF-3 , phosphorylated IRF-3 Ser386 , \u03b1-tubulin , and D8 . Primary antibodies were detected using IRDye-conjugated secondary antibodies in an Odyssey infrared imager (LI-COR Biosciences). Images were analyzed using Odyssey software, and data were obtained after integration of at least three independent experiments.Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease and phosphatase inhibitors (Roche), as well as 250 U/ml benzonase (Sigma). Lysates were rotated for 30 min at 4\u00b0C and subsequently denatured for 5 min at 95\u00b0C in the presence of loading buffer. Samples were resolved by SDS-PAGE and transferred to nitrocellulose membranes  using a Trans-Blot semidry transfer unit (Bio-Rad). Membranes were blocked in 0.1% Tween/phosphate-buffered saline supplemented with 5% skimmed milk (Sigma) and subjected to immunoblotting with the following primary antibodies at the indicated dilutions: phosphorylated STING Ser5 cells per well in the presence of PMA (50 ng/ml). After 48 h, the cells were infected with the indicated viruses in RPMI 1640 supplemented with 2% FCS at the indicated PFU/cell. After 6 h, the cells were transfected with HT-DNA at 4 \u03bcg/ml for 4 h or exposed to 15 \u03bcg/ml cGAMP for 6 h and lysed as described above, with the exception that lysates were mixed with NuPAGE LDS sample buffer (Bio-Rad) and directly analyzed by SDS-PAGE.Analysis of STING dimerization was performed as described previously  with mint test with Welch's correction where appropriate using GraphPad Prism statistical software.Statistical significance was determined using an unpaired Student"}
+{"text": "Haemadipsa japonica, a sanguivorous land leech species endemic to Japan, by using nine polymorphic nuclear microsatellites (nSSR) and cytochrome oxidase subunit one (COI) sequences of mitochondrial DNA (mtDNA). Analyses using nSSR revealed that H. japonica exhibited a stronger regional genetic differentiation among populations (G'ST\u00a0=\u00a00.77) than other animal species, probably because of the low mobility of land leech. Analyses using mtDNA indicated that H. japonica exhibited two distinct lineages (A and B), which were estimated to have diverged in the middle Pleistocene and probably because of range fragmentation resulting from climatic change and glacial and interglacial cycles. Lineage A was widely distributed across Japan, and lineage B was found in southwestern Japan. Analyses using nSSR revealed that lineage A was roughly divided into two population groups ; these analyses also revealed a gradual decrease in genetic diversity with increasing latitude in lineage A and a strong genetic drift in populations of northeastern Japan. Combined with the largely unresolved shallow polytomies from the mtDNA phylogeny, these results implied that lineage A may have undergone a rapid northward migration, probably during the Holocene. Then, the regional genetic structure with local unique gene pools may have been formed within each lineage because of the low mobility of this leech species.Recent molecular studies have indicated that phylogeographical history of Japanese biota is likely shaped by geohistory along with biological events, such as distribution shifts, isolation, and divergence of populations. However, the genetic structure and phylogeographical history of terrestrial Annelida species, including leech species, are poorly understood. Therefore, we aimed to understand the genetic structure and phylogeographical history across the natural range of  Thermocycling conditions were as follows: an initial denaturation of 1\u00a0min at 94\u00b0C, followed by 35 cycles of 45\u00a0s at 94\u00b0C, 45\u00a0s at 50\u00b0C , 1\u00a0min at 72\u00b0C, and a final extension of 10\u00a0min at 72\u00b0C. PCR products were electrophoretically separated on a 2.0% agarose gel and visualized using ethidium bromide in 1\u00d7 TAE; all products exhibiting a single DNA fragment were selected for sequencing. These selected products were then purified using ExoSAP\u2010IT . Direct sequencing of both sequence directions was conducted using an ABI PRISM BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) on an ABI 3500 Genetic Analyzer. The obtained DNA sequences were visually inspected, including quality check, and aligned using BioEdit 7.2.5.0  gene sequence of 503 individuals of .0 Hall, . Multipl.0 Hall,  and then2.3Haemadipsa japonica by Morishima, Suzuki, and Aizawa (N\u00a0=\u00a01\u20132) . The substitution model GTR+G+I was selected based on the Akaike information criterion (AIC). Bootstrap support was calculated with 1,000 replications. Haemadipsa picta (accession number HQ203177) was included as an outgroup based on Lai, Nakano, and Chen (http://www.fluxus-engineering.com).A phylogenetic maximum likelihood analysis of the COI haplotypes was performed using PhyML 3.0 , H.\u00a0ornata (HQ203178), H.\u00a0rjukjuana (HQ322462), and H.\u00a0montana (HQ203182). Haplotypes of H.\u00a0japonica were selected from the haplotypes with highest frequencies in each lineage, namely, H27 from lineage A, H61 from sublineage B1, and H74 from sublineage B2 . Moreover, population differentiation measures (GST and NST) were estimated and the significant difference between them was tested using PERMUT  was conducted using Arlequin ver.3.5 , observed gene diversity within populations (HO), gene diversity within populations (HS), total gene diversity (HT), and measures of genetic differentiation among the population\u2014FST  (ranging from 1 to 20). The obtained results were harvested using STRUCTURE HARVESTER   and that natural logarithm of geographical distance between populations  and allelic richness (Ar)\u2014for populations in mtDNA lineages A and B.Spatial genetic structure was assessed by testing the significance of isolation by distance  (95% HPD: 2.12\u20133.33\u00a0Myr) . Finally, the two subclades within lineage B (B1 and B2) were estimated to have diverged ~0.41\u00a0Myr (95% HPD: 0.24\u20130.62\u00a0Myr) , gene diversity (HS), and overall genetic diversity (HT) were 0.543 (0.290\u20130.681), 0.558 (0.279\u20130.688), and 0.827 (0.440\u20130.922), respectively .Nine nSSR loci for 37 ty Table . The num 0.543 0.90\u20130.681, 0.543 0.90\u20130.681,K were observed first at K\u00a0=\u00a04, second at K\u00a0=\u00a02, third at K\u00a0=\u00a015, and fourth at K\u00a0=\u00a013  and those of southwestern Japan (populations 22\u201337); the observed boundary was in central Honshu. At K\u00a0=\u00a04, each of the two regions was roughly further divided into two; a gene pool in light blue was observed in more than half of the gene pools of each population located in central Honshu ; a gene pool in red was observed in western Honshu, Shikoku, and Kyushu (populations 32\u201337). At K\u00a0=\u00a013, a geographically unique clustering pattern was observed across populations: northern Honshu (populations 1\u20135), Niigata (populations 6\u20139), northern Tochigi (populations 10\u201312), southern Tochigi (populations 13\u201315), Gunma (populations 16\u201318), southern Nagano (populations 22 and 23), around lake Biwa , Aogaki and Shikoku (populations 32 and 33), and Kyushu and Yakushima islands (populations 34\u201337). In populations 19\u201321 and 31, the unique gene pools were dominant  and geographical distance , denoting isolation by distance for Haemadipsa japonica  and allelic richness (Ar [26]) for all loci in each population varied from 0.306 (population 6) to 0.797 (population 20) and from 2.687 (population 31) to 8.014 (population 20), respectively .A Mantel test using the nine nSSR loci showed a highly significant correlation between pairwise a Figure . The exp44.1FST and G'ST, of Haemadipsa japonica, estimated using nSSR, were much higher than those of other animals, including species with low dispersal potential, such as the canyon tree frog (Hyla arenicolor), the giant water bug (Abedus herberti), and the three aquatic leeches    Table . Further) Figure . This re Figures  and 8, m Figures ; this is Figures . Long\u2010di Figures . The STR4.2Haemadipsa japonica was comprised of two mtDNA lineages (A and B) with no shared haplotypes within the lineage , Japanese hare (Lepus brachyurus), tree frog (Hyla japonica), Pelophylax frog (Pelophylax nigromaculatus), and seed parasitic weevil (Curculio hilgendorfi)  are presently not found in the Korean Peninsula nor in the continental part of northeastern China, the hypothesis of two independent colonizations from the Asian continent to the Japanese archipelago seems unlikely, although the possibility of a later extinction of these Haemadipsa species in the continental areas cannot be ruled out. Alternatively, the second explanation is likely plausible. The global glacial and interglacial cycles had already started during the middle Pleistocene . Therefore, sublineage B2 may have diverged from other populations possessing lineage B haplotypes and undergone subsequent strong genetic drift and fixation of sublineage B2 haplotypes.Our analysis showed that  Figures  and 4. T Figures . GeneticHaemadipsa japonica were geographically separated from each other and randomly distributed in certain areas of Honshu, adaptive introgression of mtDNA seems an unlikely explanation. An alternative explanation is demographic consequences. This means that, after the divergence of lineages A and B, populations possessing each haplotype may have had secondary contact with mtDNA introgressive hybridization and may have separated from each other again, which was then followed by population fragmentation and genetic drift. As previously discussed in this text, H.\u00a0japonica is considered to have low mobility; therefore, subsequent gene flow among these populations could have been reduced. As the effective population size of mitochondrial genome in hermaphrodites represents only one half of a nuclear genome , the gradual decrease in genetic diversity with latitude within lineage A may have been a result of a northward migration followed by a subsequent founder event; such northward migration has also been suggested by studies on the alpine butterfly  in the Eurasian continent , lineage A of northeastern Japan (populations 1\u201321) has almost exclusively unique gene pools (in blue and light blue) of nSSR, except for populations 19 and 20 at Haemadipsa japonica, deep divergence should be engraved within the species\u2019 mtDNA; however, this divergence was not found. Therefore, alternative explanations are that these mountain chains may have acted as geographical barriers to the dispersion of H.\u00a0japonica or that complex geographical features in these mountain regions may have served as pocket refugia during a recurrent northward migration followed by a retreat toward south near central Honshu\u2014traced by the presence of the gene pool in light blue in central Honshu , which diverged in the middle Pleistocene. After the LGM, probably during the Holocene, H.\u00a0japonica underwent a northward migration with a rapid divergence of haplotypes; the subsequent regional strong genetic structure may have been a result of the low mobility of H.\u00a0japonica.In this study, H.\u00a0japonica, besides new areas of occurrence toward which leech populations may locally expand.Since the 1980s, the populations of this leech have expanded, possibly because of the increase in mammal populations, and it has become a serious problem in several prefectures in Japan, as the leech has expanded to areas near human residence (Asada, Ochiai, & Yamanaka, None declared.K.M. and M.A. designed this study and conducted sampling in field. K.M. conducted nuclear microsatellite genotyping, sequencing of mtDNA, and analyzed data. All authors contributed to writing this manuscript.\u00a0Click here for additional data file."}
+{"text": "Role of laboratory service in patient care is well recognized in developed compared to developing countries like Ethiopia where most medical decisions are based on clinical judgment. Laboratory based medical decisions save life and resources. Thus, health facilities (HFs) having capacitated laboratories provide better health care service. Thus, this study assessed capacity of laboratories found in all nine HFs in zone one of Afar region, Ethiopia. Cross-sectional study was conducted from January to May 2015. Data were collected using questioner from medical laboratory professionals (MLPs) and using cheek list from laboratory registration books. Collected data was analyzed using SPSS. Availability of laboratory related national guidelines and standard operating procedure varies among HFs studied. In 42 selected laboratory equipment and materials assessed, their availability shows variations among HFs from 13 (30.2%) to 32 (74.4%). Among different laboratory tests recommended to be performed at health center (HC) level by World Health Organization (WHO), six tests were not performed in all HC laboratories. Moreover, 11 (31.4%) MLPs received in-service training in the past one year. Internal quality control measures were done in 3 (33.3%) laboratories. However, all laboratories were involved in external quality assurance with varied schedules. Specimens after testing and specimen with prolonged transit time were discarded using standard rejection criteria in 8 (88.9%) and 3 (33.3) laboratories, respectively. Study showed that laboratories assessed have good capacities in involving external quality assurance and having proper laboratory test request format. But capacity of laboratories assessed is limited and unsatisfactory in number of laboratory tests performed at HC laboratories, in internal quality control measure implementation, and in getting up-to-date in-service training to staff. Thus, to enhance capacity of laboratories in HC, responsible bodies shall avail basic laboratory materials and equipment, make fair distribution of MLPs, provide up-to-date training, and implement internal quality control measures in laboratories. Laboratory services, either diagnostic or screening tests, are crucial parts of the health system having great contribution for infectious and noninfectious disease prevention and management . The impIn Africa, building the capacity of laboratory services in health facilities was given low priority for long time because of different challenges which include resource constrain, poor infrastructure, low qualified human resource, weak procurement and supply systems, lack of quality standards, limited quality assurance measures, poor laboratory management, and failure to implement/develop national laboratory policies , 4, 8, 9But as to our knowledge, the status of laboratory capacity in the study area was not studied. Studying the capacity of laboratories had a lot of significance which includes support proper laboratory resource allocation, indicates capacity gaps allowing different organizations working on the area to build laboratory capacities properly, and gives guidance for policy makers to strengthen the laboratory as well as health system at large. Therefore, objective of the present study was to assess the capacity of laboratories in health facilities found in zone one of Afar regional state, Ethiopia.2, shares common international boundaries with the State of Eritrea in the northeast and Djibouti in the east, as well as regional boundaries with Tigray Regional States in north-west, Amhara in the southwest, Oromia in the south and Ethiopian Somali in the southeast. The Afar National Regional State consists of 5 administrative zones (subregions) and 32 administrative units called Weredas .Based on assessments done on 42 selected laboratory equipment and materials, all nine health facility laboratories assessed had staining rack, drying rack, microscope slides, lancets, sharp container, and gloves in their laboratories. On the other hand, 66.7%, 55.6%, and 44.4% of health facilities did not have autoclave, differential counter, and hematocrit centrifuge in their laboratories, respectively [Though certain guidelines were available in assessed laboratories, none of them had guideline for destruction of damaged and/or expired products. Moreover, 66.7% of laboratories had standardized laboratory request format, whereas 3 (33.3%) laboratories assessed had criteria for discarding specimen with prolonged transport time . All labIn Ethiopia, there is an organized regular external quality assurance (EQA) program implemented in all health facility laboratories. Generally the hospital laboratories will conduct EQA programs on health center laboratories found in their catchment area. In turn the regional laboratories will conduct EQA on hospital laboratories in their catchment area. The national public health institute laboratory found in the country capital Addis Ababa will conduct EQA programs on the regional laboratories. Mycobacterium spp. examination using acid fast staining and malaria diagnosis using Wright's or Giemsa stained blood film as well as in rapid serological tests of Human Immunodeficiency Virus (HIV) and to some extent to hepatitis B and C viruses. Moreover, 66.7% laboratories had different standard operating procedures.Majority of health facility laboratories (66.7%) assessed did not perform internal quality control measures like running controls daily, checking each reagent using known positive and negative controls, and counter check test reports with another colleague before dispatch. On the other hand, all health facility laboratories participated in the mandatory EQA program organized by the regional laboratory found in the region capital of Samara town (Ethiopia) and national public health institute of Ethiopia found in Addis Ababa. The EQA methods implemented by regional and national organizations include on site assessment and blind checking. After conducting both EQA methods, the organizations who conducted EQA will send feedbacks to the health facility laboratories based on the results. In the present study, the involvement of health facility laboratories in the mandatory national EQA program was regular in every three months. On the other hand, their involvement in the mandatory regional EQA program was not in regular base which varied from randomly performed to every 6-12-month period. The EQA measures were done inA total of 35 medical laboratory professionals were working in nine public health facility laboratories with different level of qualification . Out of In the past one year, only 11 (31.4%) medical laboratory professionals received at least one training out of four in-service training sessions assessed in the study, namely, malaria, TB, HIV, and hepatitis. Six (54.5%) medical laboratory professionals attended training which were organized at national level where the remaining 5 (45.5%) training programs attended were organized at regional and zonal levels. the study also showed that in 5 (55.6%) health facility laboratories, the last laboratory supervisions done by either regional laboratory or national public health institute were before one month. But in the remaining laboratories, the last supervision was done in more than three months back.Though all health facility laboratories had ministore or separate area within their laboratories for storing laboratory materials and reagents, record keeping of reagents delivered from store to laboratory for consumption was found only in 5 (55.6%) health facility laboratories. Moreover, there was no any system in 8 (88.9%) health facility laboratories for regular monitoring of material and reagent quantities available in their laboratory store. In 66.7% of health facility laboratories, there were a problem of obtaining adequate stock of laboratory reagents and equipment. The main reasons for under stock of materials and reagents were delay in ordering of laboratory reagents by health facilities form the central Pharmaceutical Fund Supply Agency (PFSA) in Addis Ababa and challenge in transportation system to deliver reagents from central to regional PFSA and health facility store in 55.6% health facilities studied followed by inconsistency of test demands by physicians (11.1%) and lack of information on how to obtain reagents and materials (11.1%).Incineration using locally combustible materials (in 66.7%) and burial (in 33.3%) was used as methods for solid waste disposal. For liquid waste disposal, burial and others were used. In 66.7% of health facility laboratories, safety officer was present. There was no immunization offered for new staffs in any of health facility laboratories studied. In all laboratories studied, latex examination gloves were used as protective equipment.Out of the nine health facilities studied, 2 were hospital and one was regional laboratory. The rest 6 health facilities were health centers. Based on WHO recommendation, 18 tests can be done at health center laboratory level. Among all six health centers studied, all of them do not perform 6 tests recommended to be performed at health center .Different health care system stakeholders agreed that having laboratories with organized capacity to conduct different screening and diagnostic laboratory tests is one of the major inputs for providing quality health care system . PublishOnly 22 (52.4%) materials and equipment assessed were found in all laboratories. Moreover, none of the laboratories had biosafety cabinet level-II as well as only three laboratories had basic laboratory equipment like Bunsen burner, autoclave, incubator, and ELISA. This finding agrees with a study done by Ishengoma DR et al. which reported that 43% of the laboratories did not have basic laboratory equipment . MoreoveSkilled medical laboratory professionals with the appropriate competencies and motivation are vital to the delivery of adequate laboratory services . Among 3The present study also showed that only 31.4% of medical laboratory professionals received at least one out of four in-service training  considered which is by far very low compared with the study by Hailegiorgis B et al. where 24% of laboratory professionals were involved in malaria microscopy training assessed . This vaLaboratory quality assurance systems are critical to the success of any laboratory service . With thBut the present study also showed that 66.7% of laboratories did not use standard criteria for discarding specimens with prolonged transit time. This finding, coupled with the very hot temperature of the study area, indicates that majority laboratories were not following the proper protocol to deliver laboratory service.The infection prevention and control measures of laboratories were very low as it is being manifested in the availability of guideline on infection prevention, safe disposal of sharps, and safe disposal of biohazard medical waste only in 5 (55.6%) health facility laboratories. Moreover, all of the laboratories did not have guideline for destruction of damaged and/or expired laboratory materials and reagents.Capacitated laboratories are crucial for accurate diagnosis . The pre"}
+{"text": "Tobacco tax policies have been proven to be effective in reducing tobacco consumption, but their impact can be mitigated through price-minimizing behaviours among smokers. This study explored the purchase sources of tobacco products and the price paid for tobacco products in six EU member states.Data from Wave 1 of the EUREST-PLUS ITC Europe Survey collected from nationally representative samples of adult smokers in Germany, Greece, Hungary, Poland, Romania and Spain (ITC 6E Survey) were used. The ITC 6E Survey sample, conducted in 2016, randomly sampled 6011 adult cigarette smokers aged 18 years or older. Information on purchase sources of tobacco was examined by country. The difference in reported purchase price by purchase location (store vs non-store/other) was analysed using linear regression for each country.Tobacco purchasing patterns and sources varied widely between countries. Non-store/other purchases were very rare in Hungary (0.1%) while these types of purchases were more common in Germany (5.1%) and Poland (8.6%). Reported prices of one standard pack of 20 cigarettes were highest in Germany (4.80\u20ac) and lowest in Hungary (2.45\u20ac). While non-store purchases were only made by a minority of smokers , the price differential was considerable between store and non-store/other sources, up to 2\u20ac per pack in Greece and in Germany.The results suggest a huge variation of purchasing sources and price differentials between store and non-store purchasing sources across the six EU member states examined. While the cross-sectional data precludes any causal inference, supply chain control through licensing as introduced in Hungary and the lack of such measures in the other countries might nevertheless be a plausible explanation for the large differences in the frequency of non-store purchases observed in this study. Approximately half of smokers die prematurely, resulting in an average loss of 10 years of life2. However, high smoking rates persist with one in four EU citizens still smoking3. Raising cigarette prices through tobacco taxes is considered to be the single most effective intervention to reduce demand for cigarettes5. According to a recent simulation study6, an average price increase of 10% throughout the EU would lead to an average increase in revenues by 6.8% and the average tobacco taxation benefit of all EU countries would increase by US$ 6.6 million. Most importantly, such a rise in cigarette price would significantly reduce cigarette consumption and the number of deaths caused by smoking6.Smoking is the largest avoidable risk for premature death in the European Union (EU), responsible for 0.7 million deaths each year7. Fifty of 53 countries in the WHO European region have ratified the WHO FCTC8 including all countries that participate in the EUREST-PLUS project. In the EU, regulations of the Council Directive 2011/64/EU provide for a minimum amount of excise duty of 90\u20ac per 1000 cigarettes and minimum excise duties of 60% of the weighted average retail price (unless excise tax is higher than \u20ac115/1000 cigarettes)9. WHO recommends a tax rate of at least 75% of cigarette retail prices, which \u2014 based on EU reported rates from July 2016 \u2014 is achieved by 26 of the 28 EU member states10. However, considerable price differentials between different tobacco products as well as between countries remain, providing smokers with cheaper alternatives and potentially weakening the health impact of tax policies.The World Health Organization (WHO) Framework Convention on Tobacco Control (FCTC) Article 6 includes the recommendation that each Party implements taxation and pricing policies to reduce the demand for tobacco, taking into account the sovereign right of the Parties to determine and establish their own taxation policies14. Given the scarcity of comparative studies of tobacco purchasing patterns in the EU thus far, the purpose of this study was to explore purchasing behaviours, i.e. where smokers buy tobacco and the price of tobacco products, in six EU countries.Previous studies on purchasing patterns have shown that significant numbers of smokers engage in cost-reducing tobacco purchasing behaviours in several countries15. One objective of EURESTPLUS is to monitor and evaluate the impact of tobacco control policies through the creation of a longitudinal cohort of adult smokers in 6 EU countries . This cohort study\u2014the EUREST-PLUS ITC Europe (ITC 6E) Survey\u2014is part of the ongoing International Tobacco Control (ITC) Policy Evaluation Project16.This study was conducted in the framework of the EUREST-PLUS project (European Regulatory Science on Tobacco: Policy implementation to reduce lung diseases)17.Data were collected between June and September 2016 from nationally representative samples of adult cigarette smokers aged 18 years or older in six EU countries . The geographic strata were NUTS  regions crossed with degree of urbanization . Approximately 100 area clusters were sampled in each country, with the aim of obtaining 10 adult smokers per cluster. Clusters were allocated to strata proportionally to the size of the population aged 18 years and older. Within each cluster, household addresses were sampled using a random walk design. Where possible, one randomly selected male smoker and one randomly selected female smoker were chosen for interview from a sampled household. Screening of households continued until the required number of smokers from the cluster had been interviewed. All interviews were conducted face-to-face by interviewers using tablets (CAPI). Further details on methods used in the ITC 6E Survey can be found elsewhereThe study was approved by the Research Ethics Board of the University of Waterloo, Ontario, Canada and by local ethics boards within the study countries. Participation in the study was contingent on provision of individual informed consent, which was obtained either in written or verbal form according to local ethical requirements. The EUREST-PLUS Project is registered in Clinicaltrials.gov with trial registration number NCT02773836.18. Prices per cigarette or cigarette equivalent were then multiplied by 20 to obtain the price per standard pack of 20 cigarettes. All prices were converted to Euros for Hungary, Poland and Romania, based on historical exchange rates from July (using the online currency converter http://www.xe.com).Respondents were asked about the last time they purchased their tobacco products and the purchase source, the product purchased, and the price they paid. The purchase sources were ascertained by asking \u2018Where did you last buy cigarettes or tobacco for yourself?\u2019 with response options: \u2018Large grocery store/supermarket\u2019, \u2018Small grocery store/convenience store\u2019, \u2018Bar, restaurant, or entertainment establishment\u2019, \u2018Duty-free shop \u2019, \u2018From someone else selling cigarettes independently and/or illegally\u2019, \u2018The internet\u2019, \u2018Newsstand\u2019, \u2018Tobacconist\u2019, \u2018Vending machine\u2019, \u2018Kiosk\u2019, \u2018Gas station\u2019, and \u2018Other\u2019. For some analyses these purchase sources were categorized into \u2018store\u2019 vs \u2018non-store/other\u2019, for which the options: \u2018Duty-free shop\u2019, \u2018From someone else selling cigarettes independently and/or illegally\u2019, and \u2018The internet\u2019 and \u2018Other/don\u2019t know\u2019 where categorized as \u2018non-store/other\u2019, and all other options were collapsed into the \u2018store\u2019 category. For the product purchased, respondents were asked whether the last time they bought cigarettes for themselves they purchased: \u2018A carton of factory-made cigarettes\u2019, \u2018A pack of factory-made cigarettes\u2019, \u2018Rolling tobacco\u2019 or \u2018Both packages of factory-made cigarettes and rolling tobacco\u2019. The respondents were then asked to indicate the price they paid per carton or pack of factory-made (FM) cigarettes, as well as the size of the carton (number of packs) and/or pack (in number of cigarettes), or the price they paid for roll-your-own (RYO) tobacco, respectively, and the size of the RYO unit (in grams). Based on this information, the price per cigarette was calculated. To obtain prices for RYO, it was assumed that 0.75 g of RYO was equivalent to 1 cigaretteRespondents were described according to sociodemographic characteristics , and smoking behaviour. Age was categorized into four age groups . Degree of urbanization was classified as rural, intermediate or urban. Education was categorized into low , moderate , and high . Monthly household income information was collected using local currencies and was used to classify respondents into low, moderate or high income using country specific thresholds . Smoking behaviour was described by frequency of smoking , smoking intensity measured as number of cigarettes smoked per day , and type of cigarettes smoked .Purchase patterns and price paid were examined separately by country with cross tabulations, using the Rao-Scott chi-squared test or t-test, respectively, to test for statistically significant differences across countries. In an analysis using pooled data, a country by purchase location interaction term was included to test for differences across countries. The difference in reported purchase price by country and purchase location was analysed using a linear regression model for each country. Data were analysed using SAS 9.4. Data were weighted to ensure results were representative of the population of smokers in each of the 6 EU countries. All statistical analysis accounted for the complex sampling design of the ITC 6E Survey.The sociodemographic characteristics of the sample of smokers in each country are shown in Except for Hungary (46%), more than 70% of smokers in each country smoked FM with the high proportions in Romania (94%) and Poland (80%). Hungary had the highest proportion smoking RYO (46%), followed by Greece (27%), Spain (18%) and Germany (11%) (Rao-Scott chi-squared test p<0.001).With the exception of Hungary, most smokers reported last purchasing FM cigarettes by the pack (from 69% of German smokers to 92% of Romanian smokers). In Hungary, 48% of smokers last purchased FM cigarettes by the pack while 46% last purchased RYO. Carton purchases were relatively rare in most countries , except for Germany, where 11% of smokers reported last purchasing cigarettes by the carton.This is the first study that explores and compares cigarette purchasing patterns in several EU member states. This study of smokers from six EU member states showed large differences with regards to cigarette purchasing patterns between countries. While the vast majority of tobacco purchases were made in stores, the types of stores differed across countries with tobacconists being the most used source in Hungary and Spain compared with kiosks in Greece, convenience stores in Poland and Romania, and a wide range of sources in Germany with large stores being the most common. Illicit sources were generally rare, with no purchases from illegal sources in Hungary, and most common in Poland.10. While non-store purchases were only made by a small minority of smokers , the price differential was found to be considerable. The average absolute price difference between the tobacco products bought in stores compared to non-store locations was highest in Greece and Germany, with about 2\u20ac difference per pack.There were significant differences between the average prices of tobacco products by last purchase location (store vs non-store) in four of the six countries. The average price of tobacco products purchased from stores was highest in Germany and was lowest in Hungary. The average prices from store-sources were roughly in line with the weighted average prices reported by the EU12, United Kingdom14, and the United States11. There is evidence that smokers mitigate price increases through purchasing behaviour: studies indicate that smokers make use of available options to reduce their tobacco expenditures14, while changes in purchase patterns seem to be related to price increases14. This has important public health implications as purchasers of low/untaxed cigarettes tend to make less quit attempts compared to purchasers of full-priced cigarettes13.The findings of this study are consistent with studies using comparable ITC data from other countries showing significant portions of smokers purchasing cheaper forms of tobacco such as RYO, and/or purchasing from low-cost or tax-free sources, such as in China19. For example, Hungary implemented a law in 2012 prohibiting tobacco sales other than in national tobacco shops. Due to this law, from 1 July 2013, tobacco products can be purchased only in 7000 controlled stores, instead of more than 40000 shops in the country earlier. While the cross-sectional nature of this study precludes any causal interpretation, this law could nevertheless be a likely explanation for the extremely high prevalence of cigarette purchases at tobacconists and the very low non-store purchases observed in Hungary. Rather than using lower-cost or illicit sources, price-minimizing behaviours in Hungary might be reflected in the comparably high prevalence of purchases of cheaper RYO.Even though reduction in the number of legal retail outlets is not mentioned in the WHO FCTC as a measure to influence demand for tobacco, controlling the supply chain is nevertheless seen as important for efficient and effective tax administration20. WHO recommends a simple tax system whereby excise taxes on all tobacco products represent a minimum of 75% of the retail price21. However, as higher prices give smokers the incentive to look for cheaper tobacco products (e.g. RYO) and brands, it is essential to reduce the difference between the prices of different products and brands. In addition, measures should be taken to combat illicit trade and to control the supply chain. This would not only ensure that taxation measures unfold their full public health impact but also would make tobacco tax revenues reliable and stable.Tobacco taxation has been recognized as the most effective tool to curb smokingThe strength of this study is that large samples of current smokers with internationally standardized questionnaires could be analysed to explore tobacco purchase patterns. The cross-sectional nature of the data however precludes any causal inference, especially with regards to the impact of policies on prices paid or purchase sources. Some caution is also warranted regarding the validity of the data because the self-reported measures are prone to recall and social desirability bias. In particular, the use of illicit sources of tobacco could be underreported. Future studies using longitudinal data should further examine changes in prices or purchase sources, in relation to smoking behaviour and cessation.The results of this study suggest large variations in purchasing sources between countries as well as price differentials between store and non-store tobacco purchases across the six EU member states examined. RYO use also varied across countries. Due to the lower tax levels of RYO tobacco, smokers in this study may have reduced their tobacco expenditures by choosing to smoke RYO tobacco instead of factory-made cigarettes. To ensure that national price and tax policies yield maximal potential, countries should harmonize tax rates across tobacco products. Smokers also purchase tobacco from illicit tax-free or low-cost sources. Even though non-store purchases were rare in all countries, price differentials between store and non-store purchases were considerable. Low-cost non-store purchases can undermine national tax and price policies. While the cross-sectional study design does not allow any causal inferences to be made, the results are nevertheless consistent with the interpretation that Hungary\u2019s strategy to implement a system of national retailer licensing may have prevented non-store purchases at potentially lower prices."}
+{"text": "Existing structural components require strengthening after a certain period of time due to increases in service loads, errors in design, mechanical damage, and the need to extend the service period. Externally-bonded reinforcement (EBR) and near-surface mounted (NSM) reinforcement are two preferred strengthening approach. This paper presents a NSM technique incorporating NSM composites, namely steel and carbon fiber-reinforced polymer (CFRP) bars, as reinforcement. Experimental and analytical studies carried out to explore the performance of reinforced concrete (RC) members strengthened with the NSM composites. Analytical models were developed in predicting the maximum crack spacing and width, concrete cover separation failure loads, and deflection. A four-point bending test was applied on beams strengthened with different types and ratios of NSM reinforcement. The failure characteristics, yield, and ultimate capacities, deflection, strain, and cracking behavior of the beams were evaluated based on the experimental output. The test results indicate an increase in the cracking load of 69% and an increase in the ultimate load of 92% compared with the control beam. The predicted result from the analytical model shows good agreement with the experimental result, which ensures the competent implementation of the present NSM-steel and CFRP technique. Strengthening of existing reinforced concrete (RC) structures is a necessity due to the destructive environmental conditions, increased service loads, as well as errors in design and during construction. Externally-bonded reinforcement (EBR) and near-surface mounted (NSM) reinforcement are the leading strengthening techniques used for strengthening existing RC structures ,2,3,4,5.et al. [et al. [Several experimental investigations on flexural behavior of RC beams strengthened with NSM bars or strips using FRP materials have been introduced ,21,22,23et al.  investig [et al.  used NSMIn this paper, RC beams strengthened with the NSM composites technique and their structural performance was investigated by subjecting the beams to static loading. The studied parameters include the number of NSM grooves, type of NSM strengthening materials (CFRP and steel bars), and NSM reinforcement ratios. Steel is used as the strengthening material to ensure a cost-effective solution which is also structurally efficient , an epoxy adhesive, was used to bond the strengthening materials to the concrete substrate. Sikadur\u00ae 30 has two components, namely component A and component B. Component A is white in color, while component B is black. These two components were mixed in a ratio of 3:1 until a uniform grey color was achieved. The density was 1.65 kg/L at 23 \u00b0C after mixing. The bond strength according to DIN EN 24624 is 21 MPa with steel and 4 MPa with concrete. The compressive, tensile, shear strength, and the modulus of elasticity of the adhesive are shown in Beam specimens were cast using normal concrete. Crushed granite with a maximum size of 20 mm was used as coarse aggregate. Natural river sand was used as fine aggregate. Fresh tap water was used to hydrate the concrete mix during the casting and curing of the beams, cubes, prisms, and cylinders. The concrete mix design was in accordance with the DOE method  as shownbd \u00d7 1.5 bd (where bd is the diameter of the tension reinforcement) into the concrete cover of the beam specimens at the tension face in the longitudinal direction. The groove was made using a special concrete saw with a diamond blade. A hammer and a hand chisel were used to remove any remaining concrete lugs and to roughen the lower surface of the groove. The groove was cleaned with a wire brush and a high-pressure air jet. The details of the groove are shown in In the NSM-steel technique, strengthening bars were placed into grooves cut in the concrete cover of the RC beams and bonded using epoxy adhesive groove filler. The installation of the strengthening steel bars began with cutting groove with the dimensions 1.5 The instrumentation of the beams is presented in The flexural capacities of tested beams were evaluated by a static four point bending test, causing the larger portion of the beams to take maximum stress. The key factors considered in this studies are cracking load, ultimate load, crack width and spacing, concrete compressive strain, tensile strain in the main steel reinforcement, sectional strain, and mode of failures. The experimental performance of all tested beams in terms of flexural load capacities are shown in versus midspan deflection curves for beams CB, N-1, N-2, N-3, N-4, and N-5 are presented in i.e., flexure failure.The load versus crack width of the beam specimens are presented in The load The failure modes of all the beams tested in this study are presented in versus concrete compressive strain at the top fiber of the beams is presented in The load Demec gauges were attached along the depth of the beam at midspan  and measThe influence of NSM reinforcement on the performance of the strengthened RC beams is presented in The effect of NSM reinforcement type on the performance by strengthening is presented in The influence of number of NSM groove on the NSM strengthening of RC beams are presented in Failure of a strengthened beam by concrete cover separation is governed by two factors . The firThe analytical model for predicting concrete cover separation is illustrated in AI is moment of inertia of the section (b \u00d7 Smax):AM is the moment at point A, b is beam width, Smax is the maximum crack spacing, NSMd is the distance between the center of gravity of the NSM bars and the concrete compression fiber, y is the depth of the neutral axis, crI is the cracking moment of inertia, cf\u2019 is the compressive strength of concrete and BM is the moment at point B.n is the modular ratio.The tensile stress at the critical point A (From Equations (4) and (5) the following equation was obtained:ctf and the bending moment at point B (BM) is heading towards the beam failure load:Concrete cover separation occurs when the tensile strength at point A attains the ultimate tensile strength of the concrete eI) [The deflection prediction of flexural RC member can be calculated using the effective moment of inertia (Ie) , which iP is the predicted load, L is the total span length and La is the shear span length.Therefore, the midspan deflection of RC beam specimens in this study was estimated  using thk1 is the bond coefficient , k2 is the strain distribution coefficient , As is the area of the tension steel, The maximum flexural crack spacing is reliant on the modular ratio and the position of the neutral axis of the composite section. Thus, to predict the crack spacing for the composite strengthened beams in this study the following analytical model  was usedCw) is expressed by the following equations:The crack width approach  used to The structural characteristics obtained from the experiments were compared with the predicted results as acquired from the above presented analytical models in order to clarify the authenticity of the investigation. It is worth mentioning that along with the experimental study, the analytical models are also useful tools for predicting the behavior of composite structural elements. To verify both approaches, the concrete cover separation failure load, deflection, and maximum crack spacing were evaluated.The comparison between the ultimate deflection from the experimental tests and the predicted model is shown in The NSM strengthening technique with steel bars is a very cost effective method to improve the flexural strength of RC beams.The strengthened beam specimens expressed a bi-linear response to loading, characterized by cracking and ultimate stages. The cracking stage of the strengthened beams followed a linear elastic shape identical to the control beam.The NSM steel or CFRP bar technique increased the first crack load. Strengthened beams exhibited smaller crack spacing than the control beam. Thus, beams strengthened with NSM bars develop more and finer cracks with closer spacing than unstrengthened beams.2 improves the first crack load from 27% to 69% and the ultimate load capacity from 43% to 84%, respectively. NSM-CFRP increases the first cracking and ultimate load of 59% and 92%, respectively. After the yielding of internal reinforcement, NSM reinforcement controls crack widths until failure of the beam.Increasing the amount of NSM steel reinforcement from 100 to 226 mmThe strengthened beams failed in premature debonding. The ultimate load was reached in the instant of debonding and directly afterwards, after which imposed loading decreased rapidly. The predicted results from the analytical models complied very well with the experimental results.Based on the experimental and analytical study conducted, the effect of different variables on the flexural strength of RC beams strengthened with NSM steel and CFRP bars have been presented in this paper. The influence of varying NSM reinforcement ratios on the strengthened beams is studied. The flexural capacity, deflection, mode of failure, concrete top fiber strain, tension steel strain, and crack width of each of the tested beams were analyzed. The following conclusions can be drawn:"}
+{"text": "We report a case study of fat embolism seen on ultrasound at right internal jugular vein during central venous cannulation in a patient diagnosed with fat embolism syndrome. This case demonstrates the importance of ultrasound for evaluation of trauma cases with suspicion of fat embolism.A 23-year-old trauma patient with closed fracture of left femoral shaft and left humerus presented to our emergency department (ED). 11\u00a0h after admission to ED, patient became confused, hypoxic and hypotensive. He was then intubated for respiratory failure and mechanically ventilated. Transesophageal ultrasound revealed hyperdynamic heart, dilated right ventricle with no regional wall abnormalities and no major aorta injuries. Whole-body computed tomography was normal. During central venous cannulation of right internal jugular vein (IJV), we found free floating mobile hyperechoic spots, located at the anterior part of the vein. A diagnosis of fat embolism syndrome later was made based on the clinical presentation of long bone fractures and fat globulin in the blood. Despite aggressive fluid resuscitation, patient was a non-responder and needed vasopressor infusion for persistent shock. Blood aspirated during cannulation from the IJV revealed a fat globule. Patient underwent uneventful orthopedic procedures and was discharged well on day 5 of admission.Point-of-care ultrasound findings of fat embolism in central vein can facilitate and increase the suspicion of fat embolism syndrome.The online version of this article (10.1186/s13089-019-0116-9) contains supplementary material, which is available to authorized users. Fat embolism is a relatively under-recognized condition in patients with long bone fractures. When symptomatic, it can lead to serious effects on vital organs and even death. Diagnosis of fat embolism remains a challenge to clinicians today, as multiple diagnostic criteria exist. We report a case where the use of point-of-care ultrasound was able to visualize fat deposited at right internal jugular vein and increase the index of suspicion in diagnosis fat embolism syndrome. This case illustration demonstrates the importance of ultrasound as a tool to evaluate trauma cases with suspicion of fat embolism.A 23\u00a0years old with no known medical illness presented to our emergency department after a head-on collision. He sustained closed fracture of the left femur Fig.\u00a0a, closed2 given via non-rebreathing mask. He was intubated and mechanically ventilated. Extended Focused Sonography for Trauma was normal. Bedside transesophageal ultrasound revealed hyperdynamic heart, dilated right ventricle with no regional wall abnormalities and no major aorta injuries. Computed tomography of the whole body was also normal.11\u00a0h after ED admission, he was noted to be tachypneic and hypotensive with severe tachycardia. ABG showed hypoxemia and metabolic acidosis, despite ODuring central venous cannulation of the right internal jugular vein (IJV), we found multiple mobile and floating hyperechoic shadows deposited at the anterior part of the right IJV Fig.\u00a0c. A diagIn the intensive care unit (ICU), patient\u2019s hemodynamic and respiratory status improved after continuous fluid and vasopressor therapy. Patient underwent multiple uneventful orthopedic procedures and was discharged well on day 5 of admission.Fat embolism is defined as evidence of fat globules found in the lung parenchyma and blood circulation usually occurring in trauma patients with long bone fractures . A smallIn a retrospective study from 1979 to 2005 at the United States, Stein et al. reported incidence of fat embolism syndrome (FES) to be 0.12% to 0.7% in patients with fractures. Multiple fractures are found to increase the risk of FES compared to isolated fractures with femur being the most common cause [2\u2009<\u200960\u00a0mmHg) on FiO2 of 0.4. Minor features are fever >\u200938.5, tachycardia HR\u2009>\u2009110 beats/min, retinal changes (fat or petechiae), renal changes , sudden drop in hemoglobin, sudden thrombocytopenia, high erythrocyte sedimentation rate, jaundice and fat globules in the sputum.Diagnosis of fat embolism syndrome remains a challenge to clinicians today, as multiple diagnostic criteria exist. The most commonly used Gurd and Wilson\u2019s criteria since 1974 proposed that 2 major criteria or 1 major and 4 minor criteria suggest a diagnosis of FES . Major cThe limitations of the existing diagnostic criteria are the lack of laboratory markers or imaging tests to confirm the diagnosis of FES. Thus, the diagnosis of fat embolism is still based on clinical discretion of the treating physician after excluding other causes , 8. ThisThe improvement of the quality of transthoracic echocardiography nowadays makes it a useful tool to assess for fat emboli in the circulation and heart of patients with long bone fractures as demonstrated in multiple case reports \u201311. Fat It should be borne in mind that similar hyperechogenic spheres may also appear on ultrasound with infusion of blood products or agitated saline . AnotherBased on our experience in this case, we propose searching for fat globules at deep veins near fracture sites such as the femoral vein in lower limb fractures and the IJV in upper limb fractures. There is currently no algorithm for point-of-care ultrasound in trauma patients to look for fat emboli. We will like to suggest scanning any vein proximal to the fracture site, followed by inferior vena cava and both sides of IJV. To confirm the diagnosis of fat emboli, ultrasound can serve as a useful tool to aspirate the fat globules under direct visualization. Larger systematic studies are needed to test out this hypothesis and determine whether diagnosis of fat embolism can be established earlier. This new idea may lead to prevention of FES complication and decrease in mortality.To date, there is no proven treatment of FES, once it manifests in a patient. Prevention and high index of suspicion in high-risk patients are, therefore, needed to detect the condition early. Several measures had been identified as prophylaxis to prevent FES, but the quality of evidence is not strong. Hydration had been postulated to decrease the risk of FES due to the dilutional effect on the fat emboli and stress-related mediators . AnotherAlthough early operative fixation had been found to reduce the incidence of FES, the increased intramedullary pressure during fixation of fractures ironically increased the risk of FES . EmbolicPoint-of-care ultrasound findings of fat embolism in central vein can facilitate and increase the suspicion of fat embolism syndrome. Early diagnosis can help with prompt supportive treatment and improve outcome of this potentially fatal condition.Additional file 1. Multiple mobile and floating hyperechoic shadows deposited at the anterior part of the right IJV."}
+{"text": "There was a widespread consensus on the need to modify fiscal rules in the EU even before the COVID-19 crisis. The aim of this article is to reflect on the reform of fiscal rules from a broader perspective, looking at three different dimensions: the political economy of fiscal rules in the current political and economic environment, the renewed debate about fiscal policy roles and objectives, as well as the current incomplete nature of the European Monetary Union and the prospects for its completion. The main contribution of this paper is to analyse EMU fiscal policy and the related governance modes from a broader perspective. Furthermore, the article briefly discusses ideas for a new model of fiscal and economc surveillance based on a cooperative governance system in order to better fit with with current macroeconomic and political realities."}
+{"text": "The relationship between sarcopenia and the prognoses of patients with gastric neuroendocrine neoplasms (g-NENs) is unclear. This study was designed to explore the effects of sarcopenia on short-term and long-term outcomes of patients with g-NENs after radical gastrectomy.This study retrospectively collected data from 138 patients with g-NENs after radical gastrectomy. The skeletal muscle index (SMI) diagnostic threshold for sarcopenia was determined using X-tile software. Cox regression analyses were performed to determine the independent risk factors for 3-year overall survival (OS) and 3-year recurrence-free survival (RFS).p\u2009>\u20090.05). The 3-year OS and RFS rates were significantly worse in the sarcopenia group than in the nonsarcopenia group . The multivariate analysis revealed a relation between sarcopenia and the long-term prognoses of patients with g-NENs. A stratified analysis based on the pathological type revealed that the Kaplan-Meier curve was only significantly different in patients with gastric mixed adenoneuroendocrine carcinoma (gMANEC) ; furthermore, the multivariate analysis identified sarcopenia as an independent risk factor for patients with gMANEC (p\u2009<\u20090.05).In this study, 59 patients (42.8%) were diagnosed with sarcopenia. Among patients in the sarcopenia group and nonsarcopenia group, the incidences of total postoperative complications were 33.9 and 30.4%, incidences of serious postoperative complications were 0 and 3.7%, incidences of postoperative surgical complications were 13.6 and 15.2%, and incidences of postoperative systemic complications were 20.3 and 15.2%, respectively (all Sarcopenia is not related to the short-term prognoses of patients with g-NENs. Sarcopenia is an independent risk factor for patients with gMANEC after radical surgery. This study was designed to explore the effects of sarcopenia on the short-term and long-term outcomes of patients with g-NENs after radical gastrectomy by using data from two independent large-volume institutions.Gastric neuroendocrine neoplasms (g-NENs) are a class of tumors with significant heterogeneity that account for approximately 4% of all neuroendocrine tumors , and theIn recent years, sarcopenia has been reported to be closely related to the prognosis of patients with gastric cancer, liver cancer, colorectal cancer, and other malignant tumors \u201316. HoweThis study retrospectively analyzed the clinicopathological data from 138 patients with g-NENs treated at two institutions, with the aim of exploring the effect of sarcopenia on the short-term and long-term outcomes of patients with g-NENs after radical gastrectomy.The clinicopathological data from patients diagnosed with g-NENs at the Fujian Medical University Union Hospital (FMUUH) and the First Affiliated Hospital of Fujian Medical University (FMUFAH) from December 2009 to December 2015 were retrospectively analyzed. The inclusion criteria were as follows: (1) patients who were diagnosed with g-NENs by pathology; (2) patients without distant metastasis, as assessed by a preoperative examination; and (3) patients who underwent R0 excision. The following exclusion criteria were used: (1) distant metastasis was identified preoperatively and intraoperatively; (2) patients received neoadjuvant chemotherapy or radiotherapy before surgery; and (3) basic clinical data and computed tomography (CT) images were incomplete. One hundred thirty-eight patients with g-NENs were finally included in this study  at the level of the third lumbar vertebra (L3) by using Osirix 3.3 software  [2) of two consecutive slices was used for analysis. If necessary, the area of the selected area was manually adjusted to accurately calculate the area. The tissue discrimination threshold of skeletal muscle is \u2212\u200929 to +\u2009150 Hounsfield units (HUs) [2) was standardized to the height (m2) to obtain the L3 skeletal muscle index (SMI) (cm2/m2) [A preoperative abdominal CT scan within 1 month of surgery was considered to accurately reflect the patient\u2019s muscle status. A researcher who was blinded to the outcome measured the skeletal muscle cross-sectional area (cmwer.com) . The rests (HUs) . The mus(cm2/m2) .2/m2, the maximum chi-square log-rank value of 4.2611 was achieved. Therefore, a SMI\u2009\u2264\u200944.3\u2009cm2/m2 was defined as sarcopenia, and a SMI\u2009>\u200944.3\u2009cm2/m2 was defined as nonsarcopenia (p\u2009=\u20090.038)   was defined as the time from surgery to the last follow-up, death, or the last record in the follow-up database (such as loss of follow-up or death from other diseases). Recurrence-free survival (RFS) was defined as the time from surgery to the initial recurrence. Postoperative complications were classified according to the Clavien-Dindo criteria . Total pThe median follow-up time was 36\u2009months (range: 1\u2013102\u2009months). Physical and laboratory examinations were performed regularly after surgery, once every 3\u2009months for 2\u2009years, every 6\u2009months for the next 3\u2009years, and once a year after 5\u2009years. In addition, imaging examinations, including chest radiographs, abdominal and pelvic CTs, and endoscopy, were performed at least once a year. If necessary, additional MRI or PET studies were performed to determine whether recurrence was present.2 test or Fisher\u2019s exact test and a t-test, respectively. The OS and RFS rates were calculated by the Kaplan-Meier method, and the differences were assessed with log-rank tests. The Cox proportional hazards regression model was used to analyze the independent prognostic factors for 3-year OS and RFS rates. P values less than 0.05 were considered statistically significant.All data were statistically analyzed using SPSS 22.0 software. Continuous variables are reported as the means \u00b1 SD or medians (interquartile ranges). X-tile plots were used as a new bioinformatics tool for biomarker assessments and outcome-based cutoff point optimization , 26. Catp\u2009<\u20090.05). However, no significant differences in the other variables were observed between the two groups   were included in the sarcopenia group and 79 patients (57.2%) were included in the nonsarcopenia group. A total of 12 gNET patients, 52 gNEC patients, and 74 gMANEC patients were included in this study. Of gNET patients, 6 patients were type 1, 5 patients were type 2, and 1 patient was type 3. The comparison of clinical data between the two groups showed a higher incidence of sarcopenia in the subgroups of male patients, aged 65\u2009years, with a BMI of <\u200925 and a tumor larger than 50\u2009mm . Postoperative surgical and systemic complications occurred in 20 patients (14.5%) and 24 patients (17.4%), respectively, in the whole group. In the sarcopenia group and the nonsarcopenia group, the incidence of postoperative surgical complications was 13.6 and 15.2%, and the incidence of postoperative systemic complications was 20.3 and 15.2%, respectively . In addition, the analysis did not reveal significant differences in the incidence of specific types of complications defined according to the physical location of the complication between the two groups  , and serious complications occurred in 3 patients 2.2%). The incidence of total postoperative complications was 33.9 and 30.4%, and the incidence of serious complications was 0 and 3.7% in the sarcopenia group and the nonsarcopenia group, respectively , and the 3-year RFS rates were associated with pN and the Ki-67-positive index ; neither OS rates nor RFS rates were associated with sarcopenia in patients with gNEC   and the 2/m2 for males and a SMI\u2009<\u200928.6\u2009cm2/m2 for females [2/m2 for males and a SMI\u2009<\u200932.4\u2009cm2/m2 for females as sarcopenia, and the incidence of sarcopenia in our study was 42.8% (59/138). A significance difference in survival was not observed among the female group . Therefore, we used different diagnostic criteria for men and women in this study to better evaluate the effect of sarcopenia on the prognosis of patients with g-NENs.Currently, the value of the cutoff point of sarcopenia remains controversial. The most commonly used definitions were provided by Prado et al.  and Mart females . HoweverThe effect of sarcopenia on short-term postoperative outcomes in patients with malignant tumors remains controversial. Previous studies have confirmed that sarcopenia is associated with the postoperative short-term prognosis in patients with multiple malignant tumors , 15, 35.In recent years, studies have confirmed that sarcopenia is closely related to the long-term prognoses of patients with multiple malignant tumors , 14, 16.This study had some limitations. First, because most patients with gNET received endoscopic treatment, the number of patients with gNET included in this study was limited, which may cause bias. Second, this study employed a retrospective case-control design and was conducted in an Asian population; therefore, the results must be confirmed by prospective studies and data from Western countries. Third, the proportion of female patients in this study is relatively small , and thus the prognostic effect of sarcopenia on female patients with g-NENs must be further analyzed in a study with a larger population. We plan to conduct related studies in the future. Fourth, this study did not analyze the effects of postoperative adjuvant chemotherapy and postoperative sarcopenia caused by the gastrectomy status and tumor aggressiveness on long-term outcomes, which may also bias the results. Fifth, Due to the relatively few cases of stratified analysis of pathological subtypes, we did not identify sarcopenia scores related to tumour type gNET, gNEC and gMANEC, and the prognostic value of sarcopenia for g-NENs may be biased. In the future, a larger sample size is needed to determine the best cut-off point of sarcopenia with different pathological types, and to verify the prognostic effect of sarcopenia on different pathological types of g-NENs. Nevertheless, to our knowledge, this study is the first to explore the effects of sarcopenia on the short-term and long-term outcomes in patients with g-NENs by using data from two independent large-volume institutions, thus providing a reference for future clinical trials.2/m2 for males and a SMI\u2009<\u200932.4\u2009cm2/m2 for females were identified as the optimal cutoff points for sarcopenia in patients with g-NENs. Sarcopenia was not significantly associated with postoperative complications in patients with g-NENs. Sarcopenia is an independent risk factor for the long-term prognosis of patients with gMANEC undergoing radical gastrectomy. Further multicenter prospective studies are needed to confirm the prognostic value of sarcopenia in patients with g-NENs.In the present study, a SMI\u2009<\u200944.3\u2009cmAdditional file 1 : Supplemental Table\u00a01. Clinicopathological characteristics of patients treated at FMUUH and FMUFAH.Additional file 2 : Supplemental Table\u00a02. Univariate and multivariate analyses of factors associated with 3-year overall survival (OS) and recurrence-free survival (RFS) rates in patients with gNEC.Additional file 3 : Supplemental Table\u00a03. Four cutoff points tested as thresholds to define sarcopenia and the prevalence of sarcopenia.Additional file 4 : Supplemental Figure 1. Computed tomography (CT) image captured at the third lumbar vertebral (L3) level. The following skeletal muscles are outlined in red: rectus abdominis; psoas, quadratus lumborum, paraspinal, transverse abdominal, external oblique, internal oblique, and rectus abdominis muscles. This male patient with sarcopenia had an L3 muscle index of 52.35\u2009cm2/m2.Additional file 5 : Supplemental Figure 2. The cutoff points of the skeletal muscle index (SMI) for sarcopenia defined by X-tile software. (A) X-tile plots for males  and (B) females  are shown.Additional file 6 : Supplemental Figure 3. Kaplan-Meier analysis of the 3-year overall survival (OS) and recurrence-free survival (RFS) rates of patients with gastric neuroendocrine neoplasms (g-NENs) stratified according to the presence of sarcopenia diagnosed by the skeletal muscle index (SMI) cutoff points  defined by Martin et al. (A-B) and pathological types: (C-D) gastric neuroendocrine tumor (gNET), (E-F) gastric neuroendocrine carcinoma (gNEC), (G-H) gastric mixed adenoneuroendocrine carcinoma (gMANEC)."}
+{"text": "Combinatorial therapies are under intense investigation to develop more efficient anti-obesity drugs; however, little is known about how they act in the brain to produce enhanced anorexia and weight loss. The goal of this study was to identify the brain sites and neuronal populations engaged during the co-administration of GLP-1R and CCK1R agonists, an efficient combination therapy in obese rodents.We measured acute and long-term feeding and body weight responses and neuronal activation patterns throughout the neuraxis and in specific neuronal subsets in response to GLP-1R and CCK1R agonists administered alone or in combination in lean and high-fat diet fed mice. We used PhosphoTRAP to obtain unbiased molecular markers for neuronal populations selectively activated by the combination of the two agonists.Calcrl+ neurons as treatment targets.The initial anorectic response to GLP-1R and CCK1R co-agonism was mediated by a reduction in meal size, but over a few hours, a reduction in meal number accounted for the sustained feeding suppressive effects. The nucleus of the solitary tract (NTS) is one of the few brain sites where GLP-1R and CCK1R signalling interact to produce enhanced neuronal activation. None of the previously categorised NTS neuronal subpopulations relevant to feeding behaviour were implicated in this increased activation. However, we identified NTS/AP Collectively, these studies indicated that circuit-level integration of GLP-1R and CCK1R co-agonism in discrete brain nuclei including the NTS produces enhanced rapid and sustained appetite suppression and weight loss. \u2022GLP-1R/CCK1R co-agonism\u00a0produces greater than additive feeding and weight-suppressive effects.\u2022GLP-1R/CCK1R co-agonism activates a greater than additive number of neurons in only a few brain areas, including the NTS.\u2022Previously categorised NTS and ARH populations are not increasingly activated in response to the combinatorial treatment.\u2022Calcrl+ neurons are activated by GLP-1R and CCK1R co-agonism but not in response to mono-agonism.NTS/AP \u2022AC187 blunted the feeding-suppressive effect of GLP-1R and CCK1R co-agonism. Adcyap1r1adenylate cyclase activating polypeptide 1 receptor type 1AGRPagouti-related peptideAParea postremaARHarcuate nucleus of the hypothalamusCalcrlcalcitonin receptor-like receptor geneCCKcholecystokininCCK1Rcholecystokinin receptor type ACCK2Rcholecystokinin receptor type BCeAcentral amygdalaCLRcalcitonin receptor-like receptorCTcalcitonin receptordERdifferential enrichment ratioDLPBNdorsolateral parabrachial nucleusDMHdorsomedial nucleus of the hypothalamusDVCdorsal vagal complexERenrichment ratioGIPglucagon-like insulinotropic peptideGLP-1glucagon-like peptide-1,GLP-1Rglucagon-like peptide-1,GLP-1Rglucagon-like peptide-1 receptorMBHmediobasal hypothalamusNPYneuropeptide YNTSnucleus of the solitary tractPOMCpro-opiomelanocortinPVHparaventricular nucleus of the hypothalamusPYYpeptide YYTHtyrosine hydroxylaseVMHventromedial nucleus of the hypothalamus.1Obesity is a global public health challenge with limited and only mildly efficient non-surgical therapeutic options. Available pharmaceutical interventions produce only a fraction of the weight loss induced by bariatric surgery. One of the most pressing research questions in the field is to understand the mechanisms underlying appetite suppression following bariatric surgery to develop pharmacotherapies mimicking these effects. Several groups have approached this question using combinatorial therapies simultaneously targeting multiple appetite-suppressing pathways. These strategies enable enhanced efficacy through combined activation of independent pathways and/or prolonged suppression of counter-regulatory responses. In fact, the combination of gut-derived peptides  and/or pancreatic hormones (glucagon and amylin) used at subthreshold doses can produce synergistic reductions in food intake and body weight in rodents ,3 and GLMany successful combinatorial therapies use newly developed GLP-1 receptor (GLP-1R) agonists with enhanced bioavailability and established safety. Chronic administration of GLP-1R agonists reduces food intake and body weight in lean and high-fat diet (HFD)-fed rodents . Due to One gut-derived hormone that has received much less attention in a combinatorial setting is cholecystokinin (CCK), which robustly produces satiation but fails to decrease food intake over 24\u00a0h or improve energy balance in chronically dosed rodents due to compensatory increases in meal frequency ,13. Howe22.1ad libitum unless otherwise stated. Food intake studies were conducted on single-housed C57/Bl6 males  maintained in individually ventilated cages. In all of the studies involving high-fat-fed mice, the mice were maintained on a high-fat diet (HFD) containing 45% energy as fat  3 weeks prior to the beginning of treatments. For pica studies, 8-week-old male Sprague Dawley rats were purchased from Envigo . For immunofluorescence studies, we used 8- to 9-week-old Glp-1r-Cre:R26-tdYFP males , Pomc-GFP males (stock 009593), and Npy-GFP males (stock 006417) purchased from the Jackson Laboratories. All of the scientific procedures on animals were approved and conducted in accordance with the UK Animals (Scientific Procedures) Act 1986.All of the studies were conducted in males 9- to 10-week-old rodents at the beginning of the treatments. The rodents were maintained in temperature- and humidity-controlled rooms on a 12\u00a0h:12\u00a0h light\u2013dark cycle. The animals had free access to water and were fed a standard laboratory chow diet 2.2Exenatide and CCK-8 were purchased from Bachem. AC3174 , AC170222 (a CCKR1-selective agonist), and AC170236 (a CCKR2-selective agonist) were synthesised at Amylin Pharmaceuticals and provided via MedImmune/AstraZeneca through its acquisition of Amylin Pharmaceuticals as previously described . AC187 w2.3All of the mice were accustomed to being handled for a minimum of 7 days prior to the beginning of the experiments. Before treatment administration, groups of mice matched for body weight were food deprived for 6\u00a0h during the light phase with free access to water. The mice were randomised into treatment groups and received an i.p. injection (10\u00a0\u03bcL/g of body weight) of either saline, CCK-8 (2.5 or 10\u00a0\u03bcg/kg), exenatide , AC3174 (1\u00a0\u03bcg/kg), AC170222 (2.5\u00a0\u03bcg/kg), AC170236 (2.5\u00a0\u03bcg/kg), or a combination of these drugs. Food intake was recorded for the following 2\u00a0h. The animals received all of the treatments in a crossover manner. For studies involving pre-treatment with AC187, HFD-fed mice received 10\u00a0ml/kg vehicle  or AC187 (100\u00a0\u03bcg/kg) 30\u201345\u00a0min before receiving either saline or the combination of AC3174 (1\u00a0\u03bcg/kg) and AC170222 (10\u00a0\u03bcg/kg).2.4The mice were acclimatised for 7 days to BioDAQ cages , randomised into treatment groups, and received 2 i.p. injections (10\u00a0\u03bcL/g of body weight) per day 1\u00a0h after the onset of the light (8 AM) and 1\u00a0h before the onset of the dark (6 PM) of either saline, AC3174 (3\u00a0\u03bcg/kg), AC170222 (30\u00a0\u03bcg/kg), or combinations of these drugs (3\u00a0\u03bcg/kg AC3174\u00a0+\u00a030\u00a0\u03bcg/kg AC170222). Body weight was measured daily. Food intake was continuously and automatically recorded for the following 5 days. Eating bouts between 0.01\u00a0g and 2\u00a0g were included in the data analysis. Meals were defined by an inter-meal interval of at least 300\u00a0s and a minimal food consumption of 0.02\u00a0g.2.5Following their acclimatisation week, the male Sprague Dawley rats were given chow and kaolin diets . Chow and kaolin were provided in adjacent separate compartments in a divided food hopper. The rats were randomised to appropriate drug treatment groups based on 24\u00a0h chow intake, 24\u00a0h kaolin intake, and body weight (n\u00a0=\u00a09). Following an overnight fast in a clean cage, AC3174 and/or AC170222 were i.p. administered at 3\u00a0\u03bcg/kg and 30\u00a0\u03bcg/kg, respectively. Cisplatin  was i.p. administered at 10\u00a0mg/kg as a positive control. Cisplatin was dissolved in sterile saline and sonicated for several minutes. Chow intake and kaolin intake were recorded at 4\u00a0h and 24\u00a0h post-injection. A final body weight was also recorded at 24\u00a0h.2.6The mice were treated as previously described with saline, AC3174 (1\u00a0\u03bcg/kg), AC170222 (10\u00a0\u03bcg/kg), or a combination of AC3174 and AC170222. At 80\u00a0min after the injection, the mice were anaesthetised and perfuse fixed with PBS and 4% PFA. Their brains were removed and stored in 4% PFA and 30% sucrose at 4\u00a0\u00b0C for 2 days, then cut into 25\u00a0\u03bcm coronal sections and preserved at\u00a0\u221280\u00a0\u00b0C in cryoprotectant.2.7The brain sections were incubated in rabbit anti-c-fos primary antibody , biotinylated goat anti-rabbit secondary antibody , and ABC solution  and labelled with DAB solution. Images were obtained using a brightfield slide scanner  microscope or a light microscope  equipped with a digital camera . Image analysis and cell counting were manually performed by a blinded experimenter using ImageJ software and the mouse brain atlas from Franklin and Paxinos . For the2.8All of the brain sections were incubated with c-fos primary antibody , biotinylated goat anti-rabbit secondary antibody , and streptavidin . The brain sections were then incubated in either mouse anti-TH primary antibody  and goat anti-mouse secondary antibody , chicken anti-GFP primary antibody , goat anti-chicken secondary antibody , chicken anti-RFP , goat anti-chicken secondary antibody , or rabbit antibody anti-pS6 240/244 Alexa Fluor 594 conjugate .2.9Images were obtained using a confocal microscope . Image analysis and cell counting were performed semi-automatically using HALO software  and the mouse brain atlas from Franklin and Paxinos  was used2.10We used the PhosphoTRAP assay as previously described to identify transcripts enriched in neurons activated or inhibited by the co-administration of AC3174 and AC710222 [3, 35\u00a0mM of glucose, and 100\u00a0mg/mL of cycloheximide in methanol) on ice under 10\u00d7 magnification to collect the MBH and dorsal vagal complex . MBH and DVC tissues of 4 mice were pooled into 1 MBH sample and 1 DVC sample, leading to 4 samples by experimental conditions that were homogenised in buffer C  and clarified by centrifugation at 2,000\u00a0g\u00a0at 4\u00a0\u00b0C for 10\u00a0min. Supernatants were resuspended in 1\u00a0ml of 10% NP40 and 10\u00a0\u03bcL of 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), incubated for 2\u00a0min on ice, and centrifuged at 16,100\u00a0g\u00a0at 4\u00a0\u00b0C for 10\u00a0min. Then 25\u00a0\u03bcL of the sample was used for RNA extraction (input sample). The remaining sample was used for ribosome immunoprecipitation as described as follows.For the sample preparation, the mice were treated as previously described with saline or the combination of AC3174 and AC170222. At 30\u00a0min post-injection, the mice were sacrificed by cervical dislocation and their brain micro-dissected in buffer B  before incubation with anti-pS6 240/244 antibody  (4\u00a0\u03bcg per ip sample) and 0.1% bovine serum albumin (BSA) in buffer A (300\u00a0\u03bcL per i.p. sample). Antibody-bead conjugates were mixed end over end at 4\u00a0\u00b0C overnight. Then 300\u00a0\u03bcL of antibody-bead conjugates per i.p. were washed twice with wash buffer D . Antibody-bead conjugates were resuspended in 200\u00a0\u03bcL of homogenisation buffer C supplemented with 50\u00a0\u03bcL of 10% NP40 and 10\u00a0\u03bcL of DHPC for 1\u00a0mL of buffer C and 1\u00a0\u03bcM of ZK10 (a gift from Dr. Zackary Knight). The remaining supernatant was added to the antibody-beads conjugates, resuspended by pipetting and placed on end over end at 4\u00a0\u00b0C for 10\u00a0min. The beads were washed 4 times with 0.9\u00a0mL of ice cold wash buffer D and resuspended in 350\u00a0\u03bcL of RLT buffer (i.p. sample).For ribosome immunoprecipitation, Protein A Dynabeads (Invitrogen) were washed 3 times with buffer A . RNA quantity was assessed using a Quant-iT RiboGreen RNA Assay kit , and the samples\u2019 RNA quality was assessed using Pico chips on an Agilent Bioanalyser. The samples were cleared from DNA contamination using a TURBO DNA-free kit .For cDNA preparation and sequencing, cDNA was prepared using a Smart Seq v4 Ultra Low Input RNA kit . Library preparation was done using a Nextera XT kit (Illumina), and the samples were run on a NexSeq 500 System .ip/FPKMinput). We then calculated the differential enrichment ratio (dER) for each transcript by dividing the mean ER in the treatment (AC3174\u00a0+\u00a0AC170222) group by the mean ER in the control  group. The dER was used to identify transcripts enriched in neurons activated or inhibited by the AC3174\u00a0+\u00a0AC170222 treatment. Sequencing data are available on GEO (number pending).For differential enrichment ratio calculations, for each sample, we first calculated the enrichment ratio (ER) of the i.p. sample over the input sample (FPKM2.11Brains were removed at termination and snap-frozen on crushed dry ice and stored at\u00a0\u221280\u00a0\u00b0C until cryosectioning. The brains were divided into forebrains and hindbrains at the level of the pons and mounted on a pre-cooled cryostat sample holder with Tissue-Tek O.C.T. Compound (Sakura Finetek). Series of 12\u00a0\u03bcm thick coronal sections were cut on a cryostat  and collected on Superfrost Plus microscope slides (Thermo Fisher Scientific) and stored at\u00a0\u221280\u00a0\u00b0C until use. Serial sections were cut through the ARH with a sampling distance of 216\u00a0\u03bcm, collecting approximately 5 samples per animal. Similarly, serial sections were cut through the AP and NTS with a sampling distance of 72\u00a0\u03bcm, collecting approximately 5\u20138 samples per animal from each region.2.12RNAscope in situ hybridisation was performed on brain cryosections using an RNAscope Fluorescent Multiplex Assay V2 (Advanced Cell Diagnostics) to simultaneously visualise up to 3 mRNA targets using mouse-specific probes directed against genes listed in 2.13All of the treatments were systematically randomly assigned and administered to weight-matched groups of mice by a blinded researcher. For feeding, body weight responses, and histological analyses, data were analysed using a two-way ANOVA corrected for multiple comparisons and an alpha risk of 0.05. To identify the transcripts enriched in activated or inhibited neurons in response to each treatment, we first filtered the dataset and kept the transcripts with an average expression in the input samples greater than or equal to 2 FPKM. Statistical analyses were performed on ER values (see Methods) and treatment groups were compared using one-tailed unpaired Student's t-test with an alpha risk of 0.01.33.1We first tested the effect of a subthreshold dose of exenatide , a well-CCK1R signalling has been shown to mediate the anorectic effect of CCK . We ther., 2011; Frank A. Duca, Sakar, and Covasa, 2013). We confirmed that the HF diet blunted the anorectic response to CCK (p\u00a0=\u00a00.08\u00a0at 30\u00a0min and p\u00a0=\u00a00.12\u00a0at 2\u00a0h).We then tested whether the response to CCK1R and GLP-1R co-agonism was maintained in mice fed a high-fat (HF) diet , area postrema (AP), and NTS. This quantitative assessment confirmed the qualitative results . The com3.4The dorsal vagal complex (DVC) is a primary sensory relay for gut-derived satiation signals. In particular, DVC POMC and TH neurons have been implicated in the response to peripheral CCK , whereasGlp1r and Cckar in the DVC and ARH. In the DVC, there were virtually no cells co-expressing Cckar and Glp1r as most Cckar signals localised to the DMX .We obtained an unbiased molecular signature of MBH and DVC neurons activated or inhibited by AC3174, AC170222, and AC3174\u00a0+\u00a0AC170222 . In the p\u00a0<\u00a00.01) compared to controls (p\u00a0>\u00a00.1).A total of 25 genes were more enriched or depleted in response to the combination treatment vs the monotherapies  was enriched 15-fold in neurons activated in response to AC3174\u00a0+\u00a0AC170222 and not enriched in neurons responding to the monotherapies and therefore was further investigated as a marker for neurons responding specifically to the combination treatment. Calcrl encodes the core receptor component for CGRP, a neurotransmitter implicated in the physiological control of food intake in rodents [Calcrl+ neurons in the DVC, the expression of CCKar and Glp1r in Calcrl+ neurons, and the expression of c-fos in DVC Calcrl+ neurons in response to GLP-1R and CCK1R co-agonism. We found that Calcrl was concentrated in the AP and AP/NTS border and absent from the rest of the medulla  blockade on the anorectic response to GLP-1R and CCK1R co-agonism in the mice fed a HF diet. We used the amylin receptor antagonist AC187, which also blocks calcitonin receptor (CT) and CLR signalling, at a dose that did not affect feeding alone. This compound does not distinguish well between amylin and CT receptors and is less effective at blocking CLR signalling. Pre-treatment with AC187 blunted the acute anorectic response to AC3174\u00a0+\u00a0AC170222, indicating that targets of AC187 including CLR mediated the feeding-suppressive effect of GLP-1R and CCK1R combination therapy J. Of notAdcyap1r1 was one of the top markers of neurons inhibited by the treatment  was activated in response to this treatment, but 40% of these neurons were already activated in response to vehicle treatment, supporting the conclusion that activation of GLP-1R+ neurons in the ARH was predominantly independent of GLP-1R agonism. Together with the observation that many GLP-1R\u2212 neurons were activated in response to AC3174 in the ARH, we propose that fos activation in the ARH in response to AC3174 may not primarily be driven by GLP-1R signalling in activated neurons, but rather the product of the integration of afferent signals by many (GLP-1R+ and GLP-1R\u2212) neurons. In contract, the robust anorectic response to the CCK1R agonist was accompanied by an increase in neuronal activation across many brain sites. Remarkably, co-agonism produced increased neuronal activation in only three brain sites, most significantly in the NTS, demonstrating this site's role as the main neuronal substrate engaged in the integration of GLP-1R and CCK1R signalling. Of note, the LPBN also showed increased activation, but c-fos-positive cells were located in the dorsal portion of the LPBN, a site distinct from the ventrolateral PBN, which contains CGRP neurons implicated in aversive responses [In line with the lack of feeding response to the GLP-1R agonist alone, neuronal activation following this treatment was mild across all of the brain sites that we examined. In particular, the GLP-1R agonist did not activate hindbrain GLP-1RThe neurochemical characterisation of the activated neurons in response to the three treatments revealed that the population of neurons activated by the co-agonism was distinct from primary sensing neurons expressing CCK1R and GLP-1R, which indicated that the integration of these signals occurred at the level of a downstream integration point still within the DVC. This characterisation also increased our understanding of the regulation of MBH neuronal populations in response to the drugs and revealed that the CCK1R agonist and combinatorial treatment activated POMC neurons and inhibited AGRP neurons, with a higher effect of the combinatorial treatment, suggesting that these responses may have been involved in the increased anorectic response to GLP-1R and CCK1R co-agonism. However, in the DVC, we failed to identify the neuronal subpopulation specifically activated by the combination using this approach and excluded a contribution of DVC TH, GLP1R, and POMC neurons in this effect.Calcrl as a marker of DVC neurons activated by GLP-1R and CCK1R co-agonism. This is a druggable target, making these findings potentially translatable for developing new anti-obesity drugs. However, further research is required to determine the extent to which Calcrl signalling mediates the anorexigenic and weight-reducing effects of GLP-1/CCK co-administration. Of note, although PhosphoTRAP captured the majority of activated neurons in the MBH and DVC, this technique was not as successful in the hindbrain, with a lower enrichment in activity-dependent genes in the ip samples. This may have been because this region had virtually no activated cells at baseline and in the control group. This approach used in the MBH revealed that the GLP-1R/CCK1R combination inhibited neurons expressing PACAP receptors. Whether PACAP receptor signalling is engaged in the response remains to be established. Central PACAP signalling has previously been shown to suppress food intake [Using PhosphoTRAP as a hypothesis-generating tool to identify molecular markers for neurons regulated in the DVC and MBH specifically in response to the combination treatments, we identified d intake  mostly vd intake ,34. Howed intake . In thisIn conclusion, these studies revealed behavioural, neurochemical, and molecular mechanisms involved in the integration of two gut-derived peptides that produced enhanced appetite- and weight-suppressing effects and demonstrated the value of a novel approach to identify central druggable targets involved in the integration of combinatorial therapies.ER, SB, SW, IP, BQ, MM, NH, TD, YS, FG, FR, IP, and CB conducted the experiments. ER, BQ, BL, and CB performed data analysis. CB, GY, DB, and JT designed the experiments. CB, ER, GY, DB, and JT wrote the manuscript."}
+{"text": "Tributylphosphate diluted in Shellsol D70 and tap water were used as organic and stripping agents, respectively. Starting with SPAs with high Zn (71.7 \u00b1 4.3 g\u00b7L\u22121) and Fe (82.9 \u00b1 5.0 g\u00b7L\u22121) content, the NDSX process achieved a stripping phase with 55.7 g Zn\u00b7L\u22121 and only 3.2 g Fe\u00b7L\u22121. Other minor metals were not transferred, providing the purified zinc stripping with better quality for the next EW step. A series of five consecutive pilot-scale experiments showed the reproducibility of results, which is an indicator of the stability of the organic extractant and its adequate regeneration in the NDSX operation. Zinc mass transfer fluxes were successfully correlated to zinc concentration in the feed SPA phase, together with data extracted from previous laboratory-scale experiments, allowing us to obtain the design parameter that will enable the leap to the industrial scale. Therefore, the results herein presented demonstrate the NDSX technology in an industrially relevant environment equivalent to TRL 6, which is an essential progress to increase zinc metal resources in the galvanizing sector.Zinc recovery from spent pickling acids (SPAs) can play an important role in achieving a circular economy in the galvanizing industry. This work evaluates the scale-up of membrane-based solvent extraction technology aimed at the selective separation of zinc from industrial SPAs as a purification step prior to zinc electrowinning (EW). The experiments were carried out at a pilot scale treating SPAs batches of 57 to 91 L in a non-dispersive solvent extraction (NDSX) configuration that simultaneously performed the extraction and backextraction steps. The pilot plant was equipped with four hollow fiber contactors and 80 m The hot-dip galvanizing (HDG) process is one of the most common methods to prevent steel corrosion by providing steel components with a protective zinc coating . Acid pi\u22121 [However, the management of the spent pickling acids (SPAs) constitutes one of the environmental challenges for the galvanizing industry. SPAs after steel pickling in the HDG plants consist of free HCl, iron, zinc, and chloride ions . The pre\u22121 . It is a\u22121 .\u22121, the average being 101.6 g\u00b7L\u22121. Iron is mostly present as Fe2+. Zinc concentration varies in a similar range, the average being 95.7 g\u00b7L\u22121. The wide variety of zinc and iron concentration is the result of the diverse practices applied by galvanizers, e.g., the remaining acid can be used for stripping the zinc layer from rejected galvanized steel products [The conventional SPAs treatment consists of residual acid neutralization with lime or some other cheap alkaline agents , which i2\u2212 eq. per ton of primary zinc produced [The conventional treatment of SPAs is being substituted by innovative alternatives that can have different objectives: acid recovery, metals recovery, and the conversion of the waste into other products . In thisproduced . In addiproduced .Technologies that enable acid recovery are spray roasting, evaporation, diffusion dialysis (DD), membrane distillation (MD), electrodialysis (ED), and membrane electrolysis (ME) . MethodsHFMCs permit the non-dispersive extraction of metals by using the porous membrane to stabilize the aqueous\u2013organic interface ,45,46,472. Experiments have been performed with real SPAs that were supplied by an HDG manufacturer. The aims of this work are (i) the evaluation of the effect of the process operation variables to promote the selective zinc separation over iron, (ii) to obtain a stripping phase with enough quality for zinc electrowinning, and (iii) to assess the scale-up of the NDSX technology and define procedures for the industrial scale design. This work is focused on the validation of the NDSX technology at a demonstrative scale in real conditions, for the selective separation and recovery of zinc from SPAs generated in the HDG process. In this context, pilot-scale experiments have been performed in an NDSX plant equipped with four hollow fiber modules and a total membrane area of 80 mSamples of SPAs were provided by GALESA, which is a hot-dip galvanizer located in Spain. Zinc and iron concentration in SPA samples and stripping solution were measured by Microwave Plasma-Atomic Emission Spectrometry . Nearly all (98.2%) of the total iron content in the SPA batch was as Fe (II), as determined by UV/Vis spectrometry . Other metals that were present in low concentrations were analyzed by Inductively Coupled Plasma/Mass Spectrometry . Several studies have already determined the presence of minor metals in SPAs by atomic absorption spectroscopy ,11. Ion v/v) solution of tributylphosphate (TBP) and ShellsolD70, which is an aliphatic solvent that is used to dilute TBP with the aim of reducing the viscosity of the organic stream. Tap water was used as the stripping agent.The composition of the organic and stripping phases were selected based on the previous expertise of the research group  and acco2 [The SPA feed phase, the organic extractant phase, and the aqueous stripping phase were allocated in three tanks. Two HF modules were used for the extraction (EX) step, in which zinc was transferred from the SPA feed phase that flowed through the inner side of the fibers to the organic extractant phase that flowed through the shell side of the module. In the two back-extraction (BEX) HF modules, zinc was backextracted by the stripping water flowing through the inner side of the membranes, while at the same time, the organic extractant was regenerated and recycled to the extraction modules again. The system is equipped with filters to prevent the entry of solids in the membrane contactors and a stage of oils and fats removal. To initiate the operation, the pneumatic pumps flowed the aqueous phases (feed and stripping) through the HF modules; next, the organic phase flow started. The hydrodynamic pressure of all streams was adjusted with the back-pressure valves located at the exit of the HF modules to achieve a minimum 0.15 bar overpressure of every aqueous stream over the organic phase flowing through the same HF module at both inlet and outlet positions of each membrane contactor. This mode of operation prevents the penetration of the organic phase into the aqueous phases and maintains the aqueous\u2013organic interphase at the porous wall of the hydrophobic polypropylene membranes. Experiments were performed at room temperature. Two flowmeters were installed at the inlet of each HFMC for measuring the flowrate of the two inlet aqueous and organic streams. Optimal volumes of each phase and operating pressures were fixed based on preliminary experiments developed by the research group at the laboratory scale using HF contactors (Liqui-Cel Extra-Flow 2.5 in. \u00d7 8 in.) with an effective membrane area of 1.4 m2 . Table 5\u2212, ZnCl42\u2212, ZnCl3\u2212, ZnCl2, ZnCl+, Zn2+, Fe2+ and FeCl+, of which ZnCl42\u2212 and Fe2+ are the predominant ones. This confirms that the high ionic strength of the SPA leads to the formation of chlorocomplexes with a high stoichiometric coefficient of chlorine [The mechanism of zinc and iron extraction by TBP in chloride media has been described by several authors, as summarized by Lum et al. . All autchlorine . The spe\u22121, while the stripping phase achieved 55.7 g\u00b7L\u22121 of zinc. The process was carried out for 29 h, although zinc mass transfer was very slow in the final period, as chemical equilibrium conditions were being approached. 3+, which is in accordance with previous literature reporting the low selectivity of zinc solvation extractants over Fe3+ and the much higher selectivity of reagents over Fe2+ [\u22121 . Nevertheless, ZnCl3\u2212 and ZnCl2 species that are also present in the feed SPAs are extracted according to reactions (2) and (3), which is a factor that reduces the Zn/Cl ratio of extraction. In the stripping phase, the initial chloride and proton concentration are much lower than in the feed, and the speciation of zinc chlorocomplexes differs from the feed phase. However, in the conditions of the stripping phase at the end of the extraction/backextraction cycle, ZnCl42\u2212 is also the major zinc chlorocomplex.As mentioned in J) has been calculated from extraction and backextraction data, using Equations (4) and (5),We have selected the mass flux of zinc as a scale-up parameter for the design of the NDSX process for zinc selective separation from SPAs generated in the HDG process. Therefore, the zinc mass flux  and with higher zinc concentration (122 \u00b1 3 g Zn2+\u00b7L\u22121). All together, bench scale and pilot scale data can be fitted to the same linear equation, with a reasonably good regression parameter. The linear fitting depicted in ane area . In thatThe flux data shown in 2 of total membrane area and TBP as a selective extractant to treat industrial SPAs with relevant zinc and iron content (71.7 \u00b1 4.3 g Zn\u00b7L\u22121 and 82.9 \u00b1 5.0 g Fe\u00b7L\u22121). Zinc was recovered as metal dissolved in aqueous solution with a concentration of 55.7 g Zn\u00b7L\u22121 and reduced iron content of 3.2 g Fe\u00b7L\u22121. Favorably, other minor metals that are present in the SPAs were not transferred to the zinc-enriched stripping solution. The stable performance of the pilot plant in five consecutive batch extraction/backextraction cycles showed that the operation of the NDSX systems achieves the adequate regeneration and chemical stability of the organic extractant. Zinc extraction mechanism and mass transfer fluxes were satisfactorily correlated with previous laboratory-scale literature data. Therefore, this study defines the function that relates zinc mass transfer flux with the zinc concentration in the SPAs that is needed for design and scale-up purposes of the technology toward its prototype on-site demonstration in real galvanizing facilities. The presence of small iron concentration in the purified zinc liquor is not expected to prevent the EW recovery of secondary zinc with purity >99.5%. However, iron transfer should be minimized for zinc purities >99.9%. Future research will be focused on defining the operation conditions needed to avoid iron (II) oxidation, in order to prevent the iron (III) transfer that was observed in the present pilot plant demonstration study. Our study demonstrates the potentiality of NDSX technology for recovering zinc from residual SPAs generated in HDG, at a pilot scale never tested before, which could be reused either by galvanizers or as supply for other secondary zinc markets.This work is aimed at developing a key enabling technology to move the galvanizing sector toward a sustainable use of metallic resources. In the frame of the LIFE2ACID project, we propose the selective recovery of zinc from SPAs generated in HDG facilities by the integration of membrane based non-dispersive solvent extraction (NDSX) and electrowinning (EW). The present study deals with the demonstration of the NDSX at the pilot scale, using a membrane-based solvent extraction plant with 80 m"}
+{"text": "Planners, who constituted a slight majority in our sample, were likely to have established pre-shopping and in-store behavior and food management and cooking skills. Extemporaneous Consumers had inferior food handling routines and were less knowledgeable and skilled in the kitchen. Regression analysis using a random-effects tobit model showed Extemporaneous Consumers were prone to waste a greater portion of the spinach product than Planners. Otherwise, both classes showed similar increases in likelihood to discard the products, as their appearance deteriorated. Their tendency to waste increased with shorter remaining shelf life for spinach but not for ground beef, and was not affected by the date label type. Results suggest an intervention that targets a general audience designed to enhance people\u2019s skills to discern edibility of food in home storage by manipulating sensory expectations from cosmetic deterioration could be impactful in efforts to curtail food waste.American households discard a significant amount of food that represent a sizable portion of their food expenditures. This study adds to our understanding of product attributes associated with food waste, with a focus on cosmetic deterioration during home storage. Specifically, we profile a sample of U.S. individuals by patterns of common food-related behaviors and determine the effects of product attributes on food waste tendencies at the point of consumption by distinct behavioral profiles. An interactive survey at the Minnesota State Fair (N = 333) was used to obtain measurements on food-related behavior and sociodemographic factors. The survey included a conjoint task to elicit food discard tendencies to construct the food waste proxy. The study considered cosmetic deterioration, date labels, implied shelf life, package size, and prices paid, in fresh, packaged spinach and ground beef products. Factor analysis and latent class modeling categorized the sample into two classes, revealing distinct food-related behavioral patterns.  Food waste has garnered much attention in local and global policy circles, as research continues to highlight its negative impacts on the environment. Evidence shows that wasted food places a huge burden on society in multiple ways, including opportunity costs of resources such as fresh water, cropland, and energy used to produce the food ,2, and mConsumers disproportionately contribute to the food currently wasted and landfilled in industrialized nations ,7. A repA growing literature shows that food waste results from multiple complex interactions and decisions that relate to food purchase and management, identifying a broad and varied set of sociodemographic, behavioral, and attitudinal factors that are both internal and external to individuals ,10. The Appearance is a universal attribute to all products and establishes the first sensory impression of the item, majorly influencing its acceptability by confirming or disconfirming consumers\u2019 sensory and hedonic expectations . IndustrOur paper contributes to the literature in two ways. First, we profile U.S. subjects based on patterns of attitudes and behaviors related to household food-related routines such as shopping, planning, waste sorting, and other \u201chome economics\u201d skills. A handful of studies have aimed to categorize consumers based on food waste amounts or tendencies, or food-related practices ,21,22. WWe designed an interactive consumer survey to collect observations on individuals\u2019 attitudes, behaviors, and socio-demographic characteristics. Included was a conjoint task that simulated food handling scenarios at home to elicit food waste proxies. For the elicitation, we selected two products where appearance and expiration date are commonly factored into their consumption decisions, bagged spinach and ground beef. The survey was administered at the 2016 Minnesota State Fair, and 333 subjects participated. We applied principal component and latent analyses to group individuals by shared underlying traits, then used regression analysis to determine the relative roles of food-related attributes and socio-demographic characteristics on food waste tendencies and differences among groups.Planners and Extemporaneous Consumers. Making up 57% percent of the respondents, the Planners class consisted of people who were likely to have steady pre-shopping planning routines, diligent in-store behavior, consistent waste sorting practices, and good cooking and food management skills. In contrast, Extemporaneous Consumers of the second class were overall prone to have poor food planning and shopping routines and reported higher likelihood, for instance, to impulsively buy more food than needed in the store. Compared to Planners, they were less involved in activities such as recycling or composting and were generally less savvy and knowledgeable in the kitchen. Our regression results showed that Extemporaneous Consumers had higher tendencies to waste than Planners in the case of spinach, but two groups responded in a similar manner to product characteristics. That is, as cosmetic appearance of the food product deteriorated, people showed higher tendencies to consume less spinach and substantially less of ground beef, suggesting higher levels of potential waste, even though the product remained edible and safe to consume. Food waste tendency was higher for spinach as the remaining shelf life was shortened, but the effect was not statistically significant for ground beef. For both products, food waste tendency was unaffected by the type of date labeling or package size.The latent class model revealed that respondents fell into two, somewhat clich\u00e9d, classes: While food waste estimation is highly sensitive to methodologies employed , the litUnderstanding contributors to food discarding habits has been a shared goal of consumer food waste research efforts, which were pioneered by Cox and Downing  in the UBecause physical measurements of household food waste are costly to obtain, many of these studies prevalently have asked the subjects to self-report amounts of food discarded using a Likert scale ,39 as prStudies have reported various demographic and psychological factors, such as motivations and barriers to reduce food waste, which are associated with household food waste tendencies. For instance, Neff et al.  note sigMeal planning and food shopping routines, as well as food handling skills, are found to be important predictors of food waste behaviors in most studies. For example, keeping a regular shopping schedule or checking inventories, as well as leftover reuse routines, are associated with smaller amounts of food waste . IndividA few studies have attempted to cluster or profile individuals by their food waste-related behavior and attitudes \u201322. RomaOf industry practices affecting individual food behavior, package size, date labeling, and price are food product attributes that have conventionally been linked to food waste behavior. For example, packages that are too big or difficult to empty are identified as causes for food waste , and shoStudies examining the role of food appearance in the context of food waste have largely focused on \u201cugly\u201d or misshapen produce and its effect on purchase intensions. An exception, Jaeger et al. , examineThe survey instrument was designed to collect information from individuals on relevant attitudinal and behavioral propensities based on the literature and to elicit food waste tendencies. The survey was administered in an interactive setting for a period of 5 days at the 2016 Minnesota State Fair, which was attended by nearly two million people during its twelve-day event. Volunteers recruited subjects for a total of about 30 minutes of their time, compensating them with a drawstring backpack with the University of Minnesota logo, a popular give-away item for human subject research projects conducted at the State Fair. Recruitment did not mention food waste to minimize self-selection bias in the study. The surveys were completed on electronic tablets. Our project staff worked closely with each respondent, explained the tasks at hand, and answered any clarifying questions. The study protocol was approved by the Institutional Review Board at the University of Minnesota.\u201cImagine you are in your kitchen to do some meal preparation using <food product>. The <food product> you took out is Product X. Thinking of all possible ways you are likely to eat <food product>, what percentage of this product are you and/or your household likely to eat? Answer: 0%\u2013 100% .\u201d The food waste proxy score was computed by subtracting the responses in percent from 100, assuming that the smaller the proportion of products likely eaten, the higher the amounts potentially discarded.To construct the food waste proxy for the study, the respondents were presented food products with varying attribute profile and asked to indicate the percentage of the presented food product they would likely consume as they are preparing meals, ranging from eating none to eating all. For each product profile, the question was posed as follows: We developed the food product profiles in consultation with food science experts to simulate realistic food handling decisions at home. In addition to cosmetic deterioration, our study focus, the following food product attributes were selected for the study from the attributes that have been associated with waste tendencies: expiration date type, days to expiration date, package size, and price purchased. We selected two products, packaged fresh spinach and ground beef, which are both sold with an expiration date and deteriorate visibly over time while their edibility is intact.appearance level of 1 suggests that the product was free from any cosmetic flaws. As the level progresses to level 3, there are multiple flaws in the form of browning in the case of beef and blemishes, spots, and wilting in the case of spinach. Respondents saw multiple images for each product from various angles. Deterioration was merely in appearance and products remained edible. Cosmetic deterioration was categorized into three distinct levels using photographic images. The Best by, Use by, and Best if used by. These particular date types have been emphasized in the recent food waste literature and \u201cBest if used by\u201d is currently proposed as the preferred food date label [Use by terms for perishable products with potential safety implications or material degradation of critical performance, and Best if used by for all other packaged foods [near, middle and far expiration date for spinach implied 1, 3, and 7 days away respectively, and for ground beef, 1, 2, and 3 days away respectively. For package size, a large spinach product weighed 10 ounces and the small 5 ounces. The large ground beef weighed 2 pounds and small size was 1 pound. Price paid varied between $1.99 to $5.99 for spinach and between $4.79 and $15.99 for ground beef. These sizes and prices were selected to ensure familiarity with attributes at typical grocery outlets.Three types of expiration dates were considered: te label . Tradinged foods . The othWe used a fractional factorial method to develop twelve profiles for each product using orthogonal design. The product profiles were grouped into three blocks of four profiles, and each subject was presented with two randomly selected blocks of four profiles, one for each product, and both for spinach if they indicated that they did not eat meat. Thus, each subject evaluated eight product profiles, and their task was interrupted with a different set of questions between the blocks to minimize response fatigue.The survey collected measurements related to shopping, purchasing, cooking and other food-related routines, which have been validated in the literature. The questions are included in the These food-related behavioral factors were then used in a latent class analysis along with measurements of composting and recycling practices in the household . The behavioral factors are captured by class membership. We examine whether food waste tendency varies across classes of individuals identified in the latent class. The data on our elicited measurements of food waste tendency and corresponding product attributes consists of multiple panel responses from participants. Thus, a regression model that accounts for panel-level effects is suitable, and we expect differences across participants to affect their food waste tendencies beyond individual characteristics we include in the model, which suggests including individual random effects .i = 1, \u2026, n individuals who are respectively members of class s = 0, \u2026, S\u22121, where conjoint task t = 1, \u2026, ni and si is a binary indicator of class membership. The dependent variable yits is the food waste tendency measured by the likelihood of product in conjoint task t being discarded by individual i in class s. The vector z represents a vector of product attributes which consists of the levels of cosmetic deterioration (appearance levels 1 through 3), expiration date type , days to expiration, package size , and the price of product purchased. The vector x is a vector of demographic characteristics including age, gender, income, education level, race, household size, and presence of children in the household. \u03b2, \u03b3, and \u03b4 are parameters. The coefficients on the interaction terms with the class indicator s will indicate whether food waste tendency varies across classes. The individual-level random effects, uis, are independently and identically distributed following a mean-zero normal distribution with a standard deviation of \u03c3u, and the error term \u03b5its is independent and identically distributed following a mean-zero normal distribution with a standard deviation of \u03c3e.We estimate the following regression model:We begin by describing the overall demographic composition of the sample compared to the population of the state of Minnesota. The scores expressed in percentage points , calculated as 100 minus the elicited proportions of products that participants indicated as likely to consume, serve as proxy measurements of food waste tendency. Variable 1 suggest that the four measures of pre-shopping routines illustrate the essence of a person\u2019s activities before grocery shopping. Making a list food purchases has a high loading of 0.80 implying that such a behavior is highly relevant in defining the pre-shopping routines factor\u2019s dimensionality.The factor analysis applied to 35 items formed six variables gauging (i) pre-shopping routines, (ii) purchasing behaviors, (iii) reasons to discard food, (iv) self-reported amounts of food discarded, (v) cooking and food management skills, and (vi) tendencies to buy or prepare too much food to provide for the family. Variable 2 imply that the measures are able to capture the factor\u2019s dimensionality, particularly for sticking to the shopping list. When these items were combined with items in Variable 1, the results did not yield Cronbach \u03b1 or factor loadings that met the minimum requirements, suggesting pre-shopping and in-store purchasing are distinct latent variables. The Cronbach \u03b1 score of 0.79 shows that rationales for discarding food whether before or after meal (Variable 3) are internally consistent and reliable for the construct capturing activities and routines linked to food discarding. For example, amongst others, tendencies for buying the wrong item, not being able to save leftovers, or a package being broken leading to throwing away food are correlated and measure the same concept with good reliability. Self-reported amounts of food discarded are also closely related across all the different products (Variable 4), with an alpha of 0.80 indicating high reliability. Variable 5 captures a construct of food management and cooking skills which performs well according to the respective alpha and factor loadings. Finally, measures capturing agreement to preferring buying and preparing more food than running out are well correlated according to the Cronbach \u03b1 score of 0.70.While the Cronbach \u03b1 of 0.52 suggests low reliability, the factor loadings for Using the six factors from the factor analysis in addition to two other variables that represent composting and recycling frequencies, the latent class analysis tested whether a group of unobserved classes (latent) validated the association among the included variables. Planners class. We call the other class, the Extemporaneous Consumers. p-value (2). We compare these conditional probabilities in each case to assign a class membership for that specific response (3). Most probabilities are significant at the 5% level with the exception of a handful which are not used for interpretation for class membership.The relative model fit evaluated by the BIC implies that the respondents fall into two classes (BIC = 1906.97). Fifty-seven percent of the sample make up, what we denote as, the Extemporaneous Consumers class. Those who tend to agree with these routines fall into the Planners class. Similar trends are noted for in-store or during shopping behaviors. Those who are in agreement with having fewer impulses in the store or sticking to their shopping lists fall into the Planners class.The results show that respondents who tend to either \u201cStrongly Disagree,\u201d \u201cDisagree\u201d or are \u201cNeutral\u201d about their pre-shopping routines such as making lists, and checking the inventories, largely fall into the Extemporaneous Consumers report that they disagree with or are neutral about them. That is, on average, they do not throw away food because the package was bad/broken or too much food was prepared, for example. On the other hand, Planners tend to agree with throwing away food for various reasons such as being unable to save leftovers or buying the wrong item. The trend is reversed when it comes to self-reported amounts of food wasted, which include both perishables such as fruits, vegetables and meats as well as shelf-stable items such as frozen or canned goods. Planners report throwing away little amounts of food while Extemporaneous Consumers throw away an average amount.When asked about reasons for food going to waste in the household, Planners report that they prefer to buy or prepare more food than needed to provide for their families than to run out, while Extemporaneous Consumers mostly disagree with those statements. Regarding food management and cooking skills such as re-using leftovers or knowing how to store food items in the kitchen, Extemporaneous Consumers report either \u201cAverage\u201d or \u201cGood\u201d skills. On the other hand, Planners report being \u201cExcellent\u201d at these home-economics skills. Finally, Planners report steady composting and recycling tendencies compared to their counterparts in the other class who do so less than half of the time.Planners are lower than those of Extemporaneous Consumers. A greater percentage of Planners\u2019 responses indicate wasting no proportion of the food product compared to Extemporaneous Consumers, while a similar or greater percentage of response indicate tossing out the entire product.The dependent variable of our analysis is the elicited measurement of food waste tendency, which is summarized in \u03c3u) supports the specification of random effects. Because of censored observations, we estimated a random-effects tobit model in Stata/IC 15.1, where lower and upper bounds were set at 0 and 100 respectively. Extemporaneous Consumers than Planners, holding everything else constant. The coefficient on the class indicator is not statistically significant but positive in the ground beef results. The marginal effect, conditional on the food waste tendency score falling between 0 and 100, implies that given the same spinach product, Extemporaneous Consumers would on average discard 12 percentage point more of the product than Planners  in the spinach results is statistically significant (p = 0.039), indicating that food waste tendency on average was higher among As cosmetic appearance deteriorates, we see a steady increase in the likelihood of food to be discarded. A slight but distinct decline in appearance from level 1 to level 2 increases the likelihood to discard by 3.1 percentage points for spinach (p = 0.048) and 7.1 percentage points for ground beef (p = 0.001), holding all other factors constant. A further decay to level 3 increases the likelihood to 6.4 percentage points for spinach (p < 0.001) and 21.6 percentage points for ground beef (p < 0.001), suggesting that one fifth of the ground beef product would be discarded for cosmetic decay even though it remains edible.The only significant coefficient on date-related variables was the one on days to expiration in the spinach model. With each additional day closer to the labeled date, the likelihood of the spinach product to be discarded increases by 1.6 percentage point (p < 0.001), holding all else equal. Comparing the magnitude with that of appearance suggests that two additional days of shelf life have a comparable effect to offset the negative impact of the cosmetic decay from level 1 to 2. In terms of elasticities, a percentage decline in the number of days to expiration results in 0.22 percent increase in the food waste tendency, all else equal. Days to expiration had no discernable impact in the beef results. Thus, having more time until expiration is associated with reduced wastage propensities for produce but not for fresh meats. The date type did not affect the tendency to waste food for both products, which is distinct from the previous findings ,24,26.The results suggest higher wastage tendencies are associated with larger product sizes, but the results are not statistically significant (p = 0.141). This finding contrasts previous findings associating larger packaging with higher food wastage ,49. SimiSociodemographic variables were included in addition to individual random effects. The directions of impacts were largely consistent between the products, with the exception of race and household size. The tendency to waste was lower among younger respondents, all else equal, and the effect was statistically significant in the spinach results (p = 0.048). Male respondents had higher average tendency to waste by 5.8 percentage points than others in the spinach results (p = 0.067). Higher income earners had on average lower tendency to waste, which was significant in the ground beef results, suggesting a magnitude of 0.2% decline for a given 1% increase in household income (p = 0.093). Similarly, those with higher educational attainment had lower tendency to waste. The difference between those with a bachelor\u2019s degree was 7.3 percentage points in the ground beef results (p = 0.010). Non-white respondents had on average 6.3 percentage points higher tendency to discard ground beef products (p = 0.084). Household composition had no impact on food waste tendency in either product.The interactive survey tool was administered at the Minnesota State Fair implying that results from our consumer survey are subject to potential biases such as sample selection bias and measurement error ,66. To aSelf-reported details about food and food-related behaviors such as shopping habits and amounts of food that potentially goes to waste in the household could be subject to information bias and more particularly recall bias ,68. ThesPlanners, who are likely to have established pre-shopping food planning routines and steady in-store behavior. These individuals would keep track of their kitchen inventories, take a shopping list to the store, and, while in store, buy according to their list. Planners are more involved in environmental activities, such as recycling and composting, and report being knowledgeable and skilled in food management and cooking. Consequently, these Planners are aware of various reasons for why food gets discarded in their household, including buying or preparing too much food. Nevertheless, they report throwing out less food compared to their counterparts. In stark contrast, Extemporaneous Consumers are likely to have poor routines when it comes to meal planning and food shopping activities. They report being less knowledgeable and savvy when it comes to cooking and demonstrate lower food management skills in the kitchen. Compared to Planners, they tend to recycle or compost less frequently.Our latent class analysis showed that individuals\u2019 food and food-related routines can be typified into intuitive patterns. One type of behavioral patterns found in our sample were Planners and Extemporaneous Consumers, while discovering additional or more nuanced patterns.Our findings confirm other studies reporting a handful of profiles of food-related skills and behavioral patterns in our society ,21. BroaOne salient finding from our study is that individuals with different behavioral profiles respond in a similar way to food product attributes. In particular, even if food products are fully edible, consumers are still likely to reject foods that had deteriorated in appearance. Moreover, these individual responses to specific attributes varies across products. The amount rejected for bagged spinach as it became visibly more wilted was modest, compared to the amount of rejection of ground beef as it displayed freezer burns and a few brown spots. Food waste tendency was much more responsive to remaining shelf life of the product indicated by date labels for spinach than for meat. For either product, date type, price, or package size had little impact, but this finding could be a result of the hypothetical nature of our study.Previous studies suggest challenges with designing effective policy interventions to curtail household food waste, in part because practices that cause food waste are too fully integrated into daily routines to be changed with additional educational campaigns . But theOur findings suggest that people determine acceptability of food based on product attributes in largely the same way regardless of their food-related aptitude. However, it would be effective for policy interventions to be mindful of variation in the populace of their general food-related aptitude and proactively respond to segment-specific response in their implementation. For example, communications from campaigns need to be accessible to those who are generally less knowledgeable or familiar with at-home food handling.S1 Appendix(DOCX)Click here for additional data file."}
+{"text": "SeviL is a recently isolated lectin found to bind to the linear saccharides of the ganglioside GM1b (Neu5Ac These proteins are found to regulate such processes as development, cell growth and immune responses through their ability to detect a variety of markers at the cell surface. In mammals, for example, large families of proteins are known that bind sialic acid (Siglecs)2 or galactose 3, and these are of enormous medical interest for their roles in cell signalling and cancer. Although mammalian proteins have inevitably drawn the lion\u2019s share of attention, marine invertebrates have also proved a rich source of lectins with novel properties that may be exploited in biotechnological or clinical applications. Many of these lectins remain uncharacterised at the protein level, but at least some of them facilitate innate immunity responses by acting as pattern recognition receptors (PRR)6. While the natural function of these proteins appears to be anti-bacterial, proliferative neoplastic disease is prevalent in marine bivalves7 and a number of lectins from such species have been found to induce apoptosis highly selectively in cancerous cells8. There is considerable interest in developing the medical potential of lectins from various sources including plants9. Among many others, a lectin from the edible mushroom Boletus edulis named BEL 11. Glycan recognition plays important roles in all forms of life, which have consequently evolved many diverse proteins (lectins) with the function of binding specifically to selected carbohydrate moieties14. These proteins are unusual in binding a sugar ligand at each of the three sub-domains , the protein tyrosine kinase receptor for nerve growth factor (NGF) found in lipid rafts20. GM1 is however only one member of several series of glycolipids that differ from each other only in the addition of a single monosaccharide, and which play different biological roles. Decoding the pattern of gangliosides present at the cell surface therefore requires strict sugar binding specificity, which remains only partly explained.Gangliosides are glycosphingolipids built from ceramide and a saccharide chain, usually carrying one or more sialic acid residues. They have numerous roles in cell signalling and signal transduction, and misregulation of their production may promote cancer21. For many years subsequently, polyclonal rabbit anti-asialo-GM1 antibodies were used to ablate NK cells in studies of innate immunity, before a lethal off-target effect was demonstrated on basophils, which also play an important role in immune responses22. Anti-GM1 IgG antibodies are associated with Guillain-Barr\u00e9 syndrome (GBS), an autoimmune-mediated neuropathy, but these human proteins also show a broad range of reactivity with other gangliosides23. Although commercial antibodies have played an important role in molecular biology, recent tests of such preparations have found some alarming results24. Even widely-used products have been found to give false positive or false negative results, leading to a drive towards validation, using gene knock-outs where possible. Lectins provide an obvious alternative solution to the problem of detecting target saccharides, if a protein of suitable specificity can be found. Such proteins offer tremendous cost savings if they can be produced in bacteria, and as pure proteins they allow much more precise characterisation than a polyclonal antibody.Conversely, the use of saccharide biomarkers as diagnostics or for cellular control requires specific and thoroughly tested molecular tools. Asialo-GM1 is found in the membrane of mammalian natural killer (NK) cells, and in 1992 it was shown that antibodies targeting it can be used as an immunosuppressant16. The 129 residue sequence of SeviL is unrelated to the 149 residue MytiLec, but shows three weakly-conserved tandem repeats of about 40 residues, each with a tryptophan residue at an equivalent position. This is highly suggestive that SeviL resembles the classical R-type lectin structure, with a QxW motif in each of the three subdomains25. The lack of overall sequence similarity, especially around the possible ligand binding sites, and the small size of the protein relative to other The natural function of SeviL remains unknown, but if SeviL is added to mammalian carcinoma cells expressing asialo-GM1 then it activates apoptotic pathways, causing cell death10. The structure was solved by molecular replacement using the program BALBES27. Crystallographic data and refinement statistics are given in Table\u00a0Gene cloning, protein expression and crystallisation were carried out as described in Supplementary Information. Apo-SeviL crystallised in space-group mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimThe mentclass2pt{minim2, but too small for PISA28 to predict a stable dimer with confidence. Contacts between the two subunits are listed in STable\u00a029.The two monomers in the dimer are closely associated by hydrophobic interactions and hydrogen bonds involving 11 residues on each subunit. Gln\u00a012 and Phe\u00a0126 lie close together, and their side-chains lie opposite their symmetry mates at the dimer interface. Based on the crystal structure, a double mutant was designed (Q12R/F126K) that was expected to fold in a monomeric form, missing important contacts at the dimer interface. The mutant protein proved stable enough to be expressed at a high level, and purified in the same way as the native protein. Analytical ultracentrifugation confirms the native protein is a tightly-bound dimer in solution, but the dimer is almost undetectable for the Q12R/F126K mutant  is lectin LSL from the mushroom Laetiporus sulphureus (PDB 1W3A)31. This is a hexameric pore-forming lectin with an N-terminal 32. Strong structural similarity is also seen to the lectin domain of interleukin-33 (PDB 5VI4)33, an agonist cytokine that is mainly expressed by epithelial, endothelial and fibroblast cells, and an attractive therapeutic target34. Overall however, the sequence and structure of SeviL provide few clues to its biological function.Although SeviL shows only weak sequence\u00a0similarity to known structural models, using DALIThe saccharides of asialo-GM1 , as well as lying close (3.0\u00a0\u00c5) to O6 of Gal\u00a04. The side-chain of Arg\u00a026 approaches within 3.6\u00a0\u00c5 the O3, O4 and O5 atoms of NGA-3, forming two hydrogen bonds with O4. A single hydrogen bond to the ligand involves the main-chain of SeviL, with Asp39 donating a bond to O6 of Gal4 through its nitrogen atom. On the opposite side of the ligand from Arg\u00a026 and Asp\u00a028, the aromatic side-chains of Phe\u00a032 and Tyr\u00a037 lie close together, providing a hydrophobic surface that touches each hexose except the glucose residue. The N-acetyl group makes no strong interaction with the protein, pointing into the bulk solvent, suggesting it plays no part in ligand specificity.Two copies of the tetrasaccharide ligand are observed in the asymmetric unit, and each hexose unit is clearly visible in the electron density map. The glucose (BGC-1) appears to be more flexible, making no direct contact with the protein and having weaker electron density than the remainder of the ligand. For one copy of the saccharide in the asymmetric unit, the glucose residue makes slight crystal contacts which possibly reduce its movement within the crystal lattice, but apparently have little effect on the overall ligand conformation (see below). The sugar binding site is centred around the position of one of the chloride ions identified in the apo-model, with the N-acetylgalactose residue (NGA-3) displacing the halide (and the Arg\u00a026 side-chain of a neighboring protein molecule) from the pocket formed by Phe\u00a021, Phe\u00a032 and Tyr\u00a037. Contacts between asialo-GM1 and SeviL are listed in STable 41. Even though the glucose residue forms fewer contacts with the proteins, and has weaker electron density in the final 2mFo-DFc map, it appears to be little more variable in conformation than the other residues of the tetrasaccharide. No torsional restraints were applied on the bonds between saccharide residues in the refinement. Modelling the GM1 saccharide into the binding site of SeviL (based on the known complex structures) shows that the sialic acid group connected to GAL-2 would cause a severe steric clash with Phe\u00a032  was used to fit by least-squares the GAL-2 residue of each complex with that of asialo-GM1 bound to SeviL. Overlap of the different ligands shows that, despite the very different binding modes, the saccharides of each complex show remarkably similar conformations Fig.\u00a0B. ComparAgrocybe aegerita is found to involve a hydrogen bond network formed by two water molecules held between the saccharide and arginine and glutamate side-chains39. The human protein galectin-3 also binds TF antigen and related sugars including the GM1 saccharide, with arginine and glutamate residues contacting the ligands indirectly through water molecules in a similar fashion to AAL42. SeviL is notably different, despite binding very similar ligands, in that no water molecules are found between the protein and bound asialo-GM1 saccharide. This close association gives SeviL a narrower range of binding partners than galectin-3, without apparently giving a significantly greater affinity of binding42.Lectins are often found to bind their substrate sugars specifically by recognizing the water structure around the ligand, so that, for example, the recognition of the Thomsen-Friedenreich (TF) antigen  with access code 26300. Peaks showing notable shifts with added ligand are shown in Fig.\u00a0In order to observe the effects of saccharide binding to SeviL in solution, the HSQC spectrum of SFigures\u00a0 and\u00a05. T43 were performed on the protein-ligand mixture to gain further molecular insights into the recognition of GM1b saccharide by Sevil . As described in the supporting information  NMR experiments44. By assuming a model of non-interacting sites the data for both ligands can be interpreted in terms of an exothermic and an endothermic binding site, with To confirm the binding specificities implied by the structural model, ITC was used to measure the affinity of SeviL for different small sugar ligands, fitting the binding curve to a simple model with one type of independent site. Experimental details are given in Methods/Isothermal Titration Calorimetry. Glucose was not seen to interact with the protein at all, but galactose, N-acetylgalactose and lactose were found to bind with mic Fig.\u00a0A,B. ITC mic Fig.\u00a0C,D. Erro SFigure\u00a0. Such th16. Mutants of SeviL, designed on the basis of the model described here, that are unable to bind the saccharide or form the dimer do not hemagglutinate  are found to be roughly equally present in the adult mammalian brain, where between them they\u00a0account for all but a few percent of the total gangliosides present51. Gangliosides are the receptors for viruses (such as SV40) and toxins (such as cholera toxin)53. The majority of cancer cells show some form of aberrent glycosylation of glycolipids at the cell surface, and this has led to considerable interest in the development of immunotherapies55. Asialo-GM1 for example is expressed on bone metastatic C4-2B prostate cancer cells, and appears to play a significant role in the progression of the disease through its interaction with the integrin 56.Gangliosides, a sub-group of glycosphingolipids, are known to be involved in a wide variety of physiological processes, including cell growth and migration, as well as apoptosishttps://pfam.xfam.org) defines 23 different subfamilies with this fold, including ricin-type lectin and fibroblast growth factor. 29. The CAZY database of carbohydrate-binding modules (www.cazy.org) also fails to include MytiLec in the family CBM13, which is defined as 57. Although the fold is common, lectins in this family are generally found to accommodate galactose or simple derivatives of it in the binding site. SeviL clearly forms a Streptomyces olivaceoviridis58; sequence and structural comparisons produced with Espript (http://espript.ibcp.fr)59 are shown in SFigure\u00a0SeviL is a recently described lectin, of uncertain biological function, isolated from mussels. Here we have shown the protein is a dimeric member of the well-known 16. The models described in this paper form the basis of further work to develop a new biosensor for asialo-GM1, a useful addition to present lectin arrays, as well as understand the biological effects of SeviL on cells that display its target saccharide.SeviL is the first lectin to be described that shows an exquisite selectivity for asialo-GM1 over the closely related GM1, suggesting it may well be a useful tool in ganglioside research, and especially in studies of NK cells, which are currently of enormous interest due to their central role in the control of cancer by the immune system. Further work is needed to investigate the biological function of SeviL, and whether it may be used to control the activity of cancer cells, or the immune cells that attack them. We have shown (using the assay described in Methods) that SeviL can trigger hemagglutination at low concentration, but only in the presence of calcium. The structure shows no evidence of any calcium binding site, such as a cluster of carboxylate bearing side-chains, indicating that the requirement for calcium is due to a secondary effect on the target cell. Eliminating saccharide binding with a single site mutation, changing Asp\u00a039 to histidine, abolishes hemagglutination. Hemagglutination is also lost in a SeviL mutant that retains sugar binding, but forms only monomers. The dimeric form therefore appears to be required for at least some of the biological effects of the native protein, which include stimulation of apoptotic pathways in cells that SeviL can bindc(s) analysis module in the program SEDFIT60. Frictional ratio (f/fo) was allowed to float during fitting. The c(s) distribution was converted into a molar mass distribution c(M). Partial specific volume of the protein, solvent density, and solvent viscosity were calculated from standard tables using the program SEDNTERP61.The sample concentration was estimated as 1.0\u00a0Experiments were performed using a MicroCal iTC200 (GE) instrument. The sample of SeviL  was placed in the cell, and equilibrated at 298\u00a0K. The selected ligand was dissolved in the same buffer, and injected into the protein sample  [2O. The spectral widths  of each spectrum were 24\u00a0ppm  for the 67 and SMILE68, and signal assignments were performed using the programs MagRO69 and NMRView70.SeviL labelled uniformly with 71. The dissociation constant was set in the low millimolar range in accordance with ITC results. The NOE R factor, a normalised root-mean square deviation (RMSD) value, represents a measure of the fit between experimental and theoretical data.STD NMR analysis of the interaction between SeviL and the GM1b\u00a0saccharide was performed by dialysing protein against water, freeze-drying and redissolving in phosphate-buffered saline (PBS). STD NMR experiments were carried out at 298\u00a0K using a Bruker 600 MHz DRX spectrometer fitted with a cryoprobe. The pseudo 2D pulse program stddiff.3 was used. The experiments were recorded as 32000 data points and zero-filled up to 64000 data points prior to processing; the protein resonances were selectively irradiated at 6.5\u00a0ppm by a train of Gauss pulses with a length of 50\u00a0ms. A 20\u00a0ms spin-lock pulse was applied to reduce protein signals. A saturation time of 2\u00a0s and a protein:ligand molar ratio of 1:10 were used. The STD effect of a given proton was calculated by using (I0 - Isat)/I0, where (I0 - Isat) is the intensity of the signal in the STD NMR spectrum and I0 is the peak intensity of an unsaturated reference spectrum (off-resonance). The STD signal with the highest intensity, the proton at position 4 of GalNAc (C) residue, was set to 100% and the others were normalised to this peak, allowing ligand epitope mapping. Data acquisition and processing were performed with TOPSPIN 3.2 software. The theoretical STD effects of Sevil-GM1b complex were calculated with the program CORCEMA-ST72. 20\u00a02. The plate was incubated at room temperature for 1\u00a0h, and the formation of a sheet (agglutination-positive) or dot (agglutination-negative) was observed and scored against the lectin titre.Hemagglutination assay was performed in 96-well U-shape plates as described previouslySupplementary information 1."}
+{"text": "Rich phenomena from complex systems have long intrigued researchers, and yet modeling system micro-dynamics and inferring the forms of interaction remain challenging for conventional data-driven approaches, being generally established by scientists with human ingenuity. In this study, we propose AgentNet, a model-free data-driven framework consisting of deep neural networks to reveal and analyze the hidden interactions in complex systems from observed data alone. AgentNet utilizes a graph attention network with novel variable-wise attention to model the interaction between individual agents, and employs various encoders and decoders that can be selectively applied to any desired system. Our model successfully captured a wide variety of simulated complex systems, namely cellular automata (discrete), the Vicsek model (continuous), and active Ornstein\u2013Uhlenbeck particles (non-Markovian) in which, notably, AgentNet\u2019s visualized attention values coincided with the true variable-wise interaction strengths and exhibited collective behavior that was absent in the training data. A demonstration with empirical data from a flock of birds showed that AgentNet could identify hidden interaction ranges exhibited by real birds, which cannot be detected by conventional velocity correlation analysis. We expect our framework to open a novel path to investigating complex systems and to provide insight into general process-driven modeling. Unfortunately, due to the intrinsic complexity of these systems, extracting hidden micro-dynamics from the observed data of an unknown complex system is virtually infeasible in most cases. Although conventional process-driven modeling is intelligible and provides the conceptual framework, its application to complex systems, to date, still strongly relies on human intuition with various prior assumptions.Complex systems are collections of interactive agents that exhibit non-trivial collective behavior. They have gathered a significant amount of research interest in the last several decades in a wide variety of academic fields from spin systems to human societies. In particular, the domain of physics mainly focuses on investigating the micro-level processes that govern emergent behavior in complex systems and modeling them mathematically. The Vicsek model11, DDM was employed to discover hidden parameters or dynamics from data in an automated manner. Particularly, active matter modeling greatly relies on DDM by first designing a model with intuition from observed data and then performing parameter fitting to match the data16, although many of them suffer from sparse, noisy, or discontinuous observation data.To overcome these obstacles, data-driven modeling (DDM), a methodology that finds a relationship between state variables or their time evolution from observed data, has emerged as a powerful tool for system analysis alongside the emergence of machine learning and large-scale data. In previous literature17, which models dependencies between linked agents on a graph and has enabled remarkable progress in graph analysis. Similar to18, one may depict a complex system as a dynamically changing graph in which each vertex is an agent, with links between agents indicating interactions. In this approach, the problem of modeling the micro-dynamics of single agents becomes equivalent to properly inferring the effect from other agents on a graph and estimating the state transition of each agent at the next time step. Several attempts have been separately made to employ GNNs in the prediction and analysis of specific complex systems and physical models29, but these approaches are mostly limited to the verification of a single system or a small number of agents, and more significantly, it remains difficult to interpret the characteristics of the interaction due to the neural network\u2019s notorious black-box nature. Recently, graph attention networks (GATs)30 and its applications32 showed a path to interpretable GNN by assigning attention to important neighbors, but this attention value cannot be directly interpreted with a physical meaning. For instance, in a multi-dimensional system, the interaction strength cannot be a simple scalar value since each state variable possesses its own interaction range and strength and may yield an assymteric, inhomogeneous interaction range.Among various DDM techniques, deep neural networks (DNNs) have recently shown phenomenal performance in pattern recognition and function approximation. One specialized DNN variant for graph-structured data is the graph neural network (GNN)34, AgentNet poses minimum prior assumptions about the unknown nature of the target agents. Our model jointly learns the interaction strength that affects each variable\u2019s transition and overall transition function from observed data in an end-to-end manner without any human intervention or manual operation. This is a critical difference from the conventional approach with GATs, which only assigns a single attention value per agent while our model assigns completely independent attention values for every state variable and employing separate decoders for each of them. We found that our variable-wise neural attention achieves better performances over GATs\u2019 single multiplicative attention, and enables more extensive physical interpretation for the first time that was impossible for conventional GATs such as identifying directional forces separately. Also, the visualization and inspection of the inner modules as granted by our framework enables a clear interpretation of the trained model, which also provides insights for process-driven modeling. As a prediction model, a trained AgentNet can generate an individual level of state predictions from desired initial conditions, making AgentNet an outstanding simulator of target systems including even those that exhibit collective behavior that was absent in the training data.Inspired by these recent attempts, we introduce AgentNet, a generalized neural network framework based on GATs with a novel attention scheme to model various complex systems in an physically interpretable manner. AgentNet approximates the transition function of the states of individual agents by training the neural network to predict the future state variables. Due to the rich functional expressibility of DNNs, which is practically unconstrained35, the Vicsek model1, and the active Ornstein\u2013Uhlenbeck particle (AOUP)36 model, along with application to real-world data comprising trajectories in a flock of birds37 containing more than 1800 agents in a single instance, greatly exceeding the previous range of neural network approaches for the interpretable interaction retreival39 which treated at most hundered of agents. For the simulated systems, we show that each component of AgentNet learns predictable and tractable parts of the expected transition function by comparing extracted features with ground-truth functions. For the bird flock where the exact analytical expression of the system is completely unknown, AgentNet successfully provides the interaction range of a bird, which is physiologically plausible and coincides with previous behavioral studies about the bird41.First, we show the spontaneous correspondence between the complex system and the structure of AgentNet by providing formulations of both systems. The capability of AgentNet is thoroughly demonstrated here via data from simulated complex systems: cellular automatan agents for which the state of each agent until time T is  identified and observed. The basic premise of the agent-based system is that the agent with the same state variable follows the same decision rule, and the interaction strength between two agents can be fully expressed by their state variables. This implies that any two agents with the same state variables should be interchangeable without altering the outcome.In this paper, we focus on a general agent-based system consisting of n agents as t as k state variables j number of time-dependent global external variables t to We denote the set of all m is the maximum lag for the system output and F is an overall function that could be deterministic or stochastic. If we focus on the state difference of an individual agent, we can split the overall function F into indvidual transition function f and getith agent\u2019s state vector Generally, agent modeling of a complex system aims to identify the transition function of its constituents through time steps, which can be written asith and jth agents along with the effect of 42, the voter model43, systems governed by Newtonian dynamics, and phase space dynamics driven by the Liouville equation.In this study, we assume that the system is mainly dominated by pairwise interactions and higher-order interactions are negligible. Alleviation of this assumption will be discussed in the Conclusion. This means that Eq.\u00a0 becomes4q-th state variable\u2019s interaction magnitude of the ith agent, induced by the jth agent. Now, jth agent without loss of generality. So far, we have decomposed an individual transition function into four parts; variable-wise interaction strength function v, self-interaction function f. We note that our formulation aptly applies to a physical system governed by force dynamics by interpreting v contains directional information.Although Eq.\u00a0 sums up v, f) are completely unknown, and it is infeasible to elicit these functions from observed data alone. Especially, blindness to variable-wise interaction strength function nonlinear functional form of v, f. The problem becomes harder if the system has time-correlation because it expands the range of possibly correlated variable pairs further out in the time dimension. To sum up, many of the current methodologies are not capable of DDM for complex systems without strong prior assumptions regarding the functional form. The proposed framework, AgentNet, successfully tackles this conundrum by employing DNNs to jointly learn all of the aforementioned functions by constructing corresponding neural modules for each of the functions and backpropagating errors from state predictions.In most cases, the exact analytic forms of all these functions  models for the encoder and decoder of AgentNet to capture  memory effects in the system.First, AgentNet can handle various types of state variables by minimizing cross-entropy for discrete variables and the mean squared error for continuous variables. Second, when the decision rule of a target system is stochastic, there are several ways to construct a neural network with probabilistic output44 where a softmax normalization between agents was used   model. In the CA model, each cell has its own discrete state, either We simulate CA data in the form of a 14 realized that a vast majority of the cells are irrelevant to the target cell, and thereafter concentrated its attention to a more compact region; Fig.\u00a0Figure entclass1pt{minimaentclass1pt{minima1 (VM) is one of the earliest and most prominent models to describe an active matter system, where each agent averages the velocity of nearby agents (including itself) to replace its previous velocity. At each time step, every agent updates its position by adding this newly assigned velocity with stochastic noise. In this study, every 300th agent in the simulation interacts with other agents within the range Next, we validate the capability of AgentNet for a continuous and stochastic system. The Vicsek modelThe model receives four state variables, positions x-variable attention weight x and y-variable attention only for neighbor agents, while the untrained AgentNet has no distinction between neighbor and outside cells. The predicted position distributions for these two sample agents are depicted in Fig. As a result, AgentNet for VM achieved a NLL loss of T is temperature36. Here, i due to the soft-core potential from other particles, R. In this study, we use AOUPs confined in a harmonic potential as an example system, describing the intrinsic propulsion force 52. This model is known to exhibit a collective clustering phenomenon, with the periodicity of the resulting hexagonal pattern known to be approximately 1.4R with no 53.Differing from the Vicsek model, some active matter shows a time-correlation of particle positions due to the force inherent in the particles that allows them to move. These systems are generally referred to as self-propelled particles, which can be described by overdamped Langevin equations for the position R ranging from 2.0 to 4.0, and predicts the parameters for four 1D Gaussian distributions, similar to the AgentNet for VM. Note that the internal variable, has to infer this hidden variable by eight steps of past trajectory.AgentNet for AOUP adopts an LSTM model as an encoder to enable iterative data generation. The model observes 8 steps of trajectories as input data, and the loss is calculated for the next 12 steps. The model receives four state variables, 23 along with a linear extrapolation and naive LSTM without the graph attention core as baselines. AgentNet for AOUP showed ADE/FDE of 0.041/0.064, while linear extrapolation and LSTM showed much lower performances of 0.210/0.465 and 0.158/0.316, respectively. The performance of our model also exceeds the modern architectures like GAT3+ (GAT with 3-headed attention and transformer architecture), which showed the performance of 0.065/0.087. Figure R and further exhibits a collective behavior that occurs far beyond the trained time scale. Since our model can iteratively predict future states indefinitely, we tested our model to predict a total of 42 steps, which is 30 more steps than the model was originally trained for. Surprisingly, our model predicts a precise hexagonal pattern of periodicity 7, which coincides with the theoretical value of periodicity when First, we compare the average displacement error (ADE) and final displacement error (FDE) of our model among 12 predicted steps as in previous worksx and y-directional velocity x direction 51).Moreover, we demonstrate that the attention mentclass2pt{minim40. We employed here a portion of the data from54, recorded in Raleigh, North Carolina, in 2014. Since half of the trajectories last less than 23 would significantly reduce the number of birds to consider at a given time step. To handle these disjointed yet entangled pieces of trajectories, we propose a novel inspection method that examines the data at every step of the LSTM to manually connect the hidden states from the past, exclude the nonexistent birds at a certain time, and start a new chain of hidden states from a separate neural network if an agent newly enters the scene. While several previous approaches could handle graphs with dynamic edges58, AgentNet is, to the best of our knowledge, the first attempt to deal with dynamic nodes on a spatiotemporal graph . Bird flocks are renowned for their rich diversity of flocking dynamics, for which models with various mechanisms such as velocity alignment and cohesion have been proposed in the last several decadesThe number of total birds appearing in each set varied from 300 to 1800, and each trajectory in the set started and ended at different times. The model received state variables that exist at the current time step, produced statistics of three-dimensional position and velocity, and then the sampled states were fed back into the model for the next time prediction. NLL losses were calculated at every LSTM step for existing birds.xy-plane coincides with previous literature about biological agents\u2019 visual frustum, which depends on forward-oriented sight and the relative distance from each agent59. Also, the bird\u2019s z-directional attention is relatively concentrated downwards; this predicted attention is physiologically plausible since downward-oriented visual fields are widely reported in various types of birds due to their foraging nature and the blind area from the beak60.Figure\u00a0xy-plane and xz-plane shows no particular directional tendency as attention does. Although many studies employ state correlations between agents to figure out the characteristics of interaction62, correlation might be significantly different from the interaction range itself63. Different from correlations, our model provides a causal interaction strength since the attention value is strongly connected to the predictability of future dynamics, which is quite useful for inferring and modeling the microdynamics of individual birds.Interestingly, Fig.\u00a051). This directional homogeneity strongly implies that the bird-bird interaction is more like a near-sighted version of the Vicsek model, differs from the distance-based force models like AOUP which must exhibit directional heterogeneity. In conclusion, AgentNet employed the position and velocity (heading direction) of neighboring birds into its prediction, thereby showing better prediction compared to the non-interactive baseline and qualitatively plausible interaction range.Our model with variable-wise attention can further verify important physical insights. For instance, we have found that although the scale is different, the form of attention concentration is surprisingly the same regardless of the directions . The dimension notation such as  means that the model consists of three perceptron layers with 32, 16, and 1 neurons in each layer. Also, dims. is an abbreviation of dimensions.We implemented our AgentNet model with PyTorch. Here, input dimensions are chosen as the sum of the number of state variables and additional variables, such as global variables (as in AgentNet for AOUP) or indicator variables (as in AgentNet for CS). The form of the final dimension indicates that each output of the encoder  will be processed separately. See44 for more details about transformer architecture.All of the encoding layers of AgentNet are composed of qth state variable between agents i and j, and feeds the concatenated vectors into MLP asWith these outputs and  global external variables  value and its averaged attention-weighted values (from others) and feed it into the variable-wise separated decoder. Since two tensors are concatenated, the last dimension of this tensor has twice the length of the original dimension of the value tensor. The decoder consists of . For instance, AgentNet for CS has In the stochastic setting , the decoded tensor further feeds into other layers to obtain sufficient statistics for the probabilistic distribution. In this paper, those statistics are means and variances of state variables. MLP layers for these values consist of 70. Hidden states and cell states have 128 dims. each and are initialized by additional MLPs that are jointly trained with the main module. As explained in the main manuscript, AgentNet checks at each time step whether an agent is new and present. When an agent is newly entered, new LSTM hidden states are initialized. Otherwise, hidden states succeed from the previous result.In the case of a target system with probable time correlations, we adopted long short-term memory (LSTM) as an encoder to capture the correlations30, we left everything the same as AgentNet and replaced variable-wise attention core to original graph-attention core with linear projection matrices of  and inner-product attention was used with those 128-dimensional vectors. For GAT3+, we implemented multi-headed attention (with 3 heads) with dimensions of 12 (for AOUP) and 32 (for CS), and every other module is the same as AgentNet. This choice is to .For the baseline, we employed a MLP, LSTM, and GAT model where the variable-wise graph attention module is missing. For MLP and LSTM, We doubled the number of layers and neurons of the decoder to compensate for the missing attention module, which its decoder consists of 71 with a form of 72 were used for the construction of models and training. The learning rate was set to 0.0005 and decreased to 70% of the previous value when the test loss remained steady for 30 epochs. Batch size is fixed to 32 in every experiment for AgentNet and baselines, except for the AgentNet for CS where a single batch is used due to a memory limitation. In the case of AgentNet for CS, we employed weighted NLL loss for different time steps, in which weights are inversely proportional to the frequency of the sample with a given trajectory length, to resolve the imbalance of available trajectory length. Table All training used 2 to 10 NVIDIA TITAN V GPUs, with which the longest training for a single model took less than two days. Mish activation functionSupplementary Information."}
+{"text": "Receiving a diagnosis of autism in adulthood can be a life changing event, impacting identity, relationships, and mental health. A lack of post-diagnostic support has been highlighted by autistic adults, their allies, clinicians, and service providers. It can be a source of distress for autistic adults, reinforcing feelings of social isolation and rejection. Peer support could be a cost-effective, flexible, and sustainable model to provide community-based support for autistic adults. However, there is little research on the value of peer support, despite calls from the autistic community. This qualitative study explored autistic experiences and needs post-diagnosis, identifying specific ways that peer support may benefit them, and exploring the limitations of peer support. Twelve autistic adults who had all received an autism diagnosis in adulthood completed a semi-structured interview focussing on the diagnostic experience, post-diagnostic support needed and provided, engagement with the autistic community, and post-diagnostic peer support. Thematic analysis of interview transcripts resulted in four themes: (1) Mismatch in support needed and provided; (2) Community connection; (3) Flexible and personalised support; and (4) Sustainability. Participants indicated that peer support may be a useful mechanism to support autistic adults\u2019 post-diagnosis and offers unique opportunities not available through other support channels. Though informal peer support exists, it could be more sustainable and effective if well-supported and funded. Due to broader diagnostic criteria and increased public awareness  many autThough autism diagnoses are more widely available, there are many barriers to efficient diagnostic assessment pathways, and appropriate post-diagnostic support . The aduThe lack of post-diagnostic support has been highlighted by autistic adults, the families of autistic people, clinicians, and service providers . Most adReceiving an autism diagnosis as an adult can be a life changing event . Though Diagnosis can have a profound impact on identity , and somWhile autistic/non-autistic interactions can be positive e.g., , autistiThe peer support model assumes that shared experience of a phenomenon enhances the development of an empathetic supportive relationship . In non-n = 7), the success of the participant-led discussions was interpreted by the authors as illustrating the willingness of the students to engage with one another and with this form of support. Participants highlighted that they appreciated the opportunity to offer advice and support to others. n = 7) to explore the topic of autistic peer support with a group of autistic adults who worked with and provided support to young autistic adults in an autistic-only workplace. They sought to outline an alternative model of autistic development, underpinned by common experiences, mutual understanding, and a focus on ways of being that are different, rather than deficient. In n = 9 pairs), the importance of supervision and ongoing training was emphasised, as well as reliability and consistency on the part of the mentor. Although the majority of the mentors in this study were not autistic, those in same-neurotype pairings reported benefitting from the enhanced empathetic closeness brought about by similarity of experiences between the mentor and mentee. Finally, A small number of studies have examined autism-specific peer support in adults. Most of these studies have involved support delivered through online support groups . MacLeodWhile further research examining the efficacy of a post-diagnostic peer support intervention for autistic adults is needed, a crucial first step is to ensure any peer intervention is co-designed with or led by autistic people and reflects their views, preferences, and priorities. The purpose of this study was to elicit the views of people who had received a diagnosis of autism in adulthood, exploring their diagnostic experiences, the post-diagnostic support that they needed and were provided. We focussed on what the function and focus of peer support may be, whether they felt it could act in place of other support, should exist alongside it, or not at all, and the benefits and challenges that they suggest it may face. In this study, we used a qualitative methodology to examine these views and explore how these may be incorporated into a future support system.This study used a qualitative design, with semi-structured interviews analysed thematically. Ethical approval was obtained from the University of Edinburgh Moray House School of Education and Sport Research Ethics Committee.Participants were 12 autistic adults (see Participants (7F/1NB/4M) had a mean age of 44.92 years [standard deviation (SD) = 11.94], and a mean, autism quotient (AQ) score of 41.25 (SD = 3.74). The mean age of diagnosis was 40.75 years (SD = 12.01), and mean time since diagnosis was 4.25 years (SD = 2.8). All participants identified as white British, Scottish, or European. A number code was generated for each participant and identifying details redacted from reported quotes.n = 2), over the phone (n = 1) or via video call (n = 9) depending on the preference of the participant.All participants provided written informed consent before the study commenced. Before the interview commenced, participants were told (1) that they could take a break at any time during the session for any reason, (2) they did not have to talk about anything they did not want to talk about, and (3) if they wanted to answer a question in more detail, they could go back to a question or answer it in more detail. The first author conducted interviews either in-person  did not meet their needs. They stated that support should focus on enhancing understanding on how this diagnosis applied to their lives, their relationships, and future plans, and that support should come from sources that understood their experiences.The information provided post-diagnosis was mostly in written form and described what autism is, which participants found insufficient and not helpful. The content of written resources focussed on facts about autism, whereas participants wanted to know what being autistic meant/looked like for them, and what it meant for their relationships and identity.\u201cI got a print out leaflet about Asperger\u2019s. They weren\u2019t particularly useful suggestions. I already knew about autism cause I had studied it, and I had worked with autistic kids, so that wasn\u2019t much use to me\u201d \u2013 Participant 3 .Reading the report, it is written in quite a negative way. Because it is the deficit model. So it was quite hard reading the things about myself that I am bad at. Cause I have tried my best to hide them, for the last 40 years. [\u2026] I am an intelligent person, I am a very capable person but [\u2026] this new diagnosis and this new information [\u2026] I was still not quite sure what to do with it.\u201d \u2013 Participant 11 . Some participants described the negative impact this had on their self-esteem \u2013 \u201cthe diagnosis had a knock-on effect on my confidence at that time [\u2026] I think I needed probably a bit of help to process some of that stuff.\u201d \u2013 Participant 6 .The personalised information that participants received in their diagnostic report was often framed in a very negative and pathologising way, and was difficult to process. \u201cParticipants had a good understanding of autism and its core diagnostic features in a general sense, but had difficulty understanding what this meant for them on an individual level. As autism is very heterogeneous, many struggled to identify with the information provided to them. What most participants wanted was the opportunity to discuss what the diagnosis meant to them.\u201cThere wasn\u2019t any help. You\u2019ve just been diagnosed with something which is a neurodevelopmental condition and a disability and you have had it all your life, it was quite\u2026 well it was frustrating but it also it was quite\u2026 in a way quite scary to be sort of\u2026 left unsure how to manage this in\u2026 from the rest of my life going forward\u201d \u2013 Participant 5 .Many participants reported requesting post-diagnostic support and being refused, or told none was available.\u201cI mean there is nothing available really [\u2026] I did go to my GP a few months after my diagnosis and explained\u2026but they couldn\u2019t help me. My GP was quite understanding\u2026 really understanding, really, and put me in touch with somebody fairly senior from the local adult autism team\u2026 but they weren\u2019t very helpful.\u201d \u2013 Participant 8 .\u201cif there is any support available it is very much geared towards people with much higher support needs in terms of day to day living.\u201d \u2013 Participant 8 ]. Some participants reported being perceived as too \u201chigh functioning\u201d to require support . Services did not offer support to build understanding of the diagnosis, how it fits within a person\u2019s identity, and build strategies to help them move forward.Some participants had been signposted to post-diagnostic support, however this had been designed for autistic people with high support needs or a learning disability, and was therefore not suitable for their needs [\u201cGetting a diagnosis was also a frustrating thing because [\u2026] there wasn\u2019t really anything in the report that I was given at my diagnosis or after my diagnosis that really said anything like \u2018well this is how it seems to us how your autism has affected you\u2019.\u201d Participant 5 .I had to wait a year for the post diagnostic group to start again. When I went along to that, only two of us turned up so they cancelled it so then I had to wait another four months until they set up another one, I was in crisis at that point and I really needed the support right then and there. Not\u2026 wait a few months\u2026 and then wait a year\u2026 it wasn\u2019t great\u201d \u2013 Participant 3 . Some faced geographical barriers due to living a long distance from services, especially those who did not live in cities . Some found that communication difficulties related to being autistic made it difficult to reach out to services. Referrals had also been made to services with insecure funding, and the lack of consistent or sustainable long-term funding increased anxiety when engaging with a service, which in some cases led to reduced engagement.Participants also reported barriers to accessing autism services. Some were oversubscribed with long waiting lists, so support was significantly delayed and not available when most needed .Some participants also found that receiving an autism diagnosis resulted in them losing support from health providers or third sector organisations, as it was assumed that their support was not suitable for an autistic person.\u201cI asked our  counselling service, I wanted to talk to somebody about stuff, but because I asked them \u2018I want some help talking through stuff in relation to my autism diagnosis\u2019 they said \u2018oh no, we can\u2019t help you, we are not specialist enough for that.\u201d\u2019 \u2013 Participant 11 .\u201cI did phone mental health services and ask them for help. and they said autism isn\u2019t a mental health issue. Even though that was where I got diagnosed and that I sounded quite articulate and I would be all right.\u201d \u2013 Participant 4 .In general, participants said that the most beneficial support to them would have included building a sense of identity and community, as well as self-care skills and tools to manage stress and sensory and social overload .Explaining what autism is to family, friends, and employers, and identifying and requesting beneficial adjustments could be difficult. Some participants highlighted that friends and family had difficulty in accepting and understanding their diagnosis, and it was particularly difficult when others doubted or disbelieved their diagnosis, or perceived them as so \u201chigh functioning\u201d to not require adjustments.\u201cI needed help explaining it to my family. Cause even though my mum was there (at the diagnosis) and got it explained to her, she hadn\u2019t quite\u2026 she knew what autism was but she didn\u2019t really know what it was in relation to me.\u201d \u2013 Participant 3 .\u201cI think people who weren\u2019t autistic didn\u2019t understand why it was important\u201d \u2013 Participant 6 .All participants described their difficulties processing the diagnosis, and reframing history in light of their diagnosis. Though some highlighted that diagnosis does not change who you are as a person and that you are still the same person afterward, it is still an adjustment and a lot of information to process, and it can take a long time to get used to.\u201cI guess it was\u2026 well it still is, a bit of a rollercoaster because you have to\u2026 it kind of forces you to look back on your life through a different lens. Lots of stuff made sense. There was a lot of positives, where I could look back and think\u2026 well actually I didn\u2019t fail at this thing that I didn\u2019t manage to do. There was a reason that I now understand so I can forgive myself for not going to a top university and becoming a high-flying lawyer or whatever. Because that just wasn\u2019t the right thing. And now I understand why I struggled with certain things. So that made sense.\u201d \u2013 Participant 8 .\u201cI think maybe some kind of support to just sort of deal with the transition into sort of knowing. I usually describe it like if you had your skirt caught in your knickers or something and you realised in that moment and you are like \u2018oh my god, it has been like this the whole time\u2019 and you think back to all these social situations that you thought you were so dead on with and then you look back and you were like \u2018no\u2019.\u201d \u2013 Participant 6 .\u201cInitially you had this bounce of\u2026 you have this sort of elation of knowing what things are, and being able to reframe things, but actually the problems were still there. But then actually once that has subsided then it is still the \u2018oh god, what do I do now?\u2019 sort of a feeling.\u201d \u2013 Participant 9 .Participants experienced many emotions in the aftermath of diagnosis, including fear, happiness, validation, relief, upset, and anger, but lacked an understanding person to discuss this with. Prior to their diagnosis, participants had often experienced difficulties in their social life, complex sensory processing differences, and difficulties with executive function. Many participants felt an important part of the post-diagnostic process was to develop self-compassion and understanding of what being autistic means, and reframing their identity and expectations around them being an autistic person, rather than a \u201cfailed neurotypical.\u201d Spending time with other autistic people in a peer support setting may have been particularly beneficial in understanding oneself and validating their experiences.\u201cI would have been very happy to just to be in a room with people who had that new diagnosis in common\u2026 because I have found that in trying to understand how the course of my life has run, in the light of this new diagnosis I have very often figured things out on the basis of something that somebody else has said about the course of their life. And it has not always been\u2026 \u2018oh that has happened to me too\u2019. It has sometimes been \u2018oh well that didn\u2019t happen to me\u2019 or \u2018I don\u2019t have that particular expression of the condition so\u2026 how does it work in me instead?\u2019. But it has been that prompt of having somebody else\u2019s life story, if you like, that has been really important for me understanding myself I suppose, ultimately.\u201d \u2013 Participant 5 .\u201cJust having someone kind of validate your symptoms and what you have been through, and when you have been in that mindset of \u2018no it is just me, I am just not coping well with this\u2019. Suddenly going from seeing myself as like a slightly defective neurotypical person, to actually a pretty well coping autistic person.\u201d Participant 11 .\u201cMost of the support I get is through social media\u2026there is no real official diagnosis support\u201d \u2013 Participant 7 /\u201cThrough twitter and facebook, I have found a community in which I get support. But when it comes to actual formal support in healthcare, it is a dead end for me\u201d \u2013 Participant 9 ]. They noted how useful it could be to learn about autism from other autistic people . However, not all participants enjoyed social media engagement . \u201cSome people are very angry on there [social media] or really struggling and sometimes it can be quite depressing to read those things\u201d \u2013 Participant 2 , and thought they would have benefitted from in-person peer support .In the absence of the formal support desired by autistic adults post-diagnosis, many participants reported turning to social media groups to connect with other autistic people . This communicative ease and mutual understanding may create a comfortable and supportive environment for peer support following diagnosis, and more so than support provided by non-autistic people.Participants felt that after receiving an autism diagnosis, building connections with like-minded peers, and feeling part of a community was particularly important. Participants said that relationships with other autistic people felt easier and more comfortable than relationships with non-autistic people, and that they experienced feelings of similarity and connection with them. Participants felt that they were more able to create authentic connections with other autistic people because they had a reduced need to mask their natural autistic behaviours . It is also important to consider compatibility (or incompatibility) of communication styles and preferences of group members.Autistic people are not solely defined by being autistic and for peer support to be successful, participants felt it was important to take into account other aspects of their backgrounds  are communicating effectively to the particular person that they are mentoring\u2026you need someone who can communicate with you in a way you respond to positively. Otherwise you are just going to feel even more alientated: here I am with this new diagnosis and even other \u201cAspys\u201d or people on the spectrum don\u2019t get me either\u201d \u2013 Participant 5 .Participants stated that peer support coordinators require training and support in order to be able to run a peer support service that meets the needs of a wide range of autistic adults.\u201cIf you are going to mentor someone you would need to tell them what other support is available. Training on what they are entitled to, what issues they might experience, where to get help, that sort of thing. And where to get help if they are having mental health problems or bullying at work, or anything like that. Some kind of psychological training or coaching because you don\u2019t want to say the wrong thing\u201d \u2013 Participant 7 .\u201cBecause they [peer support coordinators] are offering psychological support, they will need training before doing it.\u201d \u2013 Participant 8 .\u201cTraining on group dynamics if they are not managed well, if there is a personality clash or something like that, places of inclusion can become quite exclusive\u201d Participant 9 .Some awareness of being a parent or carer\u2026and I don\u2019t know if they would have to go on a course to educate themselves but there are other things that non-white autistic people are party to that white autistic people wont understand\u2026little bits of racism\u201d \u2013 Participant 10 .\u201cAdditionally, participants reported that facilitators should have professional supervision to reduce the risk of burnout, and to enhance resilience and confidence.I think kind of like the supervision model is always a really good one. If you are being the person that is taking an emotional burden from somebody then you need somewhere to put it as well. So I think kind of having a forum where I would then be able to\u2026 either I had a mentor or there was some sort of group where mentors were able to chat stuff through and put stuff down, something like that would be really useful. \u2013 Participant 11 .\u201cBeing a peer support worker, there needs to be thoughts about workplace mental health, stress at work, how that affects you, the boundaries of it\u2026I guess proper like supervision, where you were able to talk about these specific issues, that would be a massive part of it\u201d \u2013 Participant 6 .Participants reported that long-term secure funding was essential for providing a stable environment for them to feel comfortable in the peer support relationship. Some participants had engaged with peer support or autistic-led support after their diagnosis, but a lack of sustainable funding had resulted in anxiety around whether support would be available to them beyond the short-term.it should be properly constituted, and perhaps even having some sort of professional workers there as facilitators who are there just to\u2026 you know manage the group and maybe the admin and booking rooms and things like that\u201d \u2013 Participant 9 .Providing administrative support to organise and host peer support is essential to ensure smoothly-run and consistent support: \u201c\u2026Many participants reported that following a diagnosis of autism, services were less likely to offer them support or be willing to provide them with care.\u201cAll of a sudden\u2026 IAPT and local community mental health services wouldn\u2019t actually take a referral because they said it wasn\u2019t within their expertise and that I should access the specialist service. But the specialist service was only a diagnostic service, not a general health and wellbeing and ongoing support service. So it actually closed things off.\u201d \u2013 Participant 9 .Additionally, some services did not consider the autism diagnosis and gave advice that was difficult to understand or apply.\u201cOne thing I have found with my chronic fatigue syndrome for example, some of the things I am expected to do for that are completely at 180 degrees from the things I am supposed to do to manage my depression, so it is very difficult to know what to do. Then when you add autism into the mix as well. It is just a further complication\u2026that has been very, very frustrating and there has been absolutely no support at any level, from the state sector, the NHS, or from the third sector, on trying to figure that out.\u201d \u2013 Participant 5 .Participants noted the importance of recognising the boundaries and limitations of peer support, and acknowledged that autistic adults may require support from other healthcare providers. This may include accessing mental health care from specialist providers. Peer support frameworks should not be used in place of specialist support: it should work alongside specialist teams to ensure that autistic adults can access the support that best suits their needs.In general, participants were very positive about the concept of post-diagnostic peer support. They felt it offered unique opportunities to engage with other autistic people, learn more about autism, and understand how their new diagnosis applied to their lives in a more meaningful way than the facts-based information that had been provided to them at their diagnosis. Participants liked the flexible nature of peer support and the opportunity to focus on a wide range of topics from an autistic perspective. Participants provided key insights into what peer support should focus on, how it should be run, and the specific benefits it may offer. According to these findings, maximising the potential of peer support will involve sustained funding, engaging with other specialist services, and training and support for facilitators. These findings are a crucial step in future studies that may examine the efficacy of a post-diagnostic peer support intervention for autistic adults.Many of the findings of this research are supported by recent literature. One of the benefits of peer support indicated by this study is the relational and emotional benefits of autistic-autistic interaction. Being with other autistic people may act as a buffer to the effects of minority stress , and beiIt is essential for autistic people to be integrally involved with creating, designing, and implementing supports to ensure that they meet their needs , and thiThis study has some limitations. First, the sample included 12 autistic adults. The number of participants for this study was selected before commencing the study and is in line with similar studies in this area see , and basThis study found that peer support may be a useful mechanism to support autistic adults after their diagnosis. Autistic adults were generally positive about the concept of peer support and the opportunity it provides to interact with others in a comfortable way, and to discuss difficulties with empathetic and understanding others. Peer support offers unique opportunities not available through other support channels, and can run alongside other specialist support if required. Peer support may be a sustainable and low cost option to fill the much-needed post-diagnostic support gap currently faced by autistic adults; however, careful planning, ongoing support and training for peer support facilitators, and centring the voices of autistic adults is essential to ensure the success of peer support programmes.catherine.crompton@ed.ac.uk.The datasets presented in this article are not readily available because of the potentially identifiable nature of the raw qualitative interview data. Requests to access the datasets should be directed to CC, The studies involving human participants were reviewed and approved by University of Edinburgh Moray House School of Education Research Ethics Committee. The participants provided written informed consent to participate in this study.CC: conceptualisation, funding acquisition, data collection and curation, formal analysis, project administration, and writing-original draft preparation. SH: formal analysis and writing-review and editing. CM: writing-original draft preparation. AS and SF-W: conceptualisation, funding acquisition, supervision, and writing-review and editing. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Sensor devices that act in the IoT architecture perception layer are characterized by low data processing and storage capacity. These reduced capabilities make the system ubiquitous and lightweight, but considerably reduce its security. The IoT-based Food Traceability Systems (FTS), aimed at ensuring food safety and quality, serve as a motivating scenario for BIoTS development and deployment; therefore, security challenges and gaps related with data integrity are analyzed from this perspective. This paper proposes the BIoTS hardware design that contains some modules built-in VHDL  and other peripheral electronic devices to provide capabilities to the perception layer by implementing the blockchain architecture\u2019s security requirements in an IoT device. The proposed hardware is implemented on FPGA Altera DE0-Nano. BIoTS can participate as a miner in the blockchain network through Smart Contracts and solve security issues related to data integrity and data traceability in an Blockchain-IoT system. Blockchain algorithms implemented in IoT hardware opens a path to IoT devices\u2019 security and ensures participation in data validation inside a food certification process. Internet of Things (IoT) has become an adaptable technology to any context for collecting and transmitting data, analyzing, or informing the stakeholders\u00a0. In all Transparency and data integrity is a critical security issue within IoT ecosystems. Improving the communication process between peer devices might focus on solving data management from several technological schemes (fog or edge computing), but the information transparency recorded is not guaranteed. A current IoT system contains various devices with embedded sensors characterized by low power, reduced memory capacity, and limited processing capabilities. These features allow identifying the origin of significant security problems. Nevertheless, any solution concerning IoT devices\u2019 security capabilities is designed from the software that governs the management data, generally in the system\u2019s management layer. For this reason, new challenges arise today in the management of informatic security in communication systems.Blockchain technology can be defined as a phenomenon from two viewpoints; financial and technological. Financial because the cooperation in a business process imposes crypto-currency and breaks the standard currency scheme\u00a0,3,4. FinAlthough BIoTS can act in any IoT ecosystem, we study its impact on food traceability in this paper. The traceability of Supply Chains (SC\u2019s) is defined as the ability to track food movement through specific stages of production, transformation, and distribution\u00a0. ScenariSC\u2019s traceability systems enable us to locate, record, and trace products in the manufacture, processing, and distribution through platforms that offer access to users\u00a0, for insBlockchain-IoT (BIoT) can be implemented on an SC in two ways. First, from software usage, it allows management of the network resources to share the information and reach the participation of the actors of the SC through the smart contracts\u00a0,16. The This work aims to identify critical security issues concerning data integrity and transparency in IoT ecosystems\u2019 communication process dedicated to food traceability. Besides, it proposes hardware specific to the blockchain architecture and includes it in an IoT sensor\u2019s architecture. This device is called BIoTS (Blockchain-IoT-Sensor) with blockchain and IoT architectural features, capable of solving security issues inside an IoT network.This article is organized as follows: According to the most frequent security vulnerabilities, in\u00a0, they weGenerally, networks based on IoT may suffer identity violation and information privacy issues, such as the services related to cloud, storage, transmission, or processing\u00a0. SecuritSecurity problems like jamming attacks are considered minor problems. Nevertheless, the message collisions and errors on the sending of the packages are solved in\u00a0, where tThe works listed in the table above define the roadmap for working in agricultural product traceability security, and helps to relate security vulnerabilities, scientific work, and threats. Technologically and conceptually, we focus on the problem and evaluate alternative solutions\u00a0,41. In tSome remarkable findings for BIoTS hardware design are; a) most IoT systems\u2019 sensors will have some features, such as; interoperability, energy, size, position, and communication. These features make the ubiquity term possible and make the system a lightweight system for they can adapt secure form between them to deploy a service in any context\u00a0. b) BiosGiven the range of services provided by objects, persons, or machines in the IoT networks, it is mandatory to equip both networks and devices with security features.The standard communication protocols define the rules and security techniques in IoT networks. Fields such as health, financial security, or food safety handle processes with sensitive data that require transparency and integrity in their handling. However, the adverse factor is that as long as the design of the IoT network is done on the LLN (Low-Power and Lossy Network) network scheme, security will have that measure; that is, the device immersed in the IoT network will not have security properties beyond those possible by its\u00a0capabilities.Identifying security problems on IoT is so extensive that it generally is made from the field of application. Furthermore, the application field imposes safety criteria focused on the user and the system architecture. This hierarchy in the identification of security problems helps to identify comprehensive solutions and technologies.Due to the technology\u2019s capacity with which the build of BIoTS device proposed in this work, it is possible to identify the problems attending the two perspectives (Application and architecture), because of its implementation answers integrally to the IoT security problems. For this reason, this section presents the findings of some security problems, keeping a mixed approach between the security requirements of the application field and the IoT architecture.The scheme in Application Field Perspective: On the left of the scheme, food safety as an application field, establishes the three layers of IoT architecture as a channel to guarantee data and product traceability in the communication process throughout the IoT system.Architecture Perspective: In the upper part, the IoT architecture  establish as the central axis in the identification and classification of security problems.The implementation of BIoTS aims to solve the security problems described here. Each layer in the IoT architecture has issues that the technology on which BIoTS solves.Perception Layer: In this layer, where the devices that interact with the medium are hosted, we list and describe some security issues that BIoTS can solve in two scenarios; (a) in sensor nodes and (b) in sensor gateways. (a) In the sensor nodes case, to make the process of sensing and interconnecting with other nodes possible, they have these components; a controller, a transmitter (for communication), a memory where the device stores the program (code), a power source, and the hardware that obtains the sensed data. At this level, you are prone to security problems such as; node subversion, node failure, node outage, passive information gathering, false node message corruption, exhaustion, unfairness, Sybil, jamming, tampering, and collisions. (b) For sensor gateways, the collection of information on WSN represents a problem because the wireless communication channel involving radio communication and its possible appears problems such as; misconfiguration, hacking, signal loss, DoS, war dialing, protocol tunneling, man-in-the-middle attack, interruption interception, and modification fabrication. As you can see, in both cases, all security problems are aimed at attacking the trust, privacy, and integrity of the transmitted data.Transport Layer: To solve some security vulnerabilities associated with the network type BIoTS acting in a blockchain-based IoT network. The networks generally used for food traceability systems are two; (a) WiFi-centric network and (b) ad hoc non-centric network. For this reason, BIoTs and your ecosystem pretend to solve some security issues as: (a) In a WiFi network, attacks such as access attacks, malicious phishing AP, and DDos/Dos attacks. (b) In IoT, an unfocused ad hoc network is a Peer-to-Peer network. The traditional problems in this nature\u2019s networks have to do with the communication channel\u2019s vulnerability\u2014attacks such as Eavesdropping, interference, vulnerable posing, cheating, Man-in-the-Middle (MitM).Application Layer: In the food safety scenario, millions of users are expected to access sensitive information on edible products. Data confidentiality and traceability is the anticipated contribution of BIoTS in the network deployed for its operation. The ecosystem is expected to contribute to security issues associated with authentication and access authorization. Besides, process safety management within a supply chain based on certification through blockchain Smart Contracts is expected.The BIoTS sensor\u2019s design requires scanning security issues that it can solve in the IoT context. However, the sensor can be used in a variety of fields. Next, the technological ecosystem in which BIoTS deploys its function is analyzed, i.e., the adaptability with an IoT environment, where there are also security gaps to overcome, is determined. The security problems map designed from the two points of view described in IoT can trace or track food products, and store and process critical data for recording the process; nonetheless, it is not entirely safe in terms of data transparency held. Blockchain, beyond the crypto-currencies and the business, can certify processes transparently through data traceability.The ability to certify the processes in food supply chains through the data integrity collected from end-to-end connection points and contained in the Blockchain-IoT architecture reveals BIoTS device\u2019s aim. End-to-end connection points in the IoT architecture indicate the sensor\u2019s path to the cloud service . Simultaneously, the path from the first stage in the traceability system to the last location . See Combining two disruptive technologies involves identifying the problems they face in applying them in food security and then finding the architectural features that make the coupling possible.Blockchain technology is defined as a disruptive technology that imposes a new paradigm that can be connecting securely to the world throughout the network. Blockchain technology can be described as a platform where the transactions and the information recorded are safeguarding through cryptography algorithms in a distributed ledger to all participants of the network\u00a0,49,50.IoT represents an opportunity to apply blockchain technology as a support to guarantee security in some respects\u00a0,52. As wA sensor is an embedded device capable of acquiring information, processing it, analyzing it, storing it, and transmitting it to a repository. It also can coordinate with other networked devices. Under this concept, we describe BIoTS-Sensor architecture features that allow us to define the necessities of functioning to design a Blockchain-IoT system. Then, we make a description of some architectural modules.Most sensors immerse in the IoT ecosystem, further measure some variables, and have some capacities to provide security. Nonetheless, it does not guarantee specific security requirements for low processing, energy, and storage capabilities.These reduced capacities make the lightweight of the IoT systems and guarantee the ubiquitous characteristic of the system. However, reducing the size of the devices (sensors) present in the IoT ecosystems conflicts with the entire system\u2019s security capabilities. For this reason, the leading security solutions presented by scientific research propose solutions on the transport layer, or (fog), and on the application layer, or (cloud), to manage security. However, the possibility of adapting the sensor hardware to an IoT architecture based on blockchain to provide the security system has not been studied so far.Exploring this possibility has technical implications at the level of architecture and resources, for example; the system is no longer light, but there are fields of application  where the robustness of the system is worth the cost, especially if the integrity and transparency of the information are guaranteed. The BIoTS-device architecture description is made from the functional features of two technologies; IoT and blockchain. In the assembly and synchronization of both architectural blocks, it is necessary to identify the security requirements, both for software and hardware\u00a0,54.Block A in As we can see in The block B, we can see the module that ought added to the sensor. Each module responds to the need created by the blockchain system. The modules are related by color; thus, the blue module of the P2P Network is designed to make possible the P2P Network in which the sensor acts as Miner. The green module of the Proof of Work (PoW) algorithm makes transaction validation possible and guarantees the block\u2019s information\u2019s immutability. The yellow module subject to the Mining process is designed to calculate hashes in the communication\u2019s cryptographic function. Finally, the orange module is designed to store the records for each validation through the Merkle tree.As we can see, the junction of these blocks, the IoT as an oriented communication system, and the blockchain as a security system, together represent a communication system with a high-security level. The agro-food traceability application field, where this solution is considered, means the traceability of both product and information.The architecture adaptation deserves to describe the hardware blocks that make the coupling possible. The following describes the Blockchain Ethereum that will govern the system and the development of the sensor module that allows it to operate in this network.BIoTS has as a challenge to adapt all its architectural modules to the functional requirements of blockchain. In this case, it is necessary to adapt two algorithms at the hardware level; (i) the SHA-256 algorithm responsible for cryptography in the communications process and (ii) the Proof of Work (PoW) algorithm responsible for the consensus process in the network.As its name indicates, blockchain is a chain of blocks that systematically stores information in a decentralized network. Each node acts as a miner, and these, generate validation through their processing capabilities. The information contained in each block is interconnected with the previous block employing a hash, making it impossible to reverse or modify data in each block. That\u2019s where blockchain\u2019s security comes from A hash function can convert an input message with a specific length into an alphanumeric array on the output called a digest. A hash function has the following characteristics\u00a0.The reverse process of reconstructing the message from the hash is almost impossible.A minor change in the input message completely changes the output.The algorithm can compress any extension of the input message for arranging the output. It is impossible to find the same hash for two different input messages.k indicates the number of zero bits to be added. The value N is then converted to its 64-bit binary representation and further added to the 448-bit intermediate value to get the 512-bit message block. This formed block is further subdivided into sixteen 32-bit word sub-blocks that input the compression function.The SHA-256 algorithm has two modules; (i) Message Block schedule and (ii) Compression function. Below is a brief description of the modules. In the message schedule module, an N-bit message gets added with bit 1, followed by zero bits until the following, Equation\u00a03:\u00a0\u00a0\u00a0\u00a0Words are prepared for each round Equation )4:for First 16 rounds do (Equation (\u00a0\u00a0\u00a0\u00a0Equation )5:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Six registers b,c,d,f,g,h are updated with the previous registers6:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0K are a set of 64 constant words (Equations (4) and (5))7:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0After 64 rounds of operation 8:if\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a09:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Final 256-bit Hash value is obtained by concatenating 32-bit values10:end if\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a011:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0Hash digest = 12:end for\u00a0\u00a0\u00a0\u00a013:end forThe SHA-256 pseudo-code\u00a0Algorithm\u00a01 in the algorithm allows for a more comfortable hardware design. Generating hashes and encrypting data will enable us to understand the software structure division and translate the functions\u2019 flow for the hardware.The consensus algorithm establishes the mining agents\u2019 computational effort to solve the mathematical puzzle that validates transactions within a blockchain network. Consensus algorithms can be categorized into two groups; proof-based consensus and vote-based consensus. In the first case, the node wishing to join the network must demonstrate higher processing and storage capabilities than the rest of the network. In the second, each node in the network is asked to propose or validate a transaction block that will be part of the validation in the rest of the network. The final decision is made only after considering the majority\u2019s results. Thus, some algorithms were analyzed theoretically and based on\u00a0,57,58 toPoS: It is based on the concept of the age of the coin, this age being known as its value multiplied by the period after its creation. In other words, the longer a node has a currency, the more privileges it will obtain in the network. For this reason, the BIoTS ecosystem for food traceability systems does not require concepts of this type.DPoS: It is based on the fact that each node in the network can select tokens according to their participation. These selected tokens create new blocks one by one as assigned and get a reward. Throughout the network, the n best witnesses who has participated in the transaction\u2019s validation and has obtained the highest number of votes are entitled to the benefit. Blockchain using DPoS is more efficient and saves more energy than PoW and PoS. However, in the BIockchain-IoT ecosystem where BIoTS is deployed, it is not expected to have enough witness nodes to validate the data BIoTS collects. A BIoTS P2P network is expected to operate with consensual data sharing.PBFT: Designed to solve transmission problems and improved to avoid exponential operations. Regarding BIoTS, it is not convenient to use it as it requires a master server to execute the validation throughout all supply chain stages.The most common consensus algorithms in Blockchain (Proof of Stake (PoS), Delegated Proof of Stake (DPoS), and Practical Byzantine Fault Tolerance (PBFT)) limit the BIoTS ecosystem for the following reasons:Most Blockchain networks are decentralized, with synchronous or asynchronous communication models, and are implemented in networks of nodes where mining agents are processor-based; consensus algorithms\u2019 behavior is subject to factors such as; Blockchain type, transaction rate, scalability, adversary tolerance model, experimental setup, latency, throughput, bandwidth, communication model, communication complexity, security attacks, energy consumption, mining, consensus category, and consensus finality. Here, we analyze some of these.After this analysis, it is concluded that some voting-based consensus algorithms can be very relevant for BIoTS performance in a Blockchain-IoT ecosystem. However, the assembly and comparison of the consensus algorithms on FPGA are required, and this work does not have that scope. The BIoTS architecture was based on PoW because this algorithm is robust (Used in Bitcoin). The hardware built is expected to participate in an open blockchain network with significant security challenges.Proof of Work consensus algorithm is a mechanism that allows users or machines to coordinate in a distributed network. This algorithm ensures that all agents in the system can agree on a single truth source, even if some agents fail. In other words, a system with PoW is tolerant of security failures.Algorithm 2 Proof of Work.1:for Loop from 1 to n do2:\u00a0\u00a0\u00a0\u00a0var 3:for n major 1 do\u00a0\u00a0\u00a0\u00a04:\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0var added = 0;5:for 7:end for\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a08:end for\u00a0\u00a0\u00a0\u00a09:end forThe process of verifying the block\u2019s transactions to be added, organizing these transactions in chronological order in the block, and announcing the newly mined block to the entire network does not take much energy and time. The energy-consuming part solves the \u201chard mathematical problem\u201d to link the new block to the last block in the valid blockchain. When a miner finally finds the right solution, the node broadcasts it to the whole network simultaneously, receiving a cryptocurrency prize (the reward) provided by the PoW protocol. We show the PoW pseudo-code\u00a0Algorithm\u00a02 .AlgoritImplementing the PoW in hardware determines, among other things, the difficulty in block validation (time invested in identifying the block legitimacy), and the active participation of blockchain network miners. To the hardware block of the SHA-256 algorithm, it is necessary to adapt a function capable of adding in each round of the cryptographic encoding a string of zeros  that will act as an identifier of block and the transaction in the whole blockchain.Generate Random Nonce: once the clock for the hash and PoW is configured and synchronized, a 32-bit register and bus are generated. The first thing is to introduce a nonce every six cycles with a feedback delay of 12 cycles.Build Block: since this is not straightforward, we use a register to track the hash through the bus. To follow the nonce, we pass the least significant 8 bits of the 32-bit register and then pair the remaining 24-bit.Difficulty: in the second of two rounds of hashing of 64 bytes each, the header of the 80-byte block is the encrypted data space. The first round gives us the average state to insert the nonce at the beginning of the record of the second 64 bytes in the correct position.Broadcast Block: subsequently, the internal hash transformation is performed to complete the register. It is still not the full SHA-256 because it involves multiple rounds. Nevertheless, this process is iterative. Here, the VHDL code in the DE10-Nano is split into phases to discriminate the SHA-256 transformations, and then unified into one block intended to do the complete hash transmission.When the PoW algorithm checks zeros\u2019 existence in the hash encoded in base 64, the average and the maximum number of hashes is known to calculate the order of difficulty that increases exponentially by the expression  and SD storage hardware structure. The BIoTS hardware structure contains two modules (yellow and green) and one software blue module (Build of Blockchain on Python). The three blocks mentioned above occupy 47 percent of the available logic units in the DE0-Nano FPGA, barely sufficient resources for implementation . In TablThis SHA-256 algorithm design contains the Proof of Work algorithm development, necessary to calculate the nonce and make participation in the consensus process possible. The I2C master component for single master buses, written in VHDL for use in FPGAs, has component reads from and writes to user logic over a parallel interface. It was designed using Quartus II, version 18.0. Resource requirements depend on the implementation. A design incorporating this I2C master to create an SPI to I2C Bridge is available.The SD interfaceable with FPGA is implemented from VHDL code. Here, we implement the standard size, but electrically, all sizes work the same way. Let us focus on SD card standard size, since that is conveniently popular nowadays. To this proposal, we install an SD card of 32 GB.BIoTS analog and digital electronics\u2019 energy consumption is calculated based on the evaluation scenario\u2019s functional performance. The elements that consume the most energy are; the peripheral elements: the Wi-Fi module and Bluetooth. For the digital elements: SHA-256 cryptography algorithm, PoW consensus algorithm, and the SD memory block. The BIoTS power source is a three-cell LI-PO type battery at 11.2 Volts, 20\u201330 c discharge, and 5000 mA/h. The BIoTS analog module consumes 220 mA/h transmitting data at a 1-min interval. The power consumption of the FPGA depends on the optimization of the configured algorithms and the read and write speed of the SD memory .Under these conditions, the battery life is 45 min. However, as transactions occur, the complexity algorithm kicks in and demands more processing power from the FPGA. Thus, the power consumption is dynamic, and the FPGA performance is proportional to the blockchain network\u2019s activity.The BIoTS performance is evaluated according to the configuration shows in The difficulty level setting of the consensus algorithm is low. We do not need to process a large number of network nodes for this demonstrative purpose, nor do we need to process a large data volume. However, we expect future work to demonstrate a Blockchain-IoT network configuration where a BIoTS sensors network interacts directly.Some values related to data transmission from BIoTS to the blockchain designed for the use case are shown in These data, as shown in The graph in The data shown in remix.ethereum.org  is the rFor the evaluation scenario, the transmitted humidity and temperature data from BIoTS are correctly encrypted and recorded in the blockchain network. Since the computational overhead increases as transactions (Measurements) and blocks grow, it is only possible to know the energy performance of BIoTS when hundreds or thousands of reads are accumulated. However, future work is expected to implement a BIoTS network and determine its performance as a Blockchain-IoT ecosystem.\u201cSensor Tampering\u201d: the attack on BIoTS is unlikely because the sensor data is hosted in the SHA-256 and PoW hardware algorithms; after this process, the information is encrypted.\u201cSensor Feed Modification\u201d: this attack is possible with BIoTS; however, the resistance is high because the BIoTS firmware is almost null, and almost all elements are hardware.\u201cSybil Attack\u201d: this attack is unlikely in a network where BIoTS acts because it has the same blockchain network\u2019s resilience. However, it all depends on the network configuration .\u201cDoS, Protocol tunneling, and man-in-the-middle\u201d: these attacks are unlikely due to the blockchain network\u2019s nature. The communication channel by cryptography and the algorithms carried in hardware is immune to external intervention.\u201cJamming and Collisions\u201d: these attacks are possible in a BIoTS network. The resistance to the attack is moderate because it can identify the hash\u2019s inputs and outputs to reproduce copies.To identify the security flaws where BIoTS is a potential solution, we study a causality and effect correlation between the nature of the security attack in an IoT ecosystem and the Blockchain Hardware\u2019s architectural characteristics implemented in BIoTS ,14,53. TThis paper proposes the hardware architecture of blockchain (Cryptography and Consensus Algorithms) to enrich an IoT device and provide it with remarkable capabilities to address security issues in the IoT ecosystem. This paper proposes a secure and trusted blockchain hardware architecture thanks to the reconfigurability of an FPGA. Our goal was to realize the implementation of Blockchain in hardware adapted to an IoT device to improve physical and virtual security mechanisms on devices in a network. This proof of concept works well and can positively impact the field of IoT-based food traceability systems. Finally, IoT devices immersed in ecosystems dedicated to the security and quality of supply chains are destined to be critical actors in products\u2019 quality and safety through complete confidence in their performance in collecting, transmission, and storing information. This concept may be the future of primary IoT devices\u2019 capabilities in food traceability systems or other application fields. The adaptation of emerging technologies in the IT security applied to various application fields opens a path to the evolution of Blockchain-IoT systems."}
+{"text": "Gastric cancer (GC) is the fifth most common malignant tumor in the world. The present study was performed to discover the potential diagnostic and therapeutic long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) of GC. Data used in this study to identify differentially expressed lncRNAs (DElncRNAs) and miRNAs (DEmiRNAs) were obtained from 187 GC tissues and 32 adjacent nontumor tissues. The total clinical data on GC included 187 cases. The above data were from the TCGA database. RStudio/Bioconductor software was used to conduct univariate analysis, the least absolute shrinkage and selection operator (LASSO) Cox, and multivariate Cox proportional risk regression for the DElncRNAs and DEmiRNAs. Clinical information was analyzed through univariate and multivariate Cox analysis. Results: five lncRNAs  and two miRNAs  were proven to be independent prognostic indicators of GC. Results of the Kaplan-Meier survival analysis showed that AC007785.3, AC079385.3, LINC01729, miR-3174, and miR-605 were significantly correlated with OS of GC. The target genes of AC079385.3, miR-3174, and miR-605 were obtained and clustered mainly on MAPK and cGMP-PKG signaling pathways. The clinical data showed that age and clinicopathologic stage were correlated with the prognosis of GC. Furthermore, AC007785.3 was associated with metastasis of GC, and miR-3174 was associated with the primary tumor condition of GC. We identified three lncRNAs , two miRNAs , and clinical factors related to the pathogenesis and prognosis of GC. Our predicted results provide a possible entry point for the study of prognostic markers for GC. Gastric cancer (GC (GC: gastric cancer)) is the fifth most common malignant tumor globally . GC was Long noncoding RNAs (lncRNAs (lncRNAs: long noncoding RNAs)) are a class of RNAs that are longer than 200 nucleotides but do not encode proteins. MicroRNAs ) are a class of endogenous small 19\u201325 nucleotide noncoding RNAs that combine with the 3\u2032-UTR of target genes, leading to inhibition of translation or degradation of the target genes . Since tThe use of circulating molecular profiles as potential biomarkers for the diagnosis and prognosis of GC is still not recognized. On this basis, to exclude effects of chemoradiotherapy and individual differences in the published studies and to avoid the intervention of blood samples, we identified differentially expressed lncRNAs and miRNAs through bioinformatics analysis. In this study, we used data from the same patients' tissues from the cancer genome atlas (TCGA (TCGA: The Cancer Genome Atlas)) data portal.https://tcga-data.nci.nih.gov/tcga/). Therefore, approval from an ethics committee was not required. The inclusion criteria were set as follows: (1) samples with common and completed lncRNA, miRNA, and clinical data. (2) Patients had not received preoperative chemoradiotherapy. As a consequence, the data used to identify the differential expression of lncRNAs, miRNAs, and mRNAs in gastric cancer were obtained from 187 GC tissues and 32 adjacent nontumor tissues. The total clinical data on gastric cancer was obtained from the corresponding 187 patients.The RNA information and clinical data used in this study were from the cancer genome atlas (TCGA) data portal  > 1.0 and a false\u2009discovery\u2009rate\u2009) < 0.05 were set as the selection criteria.Firstly, the RNA sequences were downloaded from the TCGA database, and information for each sample was added to a matrix to extract the lncRNA, miRNA, and mRNA expression data through Perl. Then, the survminer packages in the R software were used to generate forest charts. The Kaplan-Meier survival curves were plotted based on the different risk scores and expression levels of the screened lncRNAs and miRNAs. The above analysis was carried out using the RStudio/Bioconductor. A P value <0.05 was defined as a significant difference.Univariate Cox regression analysis was first used to screen the DElncRNAs and DEmiRNAs associated with the prognosis of GC. Then, the least absolute shrinkage and selection operator (LASSO) regression was performed to reduce the discreteness. A multivariate Cox proportional risk regression model was used to independently identify prognostic lncRNAs and miRNAs. Concordance index (C-index) and the receiver operating characteristic (ROC (ROC: receiver operating characteristic)) curves were taken to evaluate the model. GC patients in each dataset were divided into a high-risk group and a low-risk group according to the median cut-off points of risk. The caret package. Using the RStudio/Bioconductor method, single- and multifactor Cox regression was performed in the training group using the survival package. The rms and survival packages were then used to generate nomogram and calibration graphs. The ROC curve was used to predict the accuracy of the model. The clinical data obtained from the training group were verified by calculating the C-index of the clinical data onto the test group. The Kaplan-Meier survival curve was used to predict the survival difference, and the effect of each clinical factor on the OS  of patients was evaluated. P < 0.05 was defined as a significant difference.The clinical samples were first divided into two halves using the https://genome.ucsc.edu). The target miRNAs of the screened lncRNAs were identified with the miRcode (http://www.mircode.org) database. Possible downstream target genes of the obtained target miRNAs and the screened prognostic miRNAs, which appeared in at least two databases, were collected from the miRDB, miRTarBase, and TargetScan websites. Then, the possible lncRNA-miRNA-mRNA and miRNA-mRNA networks were drawn based on the competing endogenous RNA theory. The functions and related enrichment pathways to the possible target genes were analyzed using the DAVID tool .Detailed information about the screened lncRNAs was further searched for . Besides, the expression levels of the lncRNAs between the GC tissues and normal tissues were verified in GSE53137 through Prism 8.0  using the Wilcoxon signed-rank test. miRNAs were verified in GSE94315 and GSE78091 with the same method. According to the inclusion criteria, data from 187 GC tissues and 32 adjacent nontumor tissues were obtained from the TCGA database. A total of 2865 DElncRNAs were identified, of which 2223 were overexpressed and 642 were downregulated. A total of 261 DEmiRNAs were obtained, 215 of which were upregulated and 46 were downregulated. A total of 4555 DEmRNAs were found, among which 2268 were upregulated and 2287 were downregulated.Univariate Cox regression was performed for significant lncRNA and miRNA expression. The results showed 544 DElncRNAs and seven DEmiRNAs that were associated with OS of GC patients. Next, we used LASSO regression to further reduce the discrepant, and 35 DElncRNAs and 5 DEmiRNAs were screened. The multivariate Cox proportional risk regression model was used to verify the above results and identify independent prognostic factors. The results of the multivariate Cox proportional hazard regression analysis are shown in Among the three screened lncRNAs, only the target miRNAs of AC079385.3 were found in the miRcode database. Then, five miRNAs  were obtained by taking the intersection with the 261 DEmiRNAs in our study. Finally, 212 possible target genes of the five miRNAs were explored. The lncRNA-miRNA-mRNA network is shown in Clinical data on the 187 gastric cancer patients were taken for analysis, with 95 patients in the training group and 92 patients in the test group. The clinical features of the gastric cancer patients in the training group and test group are shown in P = 0.049), and miR-3174 was associated with the primary tumor condition of GC (P = 0.012)  is one of the most aggressive and lethal tumors in the world. Efforts have been made to provide new insights into the molecular mechanisms underlying GC development. Therefore, an indepth exploration of GC-related miRNAs and lncRNAs may provide clinicians with new targets for the treatment of this disease. Several integrated genetic studies have been conducted to elucidate the roles played by miRNAs and lncRNAs \u201319. In rAs described, to exclude effects of chemoradiotherapy and individual differences, we identified differentially expressed lncRNAs and miRNAs through bioinformatic analysis with the data of the same patient from the TCGA database in this study. Using univariate, LASSO, and multivariate Cox regression analyses and survival analyses based on 187 GC and 32 adjacent normal tissues, three lncRNAs  and two miRNAs (miR-3174 and miR-605) were proven to be independent prognostic factors of GC patients.Based on the competing endogenous RNA theory, target mRNAs of AC007785.3 (miR-3174 and miR-605) were found. Functional enrichment analysis of the target mRNAs showed that the genes clustered mainly in the plasma and play roles mainly through transcription processes. For pathway analyses, genes were associated mainly with cancer pathways and work through the calcium signaling pathway, MAPK signaling pathway, cGMP-PKG signaling pathway, and focal adhesion. Among them, the MAPK and cGMP-PKG signaling pathways were identified as being active in both the early and advanced stages of tumorigenesis, survival, promoting tumor proliferation, and metastasis in various human tumors . InhibitAC007785.3, AC079385.3, and LINC01729 are not well studied, not to mention in GC. However, this offers a new direction for treatment research of GC. As we discovered, the possible target miRNAs of AC007785.3 that were upregulated were miR-7 and miR-203b, and that were downregulated were miR-133a and miR-203a. Through differential expression analysis, AC007785.3 was shown to be upregulated in GC tissues. Considering the ceRNA theory, miR-133a and miR-203a deserve more attention. Current research has found that miR-133a could inhibit the proliferation of GC cells through targeting the expression of ERBB2, FOXP3, IGF1R, and presenilin 1 and blocking autophagy-mediated glutaminolysis \u201334. miR-miR-3174 and miR-605 were also identified as prognostic factors of GC. Among them, miR-3174 was highly expressed and proved to be a protective factor in our study, which was opposite to the research results of Li et al. Li et al. discovered that miR-3174 contributed to apoptosis and autophagy cell death defects in gastric cancer cells by targeting ARHGAP10 . This deMoreover, we also carried out an in-depth exploration of the clinical data in gastric cancer. According to the clinical data, the AUCs of the 3-year and 5-year ROC curves were 0.75 and 0.776, respectively, and the results of the disease-free survival calibration chart over 3 years were in good agreement with the results of the ideal model. Age and pathologic stage were proven to be directly connected with the prognosis of GC in our study. miR-3174 was connected with the primary tumor condition of GC.To sum up, the biological functions of the three identified lncRNAs and miRNAs are not fully understood or elucidated in gastric cancer. However, some molecular biomarkers can predict the 5-year survival rate of patients with gastric cancer, which may become a new prognostic indicator for predicting clinical efficacy. MAPK and cGMP-PKG signaling pathways might be new target therapy directions for GC. The roles of these genes are worthy of further study because of their close association with the prognosis, especially in gastric cancer. More studies are needed in the future to verify these findings.This study has some limitations. Firstly, the ethnic sources of the TCGA data population are mainly limited to Caucasian and Negroid people, and inference cannot be made about other ethnicities. Secondly, in vitro and in vivo studies on these biomarkers in gastric cancer cell lines and animal models, respectively, have not been conducted. Therefore, further experimental studies are needed to strengthen the understanding of the mechanisms underlying the involvement of these markers in the prognosis of gastric cancer."}
+{"text": "The role of thoracic surgery in the management of hyperhidrosis is well-known and thoracoscopic sympathetic interruption is commonly accepted as being the most effective treatment. However, some concerns still remain regarding the potential to develop compensatory hyperidrosis (CH), the most troublesome and frequent side effect after surgery and its management. Compensatory hyperidrosis prevention may be achieved by identifying subjects at higher risk and/or targeting nerve interruption level on the base of single patient characteristics gathered during the preoperative survey. Furthermore, the surgical treatment may consist of different techniques aimed at reversing the effects of previous sympathetic interruption. To predict CH after sympathectomy, the most interesting proposals in recent literature are a temporary thoracoscopic sympathetic block and the introduction of new and targeted preoperative surveys. If the role of nerve clipping technique vs. the definitive cutting is still intensely under debated, new approaches have been recently proposed to reduce the incidence of CH. In particular, extended sympathicotomy has been described as an alternative to overcome severe forms. Last, among the techniques developed to reverse sympathetic interruption effect, diffuse sympathicotomy (DS) and microsurgical sympathetic trunk reconstruction represent advances in this field. An all-round review of these topics is strongly needed. Our aim is to cover all the above issues point by point. Although sympathectomy represents a small part of thoracic surgery, we believe that it is worthy of interest because of the profound effect that complications for a benign condition can have on patients. Primary hyperhidrosis (PH)  is a conDespite advances in surgical techniques, CH remains the most troublesome postoperative side effect, which occasionally causes patients to regret choosing surgery and surgeons to hesitate to suggest sympathectomy as upfront treatment for PH, especially for craniofacial hyperhidrosis , 9\u201311.Postoperative CH is reported in 50\u201390% of patients depending on the Authors and is experienced \u201csevere\u201d in as many as 35% patients , 5, 12. Currently, there is no effective treatment for CH after thoracic sympathectomy. Indeed, there have been several attempts to reduce CH, such as both preoperative and postoperative strategies. Among the preoperative one, limited level sympathectomy  or lowerCompensatory hyperhidrosis onset may be prevented or mitigated through several actions ranging from prevention to re-surgery. Prevention may be obtained by identifying subjects at higher risk and/or targeting nerve interruption level on the basis of single patient characteristics. On the contrary, surgical treatment may consist of techniques aimed to reverse the effects of previous sympathetic interruption.In the past decade, no one of these solutions took over, and CH is still the major cause of dissatisfaction for patients. However, in the past few years, some innovative approaches have been described and proposed. In this article, we are going to summarize insights in preoperative and postoperative management of patients who have undergone sympathetic interruption or presenting CH, with the aim to give a snapshot of the state of the art and promote valuable innovations.Currently, there are two well established strategies to avoid or deal with CH: (1) optimization targeting of sympathetic chain level and (2) chain clipping and subsequent clips removal.In 2017, Sang and co-workers published an outstanding review to identify the appropriate level of nerve chain interruption in terms of both efficacy in PH management and CH onset reduction for palmar hyperhidrosis . Their bIn 2002, Reisfeld and co-workers  recommenHowever, without considering that de-clipping requires a further and more complex operation, Loscertales and co-workers  in a proTherefore, since the techniques described above present many drawbacks and do not guarantee success, further strategies are needed. We have selected the most interesting articles describing innovative techniques and their results to prevent or control CH in patients affected by PH.Nowadays, many questionnaires and scales for patients affected by PH evaluation are available. The most diffuse one is the Hyperhidrosis Disease Severity Scale (HDSS) from the International Hyperhidrosis Society (IHS). It is a disease-specific, quick, and easily understood 4-point scale that qualitatively investigate the severity of sweating in daily activities. The HDSS determines PH severity based on the extent of sweating-related discomfort. Its effectiveness has been verified in three remarkable studies describing a strong to moderate correlations with the Hyperhidrosis Impact Questionnaire (HHIQ), Dermatology Quality of Life Index (DLQI), and gravimetric sweat production measurements.The Hyperhidrosis Impact Questionnaire, DLQI, and Hyperhidrosis Quality of Life Index (HidroQoL\u00a9) are other surveys mostly adopted by the dermatologist to measure QoL in patients affected by PH. They are mainly used to make diagnosis and investigate outcomes and satisfaction of the treatment. However, current questionnaires are not helpful in selecting patients who are more likely to develop CH after sympathectomy.We have recently published an article  focused Therefore, we introduced a preoperative NAS survey quantifying discomfort in each body area. CH risk was derived by adding 2 points to initial score of each area and then, calculating a whole-body median score. Then, the selective clipping was tailored to reduce CH risk as follows: in case of palmar or palmar-axillary PH, if median score was <7, nerve trunk was clipped at the superior margin of third and fourth rib. On the contrary, if estimation was >7, nerve trunk was clipped only at the upper margin of fourth rib. Cephalic PH was treated by one clip application at the lower margin of second rib if CH estimate was <7. Last, diffuse PH was treated by clips application at the upper margin of third, fourth, and fifth rib regardless of any CH estimate. We investigate our survey comparing the outcomes in terms of PH management, CH onset, and satisfaction of a patient with a group of patients treated by targeted nerve interruption for each location of PH, according to the ongoing STS recommendation.Results showed that our interruption-level pattern tailored to preoperatively determined CH risk guarantees better outcomes. Further studies are needed to validate the questionnaire; however, our experience confirms that a patient tailored approach based on individual preoperative sweating pattern could be a successful method to prevent CH onset.Despite ongoing recommendations have already identified the optimal target level for nerve interruption for each skin area affected by excessive sweating, two main concerns still persist: (1) recommendations do not punctually indicate where interruption should be performed but usually suggest a range encompassing almost two intercostal spaces and (2) CH onset rate is still significant. In our opinion, the most likelihood cause is individual variability, therefore any techniques aimed to anticipate the occurrence and grade of CH in each single patient, should be encouraged.2 insulation), they blocked predetermined ganglia by the use of a percutaneous spinal needle injecting 10 ml of a mixture of ropivacaine, steroids, and epinephrine. A week after temporary blocks, patients were asked about their satisfaction in terms of PH management and CH onset. In case of good results, patients decided whether to undergo definitive sympathectomy or not. Their results were encouraging since the predictive procedure was safe and effective in the most of cases in terms of specificity (94.4%) and positive predictive rate (95.2%). This allowed the Authors to conclude that their method successfully offers patients the opportunity to experience effects of definitive surgery improving postoperative overall satisfaction.With this goal in mind, Lee et al. in the past 10 years developed a procedure to obtain a temporary nerve block to simulate definitive sympathectomy and predict after surgery outcomes of patient . In partDespite some bias, already highlighted by the Authors themselves, the procedure could be the corner stone in patients at higher risk for CH such as: cranio-facial cases, high body mass index (BMI), or diffuse hyperidrosis.Since preoperative patient evaluation for CH risk determination is a constant evolving issue, as we will address in the next paragraphs, temporary predictive chain block could be proposed in selected patients to offer an experience on which they decide and therefore improving their postoperative satisfaction.As reported above, the most significant surgical innovations aimed to reduce CH have been represented by nerve interruption targeting and by clipping introduction. However, results are still not completely satisfactory.In 2020, Han and co-workers presented their new sympathectomy for the prevention of severe CH . Their bAll the procedures and techniques previously described in this paper were focused on identifying patients at higher risk for CH; they are preventive solutions useful for lowering the postoperative excessive sweating onset by preoperative selection of patient. Instead, the opposite solution involves management of patients who have already developed CH following sympathectomy. Indeed, as already showed, ongoing methods to prevent CH are unsatisfactory and the innovative ones are still under investigation. Thus, it is mandatory to develop effective therapies to be proposed for a cure, or at least a QoL improvement of these patients. As of now, medical and non-invasive treatments for CH are unsuccessful in the most of severe cases, therefore, surgical innovations are desirable.Moon and co-workers have recently published an article addressing this topic and entitled surgical treatment of compensatory hyperhidrosis . Their bSince the sympathetic dermatomes overlap each other and are hardly identifiable, they initially proposed a limited diffuse sympathicotomy (DS) for denervation in CH. Limited DS comprised the nerve interruption at right R5, R7, R9, and R11 and left R5, R6, R8, and R10 levels. Based on good outcomes and no complications, they replaced the limited DS with extended DS consisting of all level interruption . Results are interesting and deserve discussion. First, it is remarkable that Authors could obtain an immediate and diffuse control of CH in 81% of case treated by DS. However, unfortunately, outcomes progressively got worse with 54% of cases presenting a recurrence at long-term follow-up.Instead, results were much more successful in patients who experienced extended DS that at multivariate logistic regression analysis resulted an independent predictive factor for CH resolution. As well as zone of compensatory hyperhidrosis involvement  were differently associated with persistent resolution.Some biases and the small population enrolled make this study too weak for definitive conclusions; however, in our opinion this study sheds new lights on surgical management of CH.As reported in literature, postoperative CH management with a topic or systemic therapy is unlikely successful in terms of efficacy, duration, and overall satisfaction of a patient. Therefore, many attempts to develop a surgical technique suitable to stop or at least reduce CH have been proposed. However, at present, this kind of surgery is still at the embryonic level due to the controversial results and technical difficulty. Therefore, there are two possibilities to increase the use of surgery in this field currently: the introduction of new reconstructive techniques and the use of the latest generation technology.As concerning new technologies, the most interesting issue is the advent of robotic microsurgery. Chang and co-workers  last yeaThey adopted the traditional technique consisting of SNR using a sural nerve grafting; the innovation is the introduction of the robotic microsurgical approach instead of classic thoracotomy. The background has potentially better feasibility of a robot-assisted approach, thanks to the 3D vision and wrist-like movements. Authors reported the results in terms of surgical feasibility showing complete success in 7 cases. Despite short follow-up, they were able to observe interesting improvement in symptoms compared with pre-operative condition. Thus, they concluded that robotic approach provides better visualization and better movements in suturing with 8-0 nylons. However, further studies focused on clinical outcomes are mandatory.As concerning new reconstructive techniques, Gebitekin and co-workers  in 2020 Thus, they developed the Gebitekin Technique consisting of constructing a parallel pathway to sympathetic chain with 2 healthy intercostal nerves accurately selected, prepared, and joined by a tension free anastomosis. Collecting data from their series of 15 cases, they obtained good results in terms of feasibility and clinical outcomes comprising symptoms improvements and QoL.This review originates form the growing interest on this topic probably due to the number of patients who resort to surgery for hyperhidrosis and patients already affected by compensatory sweating. This condition and the absence of evidence concerning both prevention and management of CH have prompted surgeons to develop new and alternative methods to improve sympathetic block outcomes and manage any compensatory sweating.As concerning the newest insights, the past 2 years literature offers very interesting innovations . In partMoreover, these procedures could be integrated each other; for instance, the temporary nerve block could be offered to patients who are more likely to develop CH on the basis of a tailored survey.Unfortunately, all the technique described in this review are encouraging but not definitive and need further studies. Our aim is the diffusion of knowledge of new technologies between the thoracic surgeon community so that other centers, based on what already experienced, could start new trails and add their data to consolidate what at the moment are only intuitions.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.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": "Celiac disease or gluten-dependent enteropathy is a chronic autoimmune pathology triggered by dietary gluten in genetic predisposed individuals, mediated by transglutaminase 2 IgA autoantibodies and associated with a deteriorating immune and inflammatory response. This leads to intestinal villous atrophy, impairing the intestinal mucosa structure and function of secretion, digestion, and absorption. The result is macro- and micronutrient deficiency, including fat soluble vitamins and minerals, and a consequent nutritional status depletion. A lifelong gluten-free diet is the only available treatment for celiac patients in order to assure normal intestinal mucosa and remission of gastrointestinal symptoms. However, a gluten-free diet can itself cause other nutritional deficiencies due to its restrictive nature regarding gluten-containing cereals. A group of gluten-free cereals, known as pseudocereals, is increasingly recognized as valuable options for gluten-free diets due to their high nutritional value. Amaranth, quinoa, millet, and buckwheat are examples of gluten-free nutrient-dense grains that can be used as alternatives to the conventional gluten-containing grains and improve the variety and nutritional quality of the celiac diet. Current work reviews the nutritional pitfalls of a gluten-free diet and analyses how pseudocereals can contribute to revert those deficiencies and optimize the nutritional value of this mandatory diet for the celiac population. Celiac disease (CD) is an autoimmune condition that results in intestinal mucosal injury caused by dietary gluten\u2014a generic term specific for proteins (prolamins) found in the endosperm of some cereals, specifically wheat (gliadins), rye , and barley (hordeins) . This paThe only treatment available for CD is adherence to a gluten-free diet (GFD). However, the patient often struggles with following a GFD or in choosing adequate nutritional food. This diet can result in nutritional deficiency or oversufficiency ; thus, it is valuable that patients are regularly followed by a nutritionist . Even thThis literature review focuses on foreseeing the use of pseudocereals in a GFD in order to increase dietary variety and quality for CD patients.Besides genetic predisposition and a specific immune response, CD development is dependent on gluten exposure that acts as a trigger . Gluten Once ingested, gluten is partially cleaved into gliadin peptides that pass through the intestinal mucosa epithelial barrier due to increased permeability , caused The innate immune response to gliadin occurs in the intestinal mucosa epithelial layer and increases the release of cytokines, namely interleukin-15 (IL-15), produced by enterocytes, macrophages, and dendritic cells. This results in intraepithelial leukocyte differentiation into CD8+ cytotoxic T cells that express the marker for natural killer NK-G2D cells causing epithelial cell apoptosis . The accCeliac disease can be divided into two main types, symptomatic (or classic) and asymptomatic . The claThe basis of CD treatment is a lifelong strict adherence to a GFD . For mosA GFD also reduces future health problems like osteoporosis and improves mental well-being and quality of life . ImproveThough the maximum nontoxic quantity of gluten consumption for CD patients is unknown, <10\u2009mg/per day is considered a safe amount to prevent intestinal lesions . To labeFollowing a GFD implies absolute removal of dietary gluten, obviously excluding not only all ingredients that naturally contain gluten from celiac patients' diets but also all gluten sources in food that may derive from cross contamination during processing , which can be demanding. The European Commission regulation n. 41/2009, of 20 January 2009, clarified food products composition and labeling regarding gluten content that can help patients select the right dietary options . AnotherAs for dietary diversity in a GFD, corn, potato, and rice comprise the food options more consumed by celiac patients and the most common food sources in the preparation of gluten-free products by the food industry. This restriction can originate nutritional imbalances , usuallyApart from these cereals and derivatives, oats can also be incorporated in a GFD, increasing the nutritional value not only due to its protein (10 to 15%) and lipid (5 to 9%) content but also its high fiber, thiamine, and zinc content in comparison to conventional gluten-containing cereals like wheat, rye, and barley . Oats arRecently diagnosed adults with CD usually have lower levels of body mass and body fat percentage compared tononceliac individuals  as well 2) [Concerning the role of a GFD in the nutritional status of CD patients, it is commonly observed a weight gain after a period on this diet, although they remain in the same BMI category . Few stu2) , 30, 31.2) . On the 2) .The innate and adaptive immune responses activated by gluten ingestion in CD patients result in villous atrophy and crypt hyperplasia of the small intestinal mucosa . These cCobalamin or B12 deficiency occurs in about 12 to 41% of celiac patients, with terminal ileum involvement , pancreaPatients with CD can also have deficiencies in folic acid or folate. This micronutrient, absorbed primarily in the proximal small intestine, more precisely in the jejunum , is reduPyridoxine or B6 vitamin deficiency is also common in CD patients. B6 is a vital nutrient for amino acid metabolism, hemoglobin, and neurotransmitter synthesis, and also gene expression . It is aVitamin D or calciferol has a diverse role in human physiology. Its homeostasis is crucial to maintain bone mineralization and prevent osteoporosis . VitaminIron is also a nutrient which may be in deficit in CD. It has essential functions in the transport and storage of oxygen, being the active centre of hemoglobin, vital for cellular respiration and oxygen transport, and mioglobin, for the storage of oxygen in the muscles . Iron isCD patients can also be deficient in copper, selenium, and zinc. However, supplementation is unadvised since its deficiencies reverse rapidly when patients follow a GFD .A GFD is often characterized by lower fiber ingestion comparatively to an unrestricted diet , since gA GFD can cause other nutritional deficiencies because of the low quality of gluten-free food and the insufficient ingestion of folate  that resOther deficiencies may appear or even surpluses. Kulai and Rashid  demonstrPseudocereals like amaranth, quinoa, millet, and buckwheat have been increasingly recognized as relevant for a GFD due to their high nutritional value. Rich in starch, fiber, and high biological value proteins, pseudocereals are also an excellent source of vitamins and minerals and for this reason have been studied as an alternative to diversify and optimize a GFD .The protein bioavailability in pseudocereals is higher and superior to common cereals and of similar quality as animal protein . This isThe mineral deficit associated to a GFD can be surpassed and its quality improved through the substitution of processed foods of low nutritional value by pseudocereals of high nutritional value or through mineral supplementation . PseudocAccording to the National Nutrient Database for Standard Reference , by compin vitro) were also observed, 494,2\u2009\u03bcmol GAE/100\u2009g for buckwheat, 289.6\u2009\u03bcmol GAE/100\u2009g for black quinoa, and 76.2\u2009\u03bcmol GAE/100\u2009g for amaranth, respectively [Pseudocereals not only are sources of bioactive compounds not included in the nutrient definition, as some of its vitamins, minerals, peptides, and fatty acids also exhibit bioactive properties \u201356. Thesectively .  Table ectively , 56.Lactobacillus strains reduces the number of pathogenic microorganisms, like Clostridioides or Escherichia, and increases the synthesis of short-chain fatty acids, considered good sources for symbiotic formulations with potential application for dysbiosis, inflammatory bowel disease, obesity, and CD [Although pseudocereals richness in nutrients and bioactive compounds is undisputed, it is also pertinent to mention the presence of antinutritional components, mainly phytic acid, saponins, and tannins. These can interfere with nutrients, preventing their proper digestion, absorption, or utilization. However, processing treatments such as germination, fermentation, puffing, and cooking improve pseudocereals nutritional (and organoleptic) properties and reduce these antinutrients, thus incrementing the availability or digestibility of nutrients , 59\u201361. n\u2009=\u200919, 2 males and 17 females, with a median age of 59 years, BMI of 23\u2009kg/m2, 9 years under a GFD). Participants were free to choose the cooking method but were recommended to consume quinoa flakes for breakfast as porridge or pancakes and quinoa grains prepared as rice or cooked in soups or stews. Gastrointestinal parameters  were self-reported throughout the study. Ten patients did not report any symptoms, while nine reported symptoms ranging from mild to moderate severity during the first 2 weeks (which seem to be related to the increase in diet's fibre) and one patient reported mild vomiting. Histological status of intestinal biopsies was assessed in 10 participants, being maintained or improved after quinoa consumption. Regarding blood biochemistry, a mild hypocholesterolemic effect was observed, with a slight reduction of total cholesterol, low-density lipoprotein, triglycerides, and high-density lipoprotein . As so, the authors concluded that short term consumption of quinoa is safe for CD individuals since it is well tolerated, does not exacerbate the disease, and may have a mild hypocholesterolemic effect [Although many studies  regarding pseudocereals have highlighted their health potential, standing out as the most consensual properties their antioxidant, antilipidemic, antihypertensive, and antidiabetic effects , 56, lesc effect .2) were randomly assigned to the experimental or control phases. During the experimental period, patients substituted all gluten-free products, for buckwheat products . Within the experimental phase, participants received (per day), 80\u2009g of pasta, 60\u2009g of hard tacks, 40\u2009g of biscuits, and 50\u2009g of flakes. During the control period, patients maintained their normal GFD. At the end of the first 6-week intervention or control phases, patients crossed over to the other treatment condition for the remaining 6 weeks of the study. Regarding gastrointestinal symptoms, patients experienced a significant decrease in the severity of abdominal pain and bloating during the experimental phase and, in an opposite way, a significant worsening trend for the majority of NCGS symptoms such as nausea, headache, joint/muscle pain, and attention disorders during the control period. On the other hand, during the intervention phase, a significant increase in serum magnesium and a significant reduction in the circulating levels of some pro-inflammatory cytokines (interferon gamma and monocyte chemotactic protein-1) were observed. Although these results support a health benefit of buckwheat products inclusion on NCGS individuals GFD, it is important to mention that among the buckwheat products tested in this study, only pasta was 100% buckwheat, as the other items also contained whole rice flour, corn flour, almonds flour, quinoa flour, and flaxseeds, which may also have contributed for the positive findings [Another study conducted a randomised cross-over trial, to assess the effect of buckwheat products on intestinal/extra-intestinal symptoms and biochemical parameters in 19 patients with Non-Celiac Gluten Sensitivity (NCGS). Participants  following a long GFD therapy were evaluated. After 9 to 12 months of intervention, an improvement of participants nutritional status was observed. Namely, underweight children decreased from 16.25 to 10.8% and children with short stature decreased from 10.8 to 5.4%. Additionally, biochemical analysis revealed that the initial abnormal low level of ionized calcium in the blood serum decreased from 37.8 to 10.8%. Furthermore, normalization of decreased blood serum levels of iron, copper, and zinc was observed in all patients who had a deficiency of these trace elements . Authors mentioned that amaranth products tested were well tolerated and did not report any allergic or dyspeptic reactions [Regarding amaranth ingestion, the tolerability and effectiveness of amaranth products inclusion on gluten intolerant children (eactions .Therefore, though a GFD can be restrictive, there is another naturally gluten-free cereal group or pseudocereals that possess higher contents of protein, fiber, iron, magnesium, and other bioactive compounds, such as phenolics, among others. If introduced into a GFD, pseudocereals can diminish the most common nutritional deficiencies in CD patients, apart from providing a wider food choice and a less monotonous diet for the CD patients . AlthougCeliac disease is a growing health condition that affects primarily the small intestine due to the direct toxicity triggered by the ingestion and digestion of gluten. This pathology can originate significant nutritional deficiencies in vitamin B6 and B12, folate, vitamin D, iron, calcium, zinc, and copper. Until now, the only treatment available for CD is the rigorous implementation of a GFD in order to allow the full recovery of the intestinal mucosa and improve nutrient absorption. When choosing gluten-free food, specially processed one, it is crucial to look at the macronutrient profile, namely, the high content in saturated fat and low content in fiber and micronutrients which are prevalent in these types of food.The incorporation of pseudocereals by themselves or as ingredients used by the food industry in gluten-free product development is a relevant alternative because of their richness in proteins, fiber, unsaturated fat, B complex vitamins, minerals like calcium, iron, magnesium, and other bioactive compounds. It can improve the nutritional status, well-being and health of CD patients and possibly expand their social inclusion in access to more food products and restaurants.Finally, nutritionists also have a preponderant role in the dietary education of CD patients by encouraging them to prepare their meals with high quality gluten-free ingredients for a finer nutritional quality diet and a minor reliance on low nutritional value packaged food. They should also emphasize the importance of using food guides to manage dietary patterns and understanding basic concepts of food labeling. This would improve food literacy of CD patients and consequently lead to more favorable outcomes in terms of nutritional status and health."}
+{"text": "Physical activity has many health benefits, yet a large portion of our population is not meeting recommendations. Using accelerometry and global positioning systems (GPS) to accurately measure where people are active and to identify barriers and facilitators of activity across various settings will inform evidence-based policies and interventions to improve activity levels. Criteria for sufficient accelerometry data  to accurately monitor free-living physical activity in adults and children have been widely studied, implemented, and reported by researchers. However, few best practice recommendations for researchers using GPS have been established. Therefore, this paper examined the impact of three co-wear criteria of varying stringency among a sample of children aged 10 to 16 years in Baton Rouge, Louisiana. Overall and location-based physical activity was consistent across the samples even within sociodemographic subgroups. Despite the lack of significant subgroup-specific mean differences in physical activity across the three samples, associations between sociodemographics and weight status and physical activity were significantly different depending on the device time-matching \u201cco-wear\u201d criteria applied. These differences demonstrate the critical impact co-wear criteria may have on conclusions drawn from research examining health disparities. There is a need for additional research and understanding of ideal co-wear criteria that reduce bias and accurately estimate free-living location-based physical activity across diverse populations. The benefits of regular physical activity, specifically moderate-to-vigorous intensity physical activity (MVPA), are well-acknowledged for children and adolescents, and many health behaviors track into adulthood ,2,3. PhyThe extent of evidence for environmental determinants of physical activity has rapidly grown. This is in part due to the advancement of technology; improvements in the measurement of physical activity  provide a more accurate representation of this health behavior by measuring duration and intensity , and advances in computer software  provide the tools to measure physical environmental characteristics of the land that people inhabit. Most recently, an increasing number of researchers have begun to use portable, consumer-grade geographic positioning system (GPS) devices in conjunction with accelerometers to objectively measure spatial behaviors and how people use their built environment for physical activity ,11. WhenWith technological and methodological advancements, there are also new practical challenges, limitations and questions of generalizability . CriteriAs we have seen in physical activity research using accelerometry, the application of varying inclusion criteria may lead to systematic bias in study results ,20,21. OChildren aged 10 to 16 years  were recruited from the largely urban greater Baton Rouge area to participate in the Translational Investigation of Growth and Everyday Routines in Kids (TIGER Kids) Study (USDA 3092-51000-056-04A). Consent from parents/legal guardians and assent from adolescents were obtained upon each participant\u2019s arrival at the Pennington Biomedical Research Center Translational Research Clinic for Children. Children aged 10 to 16 years and weighing < 226.8 kg (500 pounds) were eligible to participate in TIGER Kids; children were excluded if pregnant, on a restricted diet due to illness, or for having significant physical or mental disability. Parents completed a demographic form that included the adolescent\u2019s date of birth, sex, and race/ethnicity. Participant standing height and weight were measured by research staff to the nearest 0.1 cm and 0.1 kg, respectively; two measurements were taken, and the average was used for analysis (a third measurement was obtained if the two measurements differed by more than 0.5 units). During measurement, participants wore a gown and no shoes, and gown weight was subtracted to calculate final weight.Participants were asked to wear the triaxial accelerometer  and the QStarz BT Q1000XT GPS data logger on an elasticized belt around their hip. Devices were synchronized by the study team at the point of delivery to ensure tracking timestamps aligned. Participants were encouraged to wear both devices 24-h per day for at least 7 days, including 2 weekend days and received regular messages to remind them to charge the GPS and to wear their devices. Participants were instructed to remove the devices during water-based activity  as devices are not waterproof. Participants were asked to re-wear the devices if they did not wear the accelerometer for at least 4 days with 10 h of wear time. Data were downloaded into ActiLife software and assessed for valid wear immediately upon receipt. Data were collected during school and summer terms between 2016 and 2018. Accelerometers were initialized to 15-s epochs; whereas GPS location and velocity data were captured approximately every 5 s.Every GPS point was identified as within or outside the participant\u2019s home neighborhood. Home addresses of all participants were geocoded and validated with GPS data. Neighborhoods were defined using 1.2 km (\u00be mile) street network buffers around the home address, which is considered an acceptable walking distance . Data weGPS data with the derived location of activity (within or outside the neighborhood) and accelerometry data were aggregated to the minute-level and matched based on the closest date and time stamp using SAS statistical software. If more than 50% of the GPS points within the minute were within a location  then the minute was categorized as within that location. Ultimately, a single dataset was generated for each study participant that contained minute-level accelerometry data and location (binary variables denoting within or outside the neighborhood). Only raw device data were used, and no GPS or accelerometry data were imputed. Sleep time and accelerometer non-wear were identified using a previously-published algorithm ,25. LogsAverage accelerometer counts per minute (CPM) were used as a measure of physical activity. Evenson cut-points were used to define moderate (\u22652296\u20134011 CPM), vigorous (\u22654012 CPM) and MVPA (\u22652296 CPM) . These cn = 199; 66.6% of those who contributed accelerometry data). As we focus on physical activity occurring outside of school, the different weekday wear time patterns across the school year versus summer precluded pooling summer and school year data for the purpose of the current study. Adolescents assessed during the school year were more likely to be female ; otherwise, no differences were noted between those dropped versus retained sample for analysis. An additional 12 participants that reported a race other than white or African-American were excluded because this group was too small to draw comparisons. The current study is limited to 187 adolescents  who met minimum accelerometry wear time criteria of at least 3 days (including 1 weekend day) of 10 h per day of wear time  contributed accelerometry data (after 36 adolescents were asked to re-wear the device). TIGER Kids enrolled and measured participants year-round, but the current analysis is limited to adolescents who contributed accelerometry and GPS measurements outside of the summer holiday  within the school year. Across wear time criteria, only activity that occurred during co-wear was analyzed. To evaluate differences across the three co-wear criteria as well as the accelerometry-only group, the four samples defined by wear time criteria were combined into a single dataset with a categorical predictor indicating membership in the criteria sample. Demographic differences across the four wear time criteria  were assn = 187, 100%), nearly all adolescents  also met the minimum co-wear criteria, whereas 142 participants (75.9%) and 128 participants (68.4%) met the moderate and stringent co-wear criteria, respectively (On average participants had 17.3 daily MVPA minutes outside of school hours. Compared to the adolescents meeting the minimum accelerometer criteria (ectively . Demograectively . p = 0.0233); the association between race and MVPA differed across the four criteria-based samples (p = 0.0110). Across all four criteria, boys had higher levels of MVPA and participants with overweight/obesity had lower levels of MVPA; however, the magnitude of these associations differed across the criteria . Patterns were similar for moderate physical activity and vigorous physical activity, when modeled separately , adjusted mean minutes of MVPA did not differ significantly across the samples . Similarparately . The physical activity field lacks justification for and consistent use of accelerometry and GPS co-wear criteria. This study aimed to describe the impact of applying different co-wear criteria among a diverse group of adolescents in Baton Rouge, Louisiana. Estimates of overall and location-based physical activity were consistent across the criteria-based samples even within sociodemographic subgroups. However, associations between physical activity and sex, race, and weight status differed across the co-wear criteria samples.Guidance exists for analysis of accelerometry data, including what represents a \u2018valid day\u2019 of wear time and what represents a \u2018valid person\u2019 for analysis , which eWe found that the different co-wear criteria, however, produced differing conclusions about associations with physical activity. If we use the accelerometry-only group as a gold standard for associations, the sample generated from the strictest co-wear criterion was more likely to produce associations substantially different from the gold standard accelerometry-only sample. For example, the stringent criteria sample is the only one to find a significant association between race and physical activity, with African-Americans having significantly higher physical activity compared to whites. Furthermore, the largest differences in physical activity according to weight status were found in the stringent criteria sample. These differences may be due to unmeasured factors related to compliance , but also demonstrates the critical impact co-wear criteria may have on conclusions drawn from research examining health disparities. This provides new insight into GPS wear time criteria, extending and building upon earlier research that advocated for research developing and standardizing accelerometry wear time criteria ,20,21.Our findings have limitations, yet highlight potential issues for future studies. Findings are sample-specific and limited by low overall (17 min/day of MVPA) and location-specific (8 min/day within neighborhood and 10\u201311 min/day outside of neighborhood) minutes of MVPA and small standard error that limited our ability to detect differences in location-based physical activity, especially within subgroups. Furthermore, this study did not explore other environmental attributes or location-based physical activity  that may demonstrate important biases across samples.Standardizing the analysis of accelerometer-GPS co-wear data is critical to minimize measurement bias and provide a uniform platform to compare results within and between populations and studies . This st"}
+{"text": "Aims: To investigate the potential protective role of montelukast (Mont) in the pre-eclampsia rat model induced by L-NG-Nitro arginine methyl ester (L-NAME). Methods and materials: Thirty-two pregnant female albino Wistar rats were assigned to four groups: the control group: pregnant rats received vehicles; the Mont group: pregnant rats received Mont  from the 6th to the 18th day of gestation; the L-NAME group: pregnant rats received L-NAME  from the 9th to the 18th day of gestation; the Mont/L-NAME group: pregnant rats received Mont  from the 6th to the 18th day of gestation and L-NAME  from the 9th to the 18th day of gestation. Placental, hepatic, and renal malondialdehyde (MDA), total nitrites (NOx), interleukin 6 (IL-6), and tumor necrosis factor (TNF)-\u03b1 were determined. Serum alanine transaminase (ALT), aspartate transaminase (AST), creatinine, urea, 24-h urinary protein, and the placental growth factor (PGF) were measured. Histopathological examinations of the placental, hepatic, and renal tissues were also performed. In addition, placental, hepatic, and renal Janus kinase 2 (Jak2) and signal transducer and activator of transcription 3 (STAT3) immunoblotting were performed. Key findings: Mont improves oxidative stress, IL-6, TNF-\u03b1, ALT, AST, creatinine, urea, 24-h urinary protein, PGF, Jak2, and STAT3 which were all affected by L-NAME. Moreover, the histopathological assessment indicated that Mont restored the normal architecture that was markedly disturbed by L-NAME. Significance: Mont exerted the biochemical and histopathological amelioration of L-NAME-caused pre-eclampsia through its anti-inflammatory, anti-oxidant function and suppression of the IL-6/Jak2/STAT3 signaling pathway. The major cause of perinatal morbidity and mortality is pre-eclampsia (PE), affecting about 5\u20137% of pregnant women and developing after 20 weeks of gestation, resulting in maternal and fetal deaths worldwide . PE is cUnfortunately, the definite etiology of PE is unclear and without effective treatments to date. It is primarily a complex disorder in which the etiology is not due to a single cause, such as genetic, immunogenic, or environmental variables but rather to a complicated combination of numerous factors. PE can cause stroke, renal failure, pulmonary edema, liver rupture, and eclampsia if left untreated . Multi-oIt is well known that PE is linked to placental development disruption, which is accompanied by cellular, molecular, immunological, and vascular changes. Furthermore, abnormal placentation and shallow trophoblast invasion within the uterus result in incomplete spiral artery remodeling, which may lead to placental hypoxia, vascular endothelial dysfunction, inhibition of placental formation, and trophoblast immaturity. Failed decidual differentiation prior to pregnancy has been linked to impaired trophoblast invasion and its consequences ,7.Moreover, the only effective treatment for PE is delivery, which results in a newborn with intrauterine growth retardation and premature birth as well as various neonatal sequelae and elevated fetal pro-inflammatory markers . DespiteNitric oxide (NO) plays an important role in the regulation of the cardiovascular system during pregnancy. L-NG-Nitro arginine methyl ester (L-NAME), a known inhibitor of NO synthesis, can cause PE. When given to animals throughout the middle-to-late stages of pregnancy, it causes a state that mimics pre-eclampsia in humans, characterized by decreased placental perfusion, increased reactive oxygen species (ROS) generation, hypertension, and proteinuria .Montelukast (Mont) is one of the most frequently used drugs in chronic asthmatic females planning for pregnancy and is safely used during pregnancy without increasing the risk of congenital anomalies . It is aIt was reported that montelukast produced peripheral analgesia through the activation of the L-arginine/NO pathway and that L-NAME antagonized its action .Therefore, the current study was performed to investigate the protective effect of Mont in PE in rats. For this purpose, various biochemical, histological, and immunohistochemical approaches were sought to unravel the potential underlying mechanisms of action. There was no significant change in MAP between the groups on day 0. Meanwhile, on day 18, there was a significant increase in MAP in the L-NAME group compared to the control and Mont groups. The Mont + L-NAME group showed significantly decreased MAP compared to the L-NAME group . There was no significant change in the 24 h urine protein between the groups on day 0. Meanwhile, on day 18, there was a significant increase in 24 h urine protein in the L-NAME group compared to the control and Mont groups. The Mont + L-NAME group showed significantly decreased 24 h urine protein compared to the L-NAME group . In In In Compared to the control and Mont groups, hepatic p-Jak2 and p-STAT3 expressions were elevated in the L-NAME group. On the other hand, the Mont + L-NAME group showed significantly suppressed hepatic p-Jak2 and p-STAT3 expressions compared to the L-NAME group . In In Photomicrographs of the control and Mont groups showed full-term rat placenta at the level of labyrinth displaying normal interramal membrane and its constituents with normal fetal capillaries and normal maternal sinus. The L-NAME group showed distorted chorionic projection, dilated and congested maternal vessels, and fetal capillaries with inflammatory cell infiltration. The Mont + L-NAME group showed amelioration of all previously mentioned pathological changes .Hepatic photomicrographs of the control and Mont groups showed normal organization of the hepatic tissue. The hepatocytes showed vesicular nuclei radiating from the central veins and separated by blood sinusoids in normal architecture. The L-NAME group showed dilated, congested central veins and portal tracts, with more numerous apoptotic cells and inflammatory cell infiltration. The Mont + L-NAME group showed more or less normal hepatic tissue .Renal photomicrographs of the control and Mont groups showed normal organization of the renal cortex, the glomerular capillary tufts and Bowman\u2019s space. Proximal convoluted tubules (PCTs) have a narrow lumen and a highly acidophilic cytoplasm. Distal convoluted tubules (DCTs) have a wider lumen and less acidophilic cytoplasm with vesicular nuclei. The L-NAME group showed disturbed lobular architecture with dilated interlobular arteries with atrophic glomerulus, dilated PCTs (P), DCTs, and numerous apoptotic cells lining the tubules. Inflammatory cell infiltration was also detected. The Mont + L-NAME group showed the restoration of normal architecture . The hisPE, which is a multi-systemic disorder, is one of the most life-threatening pregnancy-related hypertensive disturbances. Despite the severity of PE, its precise pathogenesis is not yet fully understood and is currently an area of active research.In the current work, L-NAME, a nitric oxide synthase (NOS) inhibitor, was used to induce a PE rat model, which is well proven to be able to imitate PE-like manifestations including hypertension and proteinuria . Due to In accordance with previous studies ,17,18, wL-NAME also increased serum urea and creatinine concentrations as well as increased MDA, NOx, IL-6, and TNF-\u03b1 in renal tissue. This deterioration in renal function and the increase in oxidative stress and inflammatory parameters were associated with histopathological evidence of kidney affection . Earlier studies proved these findings. Shu et al. used different doses for the induction of PE . In addiSimilarly, L-NAME increased serum ALT and AST, hepatic MDA, hepatic NOx, hepatic IL-6, and hepatic TNF-\u03b1. The increments in indicators of liver damage, oxidative stress, and inflammatory parameters were associated with changes in the histopathological picture of the liver . Previous reports mentioned similar biochemical and histopathological changes in the liver of pre-eclamptic rats injected with L-NAME that caused intense mononuclear inflammatory infiltrate and thickening of the muscle tunica of arterial vessels, mainly in the periportal area ,23.The question is: how is PE associated with renal and hepatic injury? The pathway through which these injuries occur is still under intense debate. However, it is established that PE arises, in part, from inappropriate placentation and trophoblast invasion, causing placental ischemia and encouraging the disproportionate production of anti-angiogenic (sFlt-1) and pro-angiogenic (PLGF and VEGF) agents. This imbalance activates inflammatory cells, causing them to release auto-antibodies and inflammatory cytokines as well as causing oxidative stress, all of which contribute to generalized maternal endothelial dysfunction in various organ beds, resulting in hypertension, endothelins, blood coagulation, and organ damage .However, in the L-NAME-induced pre-eclamptic rat model, treatment with Mont rescued the elevated blood pressure and proteinuria, reduced serum urea and creatinine concentrations, AST, ALT, renal and hepatic inflammatory and oxidative stress parameters, and attenuated kidney and liver histopathological changes. These findings suggest that Mont ameliorated the nephritic and hepatic dysfunctions caused by L-NAME. Many studies have reported that Mont has nephroprotective and hepatoprotective effects by reducing oxidative stress and inflammation in other models of tissue injury, such as sepsis-induced kidney injury, cisplatin-induced nephrotoxicity, lipopolysaccharide-induced hepatotoxicity, and cecal ligation and puncture-induced sepsis ,25,26,27The Jak/STAT signaling pathway has an undeniable role in cell growth, differentiation, and survival. This pathway is also involved in the inflammatory response and immune function. The Jak family includes Jak1, 2, 3, and TYK2, and the STAT protein family contains seven members: STAT1, 2, 3, 4, 5A, 5B, and 6 ,29.STAT3 depends on the IL-6/Jak2/STAT3 signaling pathway in its activity, which may explain why there are contradictory reports on the activity of STAT3 in many diseases such as tumors and gastrointestinal problems .A pleiotropic cytokine, IL-6, is released in large amounts during infection, autoimmunity, tissue damage, and cancer. Low IL-6 levels may exert beneficial actions such as tissue regeneration, while the chronically elevated production of IL-6 may result in tissue injury .The stimulation of the Jak/STAT signaling system is an important pathogenic mechanism that causes endothelial cell dysfunction ,33. PhosIn agreement with the aforementioned studies, in the current study, L-NAME increased IL-6 levels, p-Jak2, and p-STAT3 expressions, which were accompanied by the induction of inflammation and oxidative stress in the placenta, liver, and kidney. These changes caused tissue damage, which manifested as hypertension, proteinuria, and worsening kidney and liver functions.In contrast, Mont reduced IL-6 levels, p-Jak2, and p-STAT3 expressions, which were associated with the amelioration of inflammation and oxidative stress in the placenta, liver, and kidney. This protective effect was shown by the improvement in the histopathological picture of these tissues and confirmed by the reduction in blood pressure, the protein level in urine, and the restoration of kidney and liver functions.Surprisingly, it was reported that JAK/STAT signaling regulated extracellular trophoblast growth and participated in PE development by affecting expression levels of phosphorylated STAT3 and STAT1. It was also reported that JAK/STAT3 signaling is indispensable for the regulation of the invasion of trophoblast ,39. SimiIn conclusion, L-NAME induced increased blood pressure and proteinuria as well as placental, hepatic, and renal injuries. The administration of Mont alleviated all these changes through antagonizing the inflammatory status and oxidative stress, probably through IL-6/Jak2/STAT3 signaling inhibition. Accordingly, we suggest that the inhibition of Jak2/STAT3 signaling can enhance placental, hepatic, and renal functions, preventing PE and represents a novel therapeutic strategy for PE treatment.Animal handling, treatment, and authentication were conducted in accordance with the requirements of the Institutional Ethical Committee  for the care of experimental animals as well as the NIH Guide for the care and use of laboratory animals .The National Research Centre in Giza, Egypt, provided 18 adult male Sprague Dawley rats  and 36 adult female Sprague Dawley rats . The rats were housed in the animal house of the Department of Pharmacology at the Faculty of Medicine, Minia University. For 7 days before the start of the experiment, the rats were housed under regulated conditions of temperature (22 \u00b1 2 \u00b0C) and humidity (50 \u00b1 10%) with a 12/12 h reversed light/dark cycle and ad libitum access to a standard pellet diet and tap water.Male rats were mated with all the female rats overnight. The day the spermatozoa were found in the vaginal smear was considered to be day 0 of pregnancy. The mating period lasted 15 days in total until a duplicate number of groups was achieved. Non-mated female rats during this time were considered infertile and were discarded from the study . The totPregnant rats were randomized into four groups (8 rats in each group) as follows:Control group: Pregnant rats received vehicles; distilled water i.p. and carboxymethyl cellulose p.o.Mont group: pregnant rats were given Mont  from the 6th to 18th day of gestation. Mont was dissolved in carboxymethyl cellulose .L-NAME group: pregnant rats were given L-NAME dissolved in distilled water  from the 9th to 18th day of gestation .Mont/L-NAME group: pregnant rats were given Mont  from the 6th to 18th day of gestation and L-NAME  from the 9th to 18th day of gestation.The 24 h urine of rats was collected using metabolic cages on days 0 and 18 of pregnancy to determine urinary protein. A Rat Urinary Protein kit was used to determine the level of urine protein . Measurement of blood pressure (BP) was performed using the non-invasive tail-cuff method on days 0 and 18 after the rats were pre-warmed in a heating chamber at 37 \u00b0C for 15 min. The blood pressure was measured five times, and the average result for each rat was calculated.g for 15 min , with sera kept at \u221280 \u00b0C for further biochemical analysis. Placenta, liver, and kidney were excised and washed. The tissue samples were divided into three parts: the first was stored at \u221280 \u00b0C until used, the second was embedded in paraffin for the histopathological examination, and the third was used for immunoblotting.At the end of our experiment, under light halothane anesthesia, each rat was anesthetized and then euthanized on day 19 of gestation. Blood was collected from the abdominal aorta and centrifuged at 3000\u00d7 Alanine transaminase (ALT) and aspartate transaminase (AST) were measured using commercial kits purchased from Spectrum Diagnostic, Egypt.Serum urea and creatinine were measured using kits purchased from Biomed, Egypt.\u2212) was based on the Griess reaction, the reaction of nitrite with a mixture of naphthyl ethylenediamine and sulfanilamide [To assess placental, hepatic, and renal oxidative stress biomarkers, lipid peroxidation was measured as a thiobarbituric acid-reacting substance and displayed as equivalents of malondialdehyde (MDA) using 1,1,3,3-tetra methoxy propane as a standard . Total nnilamide . Tumor necrosis factor alpha (TNF-\u03b1) was measured using ELISA kits obtained from Elabscience Biotechnology Inc., Houston, TX, USA (Cat.No.: E-EL-R2856). Interleukin-6 (IL-6) was measured using ELISA kits obtained from Nanjing Jincheng Bioengineering, Nanjing, China (Cat. No: H007). Placental growth factor (PGF) was determined using ELISA kits obtained from MyBioSource, San Diego, CA, USA (Cat.No.: MBS268190). The evaluation was undertaken according to the manufacturer instructions. w/v) non-fat milk and 0.05% Tween-20. Incubation with primary antibodies (1:1000) was completed overnight at 4 \u00b0C for: rabbit anti-Janus kinase 2 (JAK2) ; rabbit anti-pY1007/1008 JAK2 ; rabbit anti-signal transducer and activator of transcription 3 (STAT3) ; rabbit anti-pY705 STAT3 ; and \u03b2-actin . Secondary antibodies of horseradish peroxidase-conjugated polyclonal goat anti-rabbit immunoglobulin were added at a dilution of 1:5000 in the blocking buffer. Chemiluminescence was used for the visualization of bands with the help of a chemiluminescence kit using a luminescent image analyzer . Protein bands of different groups were quantified as fold change relative to the control group by applying Image J Software.Placental, hepatic, and renal homogenates  were boiled for five minutes with 2-mercaptoethanol containing loading buffer and then, applied to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to do running for two hours at 100 V. After electrophoresis, proteins were applied to polyvinylidene fluoride (PVDF) membranes. Blocking for one hour was performed in a Tris-buffered saline (TBS-T) solution which contained 5%  from all groups were stained with hematoxylin and eosin (H&E). All specimens were examined and photographed using a high-resolution color digital camera mounted on a BX51 microscope  and connected to a computer programmed with LC micro application software in the light microscopic unit of the Histology Department, Faculty of Medicine, Minia University.www.graphpad.com, accessed on 10 July 2022).Results were displayed as the mean \u00b1 S.E.M. The one-way analysis of variance (ANOVA) was carried out and followed by Tukey\u2019s test to analyze the data for statistically significant variance. GraphPad Prism software was used for statistical calculations (version 5.01 for Windows, GraphPad Software, San Diego, CA, USA (L-NAME induced increased blood pressure and proteinuria as well as placental, hepatic, and renal injuries. The administration of Mont alleviated all these changes through antagonizing the inflammatory status and oxidative stress, probably through IL-6/Jak2/STAT3 signaling inhibition. Accordingly, we suggest that the inhibition of Jak2/STAT3 signaling can enhance placental, hepatic, and renal functions, preventing PE and represents a novel therapeutic strategy for PE treatment."}
+{"text": "This study aimed to compare the effects of Linagliptin and Empagliflozin on renal function and glycaemic control in patients with type 2 diabetes mellitus (DM).We conducted a randomized, double-blind, parallel trial on patients aged 30 to 80\u00a0years with type 2 DM and HbA1c\u2009\u2264\u20099%, regardless of background medical therapy, to compare the effects of Empagliflozin and Linagliptin on albuminuria, FBS, HbA1c, and eGFR. Participants were given the mentioned drugs for 12\u00a0weeks. Statistical analysis was performed using appropriate tests in IBM\u2122SPSS\u00ae statistics software for windows version 24.P\u2009<\u20090.05), but there was no significant difference between groups regarding eGFR (P\u2009=\u20090.271). Changes in the FBS, HbA1C, and eGFR were not significantly different between groups (P\u2009>\u20090.05), but there was more decrease in albuminuria in the Empagliflozin group compared to the Linagliptin group .In total, 60 patients participated in the study, thirty patients in each group. The mean age of participants was 56.8 (SD\u2009=\u20098.15) in the Empagliflozin group and 60.9 (SD\u2009=\u20097.22) in the Linagliptin group. Before the intervention, FBS, HbA1C, and albuminuria values were significantly higher in the Empagliflozin group than those in the Linagliptin group (Regardless of baseline albuminuria, eGFR, or HbA1c, Empagliflozin 10\u00a0mg daily significantly reduced albuminuria at 12\u00a0weeks compared to Linagliptin 5\u00a0mg daily in patients with type 2 diabetes.IRCT20200722048176N1. Registered 3 August 2020.Iranian Registry of Clinical Trials,  Diabetes Mellitus (DM) is a debilitating metabolic disorder characterized by impaired insulin function, leading to chronic hyperglycemia . Two maiDiabetic nephropathy (DN) is one of the most common and severe microvascular complications of DM, occurring in about 20\u201330% of diabetic patients , 7, whicA growing concern during the course of type 2 DM is the prevention or delay of the progression of DN, especially with its increasing incidence each year. Early-stage DN causes glomerulosclerosis, compensatory hypertrophy, and late-stage DN leads to gradual atrophy . TreatmeDipeptidyl peptidase-4 (DPP-4) inhibitors are another group of medications used for glycaemic control and treatment of DN in patients with type 2 DM\u00a0, 24. DDPTo summarize, Empagliflozin and Linagliptin are two medications of different classes that have been used mainly for glycaemic control in patients with type 2 DM. Besides, Empagliflozin is one of the agents that has beneficial effects on renal function and has been used for this purpose. However, studies on the efficacy of Linagliptin are limited, and there are controversies in this regard. Therefore, to address these controversies on Linagliptin effects, this study aimed to compare the effects of Linagliptin and Empagliflozin on renal function and glycaemic control in patients with type 2 DM.We ran a randomized, double-blind, parallel trial  to compare the effects of Emplagiflozin 10\u00a0mg once daily and Linagliptin 5\u00a0mg once daily on albuminuria, fasting blood sugar (FBS), HbA1c, and eGFR in patients with type 2 DM. All patients were informed in detail about the study, and verbal and written informed consent were obtained. The study design entirely was approved by the Human Ethics Committee of the Arak University of Medical Sciences (ethics code: 1399.127.REC.ARAKMU.IR).The current study was conducted on patients with type 2 diabetes mellitus in Amir-al-Momenin hospital, Arak city, Iran, from September 2020, to May 2021. Patients aged 30 to 80\u00a0years and HbA1c\u2009\u2264\u20099% regardless of any background anti-diabetic therapy were eligible for inclusion. The dose of the background glucose-lowering drug was required to be unchanged at least 12\u00a0weeks before randomization. Exclusion criteria were: history of myocardial infarction or congestive heart failure less than three months before the study, hematuria, presence of urinary foley catheter, active urinary tract infection, and any renal diseases resulting in albuminuria at the time of inclusion.Patients were divided into two groups. One group received Empagliflozin 10\u00a0mg once daily, and the other group received Linagliptin 5\u00a0mg once daily. Both medications were added to the patients\u2019 previous anti-glycemic agents, and patients in both groups received these medications for 12\u00a0weeks. The dosage of medications was determined based on the recommendations for the treatment of patients with DM\u00a0, 29.In our study, primary outcomes were changes in albuminuria and eGFR, and secondary outcomes were changes in HbA1c and FBS after 12\u00a0weeks of treatment. The subjects were required to fast for 8 to 10\u00a0h before collecting the blood samples to evaluate FBS, creatinin for calculation of GFR, and HbA1C. Also, we used a spot morning urine sample for evaluation of albuminuria. The following formula was used for the calculation of GFR based on the creatinine levels. The calculated value\u00a0was multiplied by 0.85 if the patient was female.:For the purpose of blinding, drugs were removed from their original packages and were re-packed in look-alike packages, and neither the patients nor the instructors were informed about the contents of the packages. Patients were randomized 1:1 in a block size of 6 to receive either Empagliflozin 10\u00a0mg once daily or Linagliptin 5\u00a0mg once daily, and treatment allocation was performed by a random sequence generated by a computer. However, no allocation concealment was done.P\u2009<\u20090.05). For other variables, we used parametric tests, such as the T-test, as they were distributed normally (P\u2009>\u20090.05). We used univariate analysis of variances to assess the effects of confounders. We calculated Cohen\u2019s D to evaluate the effects of medications where we find significant differences in the changes of variables between groups. We considered P\u2009\u2264\u20090.05 as statistically significant. We used IBM\u2122SPSS\u00ae statistics software for windows version 24 for statistical analysis of the data.We calculated the mean and standard deviation (SD) for continuous variables. We used Kolmogorov\u2013Smirnov (KS) test to evaluate the distribution of values of each variable. We used non-parametric tests, including the Mann\u2013Whitney U test and Wilcoxon signed ranks test, to compare the FBS, HbA1C, and albuminuria between groups as they were not distributed normally (P\u2009=\u20090.049), but there were no other differences between groups regarding the basic characteristics, including age and gender (P\u2009>\u20090.05).In total, 73 patients were assessed for the eligibility criteria, and 12 individuals were excluded as they did not meet the eligibility criteria, and one declined to participate in the study. Therefore, 60 patients were randomized into two intervention groups . After the intervention, no significant differences were seen between groups regarding the outcomes (P\u2009>\u20090.05).Clinical characteristics of participants are shown in Table P\u2009<\u20090.001). In the Linagliptin group, GFR was significantly decreased after the intervention (P\u2009<\u20090.001). Also, FBS and albuminuria did not significantly change after the treatment (P\u2009>\u20090.05). The only significant improvement in the Linagliptin group was in the HbA1C values, as HbA1C values were significantly decreased after the course of treatment (P\u2009=\u20090.002).GFR, albuminuria, FBS, and HbA1C values were significantly improved in patients who received Empagliflozin .). After adjusting for baseline values of albuminuria and HbA1C, changes in the albuminuria were significantly different between groups in favor of the Empagliflozin group (P\u2009<\u20090.001). Changes in GFR, FBS, and HbA1C were not significantly different between groups (P\u2009>\u20090.05).The decrease in albuminuria was greater in the Empagliflozin group compared to the Linagliptin group (In this double-blind, randomized clinical trial comparing the effectiveness of Empagliflozin and Linagliptin in patients with type 2 DM, Empagliflozin emerged superior in efficacy regarding reducing albuminuria in the short term follow up of 12\u00a0weeks, regardless of their pre interventional renal function, BMI, and HbA1C, which is in line with previous studies. In a study by Lee et al., comparing the adverse kidney outcomes in patients with type 2 DM who received Empagliflozin or Linagliptin, it was found that decline in GFR was slower in patients who received Empagliflozin compared to those who received Linagliptin\u00a0. Also, tIn 2017 Groop et al. conducted a randomized clinical trial, MARLINA, comparing the effectiveness of Linagliptin versus placebo in reducing the albuminuria in patients with type 2 DM in a more extended period of follow up, 24\u00a0weeks\u00a0. In theiGlycoprotein DPP-4, which has been found in healthy individuals in two forms of circulating soluble and membrane-bound with a predominancy in proximal convoluted tubules , was alsOn the other hand, SGLT2, a transport protein in proximal convoluted tubules contributing to sodium-glucose reabsorption, can be inhibited by Empagliflozin, mediating the reduction in blood pressure intraglomerular filtration, which seems to lead to a reduction in albuminuria in a short time. Beyond alleviation of patients\u2019 hemodynamic status, histopathologic effects of SGLT-2 inhibitors could mediate a reduction in their albuminuria. In 2020, Klimontov et al. administered Empagliflozin to diabetic mice. As well as a reduction of urinary albumin-to-creatinine ratio, it showed a reduction in kidney hypertrophy, mesangial expansion, basement membrane thickening, and podocytopathy of glomeruli\u00a0 that couBeyond Empagliflozin association with eGFR preservation and reduced risk of major adverse kidney events reported in a cohort study of 379,033 participants  and EMPAAn intriguing conclusion was brought out of the EMPA-REG trial regarding the importance of early albuminuria reduction, where each 30% reduction of UACR was associated significantly with a lower hazard for major cardiovascular events . In thisThere was a heterogeneity regarding the FBS, HbA1C, and albuminuria values between the groups, which should be considered in the interpretation of the current study\u2019s findings. We used blocked randomization in our study to ensure the same number of participants would be enrolled in each group. However, there is a risk of imbalance regarding the prognostic factors while using block randomization, which might be even increased in the context of our study considering the small number of our sample size\u00a0. AdjustiThis study has several limitations suggested to be considered in future studies. First of all, it is better to follow up patients for more extended periods to meet the albuminuria-lowering effects of Linagliptin and their probable adverse complications. On the other hand, stratified randomized sampling according to potential confounders would yield more reliable findings, and a larger sample size with various ethnicities could enhance the power of the study. Also, we did not record the anti-glycemic agents that patients were taking before participating in this study, which can be a confounder for our results. Future studies evaluating pre-enrollment medications may be useful for more careful comparison of the effects of these medications. Finally, we did not perform allocation concealment and did not record the side effects in this study, and future well-designed controlled trials are needed to compare the effects of Empagliflozin and Linagliptin better.Regardless of baseline albuminuria, eGFR, or HbA1c, Empagliflozin 10\u00a0mg daily significantly reduced albuminuria at 12\u00a0weeks compared to Linagliptin 5\u00a0mg daily in patients with type 2 diabetes."}
+{"text": "ACE-1 and ACE-2 enzyme activity were measured using fluorogenic peptide activity assays. ACE-1, ACE-2, and Ang-II protein level were measured by enzyme-linked immunosorbent assay (ELISA). In both regions, ACE-1 protein and Ang-II levels correlated positively with age whereas ACE-1 enzyme activity was inversely related to age. ACE-1 protein correlated positively with Ang-II, whilst ACE-1 activity correlated inversely with Ang-II in normal aging. ACE-1 enzyme activity was elevated at an early/intermediate stage, BS III\u2013IV compared to BS 0\u2013II in the temporal cortex in AD. ACE-2 protein and enzyme activity were unchanged with aging and in AD. In conclusion, ACE-1 activity is induced in the early stages of AD independently from normal physiological age-related changes in ACE-1 protein.An imbalance in the renin\u2013angiotensin system (RAS) is associated with cognitive decline and disease pathology in Alzheimer\u2019s disease (AD). In this study, we have investigated changes in the brain angiotensin-converting enzyme-1 (ACE-1) and angiotensin-II (Ang-II), and the counter-regulatory angiotensin-converting enzyme-2 (ACE-2), in the frontal and temporal cortex during normal aging and in the early stages of AD. We studied a cohort of normal aging ( The renin\u2013angiotensin system (RAS) is involved in the systemic and neurogenic regulation of blood pressure . CirculaAlzheimer\u2019s disease (AD) is the most common cause of dementia accounting for approximately 60%\u201380% of all dementia cases. Aging is the most important risk factor for cognitive decline and the development of AD. AD is not a continuum of normal aging although studies indicate that normal aging and AD share common genetic, molecular, and cellular traits . AngioteAGTR1 blockers and ACE-1 inhibitors , or expWe still have a limited understanding of the timing of ACE-1 and ACE-2 changes in normal aging and AD within the brain. Characterizing the timing of these changes in relation to normal aging and the onset of AD, will potentially inform when therapeutic intervention will be most effective. Studies to-date, largely performed in rodents and restricted to peripheral organs, indicate that there is an organ-specific age-related imbalance in local RAS pathways resulting in overactivation of downstream disease-associated RAS pathways, with concurrent loss of counter-regulatory RAS signaling, independent of systemic RAS . FurtherIn this study, we have measured the key downstream RAS mediating enzymes  and Ang-II levels  in the FCx and temporal cortex (TCx) in a cohort of normal aging and a separate AD and age-matched control cohort that was grouped according to disease stage severity: controls (Braak tangle stage 0\u2013II), intermediate stage (Braak tangle stage III\u2013IV), and end-stage disease (Braak tangle stage V\u2013VI). We hypothesized that an age-related imbalance in brain ACE-1 and ACE-2 activity is exacerbated in the early stages of AD in association with disease pathology in AD.n = 101; 19\u201380 years age-at-death) was obtained from the Sudden Death Brain Bank, Edinburgh University, UK with local ethical approval (REC reference: 16/ES/0084). Cases had no pathological diagnosis of neurodegenerative disease. The cases were stratified into the following age groups: \u226445 years, 46\u201365 years, and >65 years for certain analyses.A cohort of normal aging cases  were obtained from the South West Dementia Brain Bank, University of Bristol, UK with local ethical approval (REC reference: 18/SW/0029). A neuropathological diagnosis of AD was made according to NIA-AA guidelines (n = 20 per group). The demographic data and neuropathological findings and the MRC identifier numbers are summarized in AD cases and age-matched nondementia controls  from the mid-FCx (Brodmann area 46) and TCx , was homogenized in 1 mL of 1% SDS (sodium dodecyl sulfate) buffer with protease inhibitors in a Precellys 24 homogeniser  as previously described . The samACE-1 activity was measured in brain tissue homogenates prepared in 1% SDS lysis buffer using an ACE-1 specific fluorogenic substrate (Abz-FRK(Dnp)-P) as described in previous studies ,22. We pThe concentration of ACE-1 protein was measured in brain tissue homogenates using a commercially available duoset ACE-1 enzyme-linked immunosorbent assay (ELISA) following manufacturer\u2019s guidelines (R&D Systems) as in a previous study . In brieACE-2 activity was measured in brain tissue homogenates using the ACE-2 specific fluorogenic substrate (Mca-APK(Dnp)), as described in our previous study . Brain hThe concentration of ACE-2 protein was measured in brain tissue homogenates using a commercially available ACE-2 ELISA duoset (R&D Systems) following manufacturer\u2019s guidelines with the following modifications: capture antibody was used at 4 \u00b5g/mL (2-fold higher than recommended); detection antibody at 200 ng/mL (2-fold higher than recommended); and strep:HRP at 1 in 20 (2-fold higher than recommended). Brain tissue homogenates were diluted 1 in 10 in PBS:1% BSA, and a serial dilution of recombinant ACE-2 (40\u20132 500 pg/mL) was used to generate a standard curve. The timings for each step and the wash steps were the same as described earlier for the ACE-1 duoset ELISA. TMB substrate (R&D Systems) was added to each well for 30 minutes at room temperature, and 2N sulphuric acid was added to stop the reaction. Absorbance was read at 450 nm for each well using a microplate reader . The concentration of ACE-2 in each sample was measured in duplicate, and the average was calculated after interpolation from the standard curve.Ang-II concentration was measured by direct ELISA using an in-house assay as previously described ,24. Brait tests, ANOVA with Tukey\u2019s post hoc analysis, or Kruskal Wallis analysis, was used for comparisons between grouped data where appropriate. Spearman\u2019s rank order was used to assess correlation. GraphPad Prism version 8 was used for statistical analyses. p Values <.05 were considered to be statistically significant.Unpaired r = 0.362, p = .0006; r = 0.408, p = .0001; ACE-1 protein level correlated positively with age within the FCx  and TCx  and negatively with ACE-1 enzyme activity across both regions  and TCx . No correlations were observed between ACE-2 enzyme activity and Ang-II concentrations in either brain region  and 46\u201365 years age group  in the FCx  or between III\u2013IV and V\u2013VI . Together, these data indicate that ACE-1 protein and Ang-II levels are elevated within the FCx and TCx in normal aging but that these changes differ from AD in which ACE-1 enzyme activity becomes overactivated in the early stages of disease.Systemic RAS, primarily known for regulating blood pressure, is often elevated in diseases involving the cardiovascular system and target organs. Locally expressed RAS pathways function both synergistically and independently from systemic RAS . It remaDespite age being a major contributor to cognitive decline and disease pathology in AD, we have only limited information regarding age-related changes in brain ACE-1 and ACE-2. Most studies to-date indicate that systemic RAS activity declines with age. Life-long therapeutic blockade of systemic cRAS has been shown to promote longevity in rodents ,28. In cIn this human postmortem study, we found that ACE-1 and Ang-II protein levels were elevated in relation to normal aging in the FCx and TCx, and ACE-1 protein correlated positively with Ang-II. In vitro studies, in human kidney tubular cells and in mouse neuronal cells, indicate that Ang-II can up-regulate the gene expression and protein concentration of renin  and ACE-In contrast to normal aging, ACE-1 enzyme activity (not ACE-1 protein or Ang-II) was elevated in the TCx in AD, supporting our previous observations ,22. For Hypertension, specifically in midlife, is a highly prevalent but modifiable risk factor for age-related cognitive decline and dementia. Several recent large meta-analyses of clinical trial data revealed that management of hypertension prevented cognitive decline and lowered the risk of dementia . DementiThe timing of intervention is likely to be a critical factor in determining the success of RAS-modulation in clinical trials in AD. This is the first study to offer insights into changes in brain ACE-1 protein level and enzyme activity in relation to age- and disease-stage in AD. Our data reveal an induction of ACE-1 activity in early-stage AD, that is, Braak stage III\u2013IV that is distinct from normal age-related changes in ACE-1 protein and Ang-II levels. ACE-1 activity can be measured in ante-mortem CSF and is elevated in AD  raising In conclusion, the present study reports age-related increased expression of ACE-1 and Ang-II protein concentrations, with a corresponding reduction in ACE-1 enzyme activity, possibly to limit further RAS activation in normal aging. In contrast, ACE-1 enzyme activity is elevated in AD, specifically at an intermediate stage of disease. Overall, these data indicate that brain RAS becomes dysregulated in the early stages of AD independently from age-related changes. The study improves our understanding of RAS in AD and may be important to inform the design and timing of future RAS-targeting clinical trials.glac083_suppl_Supplementary_MaterialClick here for additional data file."}
+{"text": "Leymus chinensis (a dominant grass) as our model plant species to assess the role of diverse fairy ring microbial communities on plant growth and nutrition.Fairy rings occur in diverse global biomes; however, there is a critical knowledge gap regarding drivers of fairy rings in grassland ecosystems. Grassland fairy rings are characterized belowground by an expanding mycelial front and aboveground by vigorous vegetation rings that develop concentrically with each growing season. We evaluated fairy ring dynamics in a field study conducted in semiarid grasslands to elucidate above- and belowground interactions driving distinct vegetation patterns. We followed this initial field investigation with a complementary greenhouse experiment, using soils collected from specific fairy ring zones  to examine plant-soil-microbial interactions under controlled conditions. We selected In our field study, plants on the ring-edge produced greater shoot biomass with higher concentrations of N and P, compared to plants inside the ring or adjacent (outside) controls. Soil microbial community biomarkers indicate shifts in relative microbial biomass as fairy rings expand. Inside the ring, plant roots showed greater damage from pathogenic fungi, compared to outside or ring-edge. Our greenhouse experiment confirmed that inoculation with live ring-edge soil generally promoted plant growth but decreased shoot P concentration. Inoculation with soil collected from inside the ring increased root pathogen infection and reduced shoot biomass.L. chinensis at the leading edge may increase pathogen accumulation, resulting in reduced growth at the center of the ring in subsequent growing seasons. Our results provide new insights into the plant-soil-microbial dynamics of fairy rings in grasslands, helping to elucidate these mysterious vegetation patterns.We propose that soil microbial activity within ring-edges promotes plant growth via mobilization of plant-available P or directed stimulation. However, as the ring expands, The online version contains supplementary material available at 10.1186/s12862-022-02082-x. Basidiomycota were detected in previous fairy ring research [Basidiomycota. Zotti et al. assessed AM fungi using next generation sequencing and suggested a positive relationship between increased AM fungi and lush vegetative growth in fairy ring edges [Fairy rings are enigmatic, frequently occur in grasslands dominated by clonal perennial gramineous plants, and are characterized by ring-like lush vegetation patterns , 2. Grasresearch , 4. The research . Yang etresearch , 6, 7. Iresearch , as AM fresearch . This wing edges . HoweverCorresponding to the lush plant ring belt, poorly performing plants near the center or outside areas are common features of fairy rings. Direct pathogenic damage or cyanide released by fungi may the main factors harming plants in the inner area of fairy rings, resulting in reduced plant growth at fairy-ring centers . Models Numerous biotic or microbial-induced abiotic changes likely drive fairy ring dynamics, such as soil physical, chemical, and/or biological properties; however, these complex interactions have not been disentangled in controlled studies. Plant\u2013microbial interactions (PMI) have received substantial attention in recent decades , and soiWe conducted two experiments to investigate (1) the distinct roles of soil microbial composition as well as potential changes to soil nutrient availability linked to fairy ring ecological patterns; (2) whether mycorrhizal fungi and/or pathogens have an influence on vegetation patterns associated with fairy rings.Leymus chinensis reseeding was carried out in 2000, with L. chinensis consistently accounting for >\u200940% of total aboveground biomass. Fairy rings in these grasslands are characterized by lush circular vegetation patches . Grazing is the primary utilization of these grasslands. Prior to our study, L. chinensis were measured in three 0.04 m2 quadrates (0.2\u00a0m\u00a0\u00d7\u00a00.2\u00a0m) in each sampling area. Plants shoots were harvested and dried for 48\u00a0h at 65\u00a0\u00b0C. Plant samples were weighed and analysed for total aboveground tissue N and P. Fifty 1-cm root segments of L. chinensis were collected to estimate root disease and AM fungal root colonization. Many potentially pathogenic organisms may damage plant roots. To provide an estimate of total microbial damage, regardless of pathogen identity, we utilized methods of Schnitzer et al. [Shoot density and biomass of r et al. , visuallr et al. .Our complementary greenhouse experiment was designed to disentangle distinct influences of soil nutrients and plant\u2013soil\u2013microbial interactions in fairy ring vegetation patterns, building on field observations. We utilized a randomized block design, consisting of fully factorial combinations of three fairy ring zone substrates  that were sterilized and three live fairy ring zone substrates to serve as inoculants  to elucidate the influences of biotic (microorganisms) and abiotic (nutrient availability) factors on fairy ring vegetation patterns. For each block, we established microcosms  with a mixture of sterilized substrate soil, sterilized fine vermiculite, and a live soil inoculum treatment . Each fairy ring was studied as an independent unit. For example, sterile fairy ring zone substrates from fairy ring A only received inoculant from fairy ring A. Fine vermiculite was added to ensure good drainage. With 3 blocks \u00d7 3 soil substrates \u00d7 3 inoculation treatments \u00d7 5 replicates, our experiment totalled 135 pots. Substrate soil was sieved (2\u00a0mm) and sterilized . Soil inoculum was sieved (2\u00a0mm) and stored at \u2212\u00a020\u00a0\u00b0C prior to use.L. chinensis were field collected in autumn of 2016. Seeds were surface sterilized by washing for 5\u00a0min with 75% ethanol and then 5\u00a0min with 10% \u201c84 disinfector\u201d . Seeds were then rinsed with demineralized water, sown in 1-cm of wet sterilized soil, and placed in lighted growth chambers  to promote germination. After 15 days, two seedlings were transplanted into each pot. All pots were placed in the greenhouse (16/8 h light/dark photoperiod) at approximately 20\u201326\u00a0\u00b0C. Each pot was watered with 100 mL demineralized water and randomly rearranged every 5 days to ensure uniform conditions. Dead or unhealthy seedlings were replaced in the first week.Seeds of 2CO3 extraction. Soil saccharase and urease were measured by culturing and soil alkaline phosphatase was measured using Solarbio, BC0280 kit. Abundances of soil microbial biomarkers were assessed via phospholipid fatty acid analyses (described above).Plant material and soil were analyzed for total nitrogen (N) content through combustion (FLASH.2000) and phosphorus (P) content through Mo-Sb-Vc spectrophotometry. In addition, soil was measured for extractable ammonium  via \u201cVegan\u201d in R .For our greenhouse experiment, effects of fairy ring zone substrates and live fairy ring zone inoculations on plant performance were analyzed by two-way ANOVA, with live soil and soil substrate as fixed factors and block as a random factor.L. chinensis occurring inside and outside of each ring were each significantly less compared to plants on ring-edges . Soil AN: AP ratio ranged from 4.96 to 7.49 with the lowest ratio occurring inside the fairy ring, increasing at ring-edge, and slightly decreasing outside the ring.Chemical properties and enzyme activities of soil at three fairy ring zones are presented in Table\u00a0p\u2009=\u20090.078).Based on NMDS, soil microbial community biomarkers were highly variable Fig.\u00a0 across aL. chinensis growing on the outside zone had 307.82% and 358.20% greater AM fungal root colonization than those grown on inside and ring-edges (p\u2009<\u20090.05)  , with ring-edge substrates always characterized by lower mycorrhizal root colonization . Inoculation with live soil collected from inside rings increased root disease in all three substrates, compared with microbial inoculation from soils collected at ring-edge or outside the ring . Our field study indicates this phenomenon is driven by microorganisms that mobilize soil P along the ring-edge. Further, our greenhouse experiment indicated microorganisms associated with the ring-edge stimulated the growth of L. chinensis directly through beneficial plant-soil\u2013microbial interactions linked to live ring-edge inoculation, as well as indirect benefit through P mobilization. We speculate loss of plant production inside grassland fairy rings relates to loss of beneficial microorganisms and/or accumulation of pathogens, as inside fairy ring plants showed greater evidence of root disease in the field experiment. Furthermore, our greenhouse experiment showed live soil collected from inside fairy rings tended to cause more extensive root damage and lower shoot biomass when used as inoculum, compared to inoculum from ring-edges or outside the ring.Our results indicate soil microbial interactions drive fairy ring patterns in steppe grasslands dominated by L. chinensis is close to 13.5 , a phytohormone that directly influences plant growth [Lepistasordida) in turfgrass and found environmental stress and nitrogen absorption were improved by AHX[Although AM fungi did not play a substantial role in fairy ring patterns, inoculation with ring-edge soil did promote plant growth while decreasing shoot P concentration, especially on soil substrate collected from outside the rings. This indicates soil microorganisms may stimulate plant growth directly, not only through improved nutrient availability. For example, plant growth promoting rhizobacteria (PGPR) regulate plant growth and metabolism through phytohormones . Soil bat growth . Choi eted by AHX.L. chinensis growing inside fairy rings suffered up to 90% greater disease severity compared to plants growing at ring-edge or outside controls. Our greenhouse experiment provides further evidence, as inoculation with soil from inside the fairy ring resulted in abundant root diseases, regardless of fairy ring zone substrates.Reduced plant production inside the fairy rings was likely influenced by several mechanisms, for example self-organization , phosphoL. chinensis in our study). Second, we found microorganisms in ring-edge soil may directly stimulate plant growth. Furthermore, these microorganisms were associated with greater alkaline phosphatase, promoting plant-available P-release in ring-edge soils. Additional biotic factors, such as AM fungal symbioses, may further explain dramatic above- and belowground differences observed across fairy ring zones, but did not appear to be a major influence in vegetation characteristics of L. chinensis across the three zones of fairy rings in our study. These insights expand our understanding of fairy rings, common but mysterious landscape features, highlighting essential links between vegetation dynamics and the microbial communities that shape grassland ecosystems. However, our findings should be viewed as a foundation for further research on fairy ring dynamics in grasslands. The fairy rings we assessed in the typical steppe of northern China suggest microbial interactions are linked with multiple plant community and soil food web outcomes and provides baseline data for several opportunities to build on by adding additional metrics, such as molecular identities of soil organisms, as well as to expand our results across additional grassland types. Only the dominant species (L. chinensis) in steppe grasslands was assessed in detail in our study, and the differences and driving factors of other species among fairy ring zones may help complete models of these complex and enigmatic ecological relationships.We provide empirical evidence that soil microorganism drive fairy ring vegetation patterns in semi-arid grasslands. First, as fairy rings expand across seasons, pathogens accumulate, with negative effects on the dominate species (Additional file 1:\u00a0Figure S1.\u00a0Leymus chinensis biomassproduction under different Stoichiometric ratios in greenhouse experiment."}
+{"text": "The purpose of this finite element study was to compare bone and cement stresses and implant micromotions among all-polyethylene (PE) and hybrid glenoid components. The hypothesis was that, compared to all-PE components, hybrid components yield lower bone and cement stresses with smaller micromotions.Implant micromotions and cement and bone stresses were compared among 4 all PE  and 2 hybrid  virtually implanted glenoid components. Glenohumeral joint reaction forces were applied at five loading regions . Implant failure was assumed if glenoid micromotion exceeded 75\u00a0\u00b5m or cement stresses exceeded 4\u00a0MPa. The critical cement volume (CCV) was based on the percentage of cement volume that exceeded 4\u00a0MPa. Results were pooled and summarized in boxplots, and differences evaluated using pairwise Wilcoxon Rank Sum tests.p\u2009=\u20090.017; A-KG: 3.6\u2009\u00b1\u20090.5\u00a0MPa, p\u2009=\u20090.014; I-KG: 3.6\u2009\u00b1\u20090.6\u00a0MPa, p\u2009=\u20090.040). There were no differences in cortical and trabecular bone stresses among glenoid components. The E-hCG hybrid component exceeded micromotions of 75\u00a0\u00b5m in 2 patients. There were no differences in %CCV among glenoid components.Differences in cement stress were found only between the I-hPG hybrid component (2.9\u2009\u00b1\u20091.0\u00a0MPa) and all-PE keeled-components (U-KG: 3.8\u2009\u00b1\u20090.9\u00a0MPa, Finite element analyses reveal that compared to all-PE glenoid components, hybrid components yield similar average stresses within bone and cement. Finally, risk of fatigue failure of the cement mantle is equal for hybrid and all-PE components, as no difference in %CCV was observed.IV, in-silico.The online version contains supplementary material available at 10.1186/s40634-022-00494-8. Total shoulder arthroplasty (TSA) is an effective and reliable treatment for shoulder arthritis , 2. AmonDespite numerous attempts to improve fixation of TSA glenoid components , glenoidHybrid glenoid components, featuring porous metal or metal-coated pegs on a PE bone-implant interface, were recently introduced to combine the benefits of cemented all-PE and uncemented metal-backed PE components . Hybrid Micromotions and stresses within bone and cement were compared among glenoid components that were virtually implanted in scapulae of three men and two women scheduled for shoulder arthroplasty Table  10]. Th. Th10]. mentclasspt{minimar et al. .Table 1PThe segmented geometries were then Imported into SolidWorks 2016  for further processing. Six three-dimensional (3D) computer assisted design (CAD) glenoid component models were created using SolidWorks 2016   following the manufacturers\u2019 surgical guidelines, resulting in a total of 30 shoulder models. A standardized coordinate system was defined based on the anatomical landmarks Fig.\u00a0. GlenoidAfter virtual positioning, optimal bone-implant fits were created with Boolean subtract functions. Cement mantles of 1-mm thickness were applied in accordance with the manufacturer\u2019s guidelines for component fixation Fig.\u00a0. All comQuadratic tetrahedral volume meshes (10 nodes-per-element) were generated for 3D anatomic and implant CAD models in Solidworks 2016 . The maximum element edge-length was 2\u00a0mm, and the mesh quality was set to a maximum of 0.1\u00a0mm difference between the 3D CAD models and the 3D mesh models. The anatomic model mesh densities  ranged from 154 000 to 222 000 elements  loading on the central region of the glenoid (F-Centre); 2) loading on the anterior region of the glenoid (F-Ant); 3) loading on the posterior region of the glenoid (F-Post); 4) loading on the superior region of the glenoid (F-Sup); and 5) loading on the inferior region of the glenoid (F-Inf) : 1) micromotions were calculated from mean resultant nodal displacements  in the superior-inferior, medial\u2013lateral, and anterior\u2013posterior directions at the bone-implant interface; 2) mean von Mises stresses within cortical and trabecular bone, and cement. Implant failure was applied according to previously suggested criteria : First, The finite element analyses were done with an implicit linear static solver , and convergence was achieved with a displacement threshold of 1.0e-06 and the maximum number of iterations set at 1.0e\u2009+\u200907. All the analyses were executed on an Intel\u00ae CORE\u2122 i7-6700 @3.40\u00a0GHz workstation equipped with 16\u00a0GB RAM and an NVIDIA Quadro K420 graphics card. Simulation wall-time durations were on average 60\u00a0min per simulation.To ensure mesh independence of the predicted stresses and micromotions across simulations, the criterion for mesh convergence was defined as a change of less than 5% in the maximum displacement between mesh densities. A mesh convergence analysis on model P1 with implant I-KG, revealed that decreases in element edge lengths beyond 2\u00a0mm yielded changes\u2009<\u20090.05% in maximum and mean displacements. Decreases in element edge lengths beyond 2\u00a0mm yielded changes of\u2009<\u20098.0%,\u2009<\u20099.2% and\u2009<\u200912.7% in respectively cortical bone, trabecular bone, and cement mean von Mises stresses. Therefore, the maximum element edge lengths were limited to 2\u00a0mm in all finite element models.P values\u2009<\u20090.05 were considered statistically significant.Descriptive statistics were used to summarize the data. Micromotions and stresses within bone and cement were summarized in boxplots across the five loading configurations as applied on the five anatomical models, thereby resulting in 25 results per outcome for each of the 6 glenoid components. Differences between glenoid components were evaluated using pairwise Wilcoxon Rank Sum tests with Bonferroni correction. Statistical analyses were performed with R version 3.1.1 . p\u2009=\u20090.017; A-KG: 3.6\u2009\u00b1\u20090.5\u00a0mPa, p\u2009=\u20090.014; I-KG: 3.6\u2009\u00b1\u20090.5\u00a0mPa, p\u2009=\u20090.040)  and the all-PE keeled components  and trabecular bone  were observed among the different glenoid components Table , Fig.\u00a07.p\u2009=\u20090.037) and I-hPG hybrid pegged-component  , compared to the A-KG all-PE keeled-component  . AlthougExcessive micromotion and strains can stimulate fibrosis on the implant surface and negatively impact bone-implant stability . In the A recent systematic review showed that modern metal-backed glenoid components have improved survival rates compared to conventional metal-backed components . Since tThe results of this study should be considered with the following limitations in mind. First, although the models account for variations in joint reaction force magnitude and bone properties of cortical and cancellous bone, the impact of soft tissue tensions and conditions was not considered. Other time-dependent biological factors, such as osseous-integration, were not considered in the analysis. Second, the cohort of 5 scapulae could be considered small, and therefore may not represent the general population. Third, bone mineral density was based on CT Hounsfield Units, and the CT scanner settings were not optimized with a calibration phantom. The scans for each patient were however obtained with the same CT scanner, of which scanning parameters and post-processing settings were standardized and consistently applied. Fourth, the present study only compared all-PE components to hybrid components and did not include metal-backed components. It would be interesting to include metal-backed components in future comparative analyses, since a recent clinical study of 37 TSA with metal-backed glenoid components found 100% implant survival at mean 7-year follow-up, despite the occurrence of metallic debris formation . Fifth, Finite element analyses reveal that compared to all-PE glenoid components, hybrid components yield similar average stresses within bone and cement. Finally, risk of fatigue failure of the cement mantle is equal for hybrid and all-PE components, as no difference in %CCV was observed.Additional file 1. Anatomical material model.Additional file 2. Estimation of contact area."}
+{"text": "This Special Issue addresses new scientific insights and technological advances in the area of gluten-free product development with the aim of controlling gluten intolerance and autoimmune diseases.p < 0.05) compared to standard products, as well as higher (p < 0.05) fat contents. Bokic et al. [This Special Issue publishes seven research papers and four review articles. In the paper by Gasparre et al. , the autc et al.  investigc et al.  aimed toPediococcus pentosaceus strain.Chochkov et al.  exploredDos Reis Gallo et al.  determinGazikalovi\u0107 et al.  producedAmorphophallus konjac  flour. The bread samples with konjac showed a high fiber content and lower levels of carbohydrates, hence lower calories.Laignier et al.  developeThe review papers deal with the use of additives to replace gluten and ensure the stability and elasticity of the dough, hence improving the nutritional quality and sensory properties of gluten-free bread . The app"}
+{"text": "In the linear regression model, higher working seniority as well as higher EE, IRR, DEP, ANX, and DDF scores and lower PA were associated with higher hopelessness. In conclusion, increased hopelessness was associated with higher burnout and alexithymia. Comprehensive strategies should be implemented to support HCWs\u2019 mental health and mitigate the negative consequences of alexithymia, burnout, and hopelessness.In the present study, we aimed to assess the frequency of and the relationships between alexithymia, burnout, and hopelessness in a large sample of healthcare workers (HCWs) during the third wave of COVID-19 in Italy. Alexithymia was evaluated by the Italian version of the 20-item Toronto Alexithymia Scale (TAS-20) and its subscales Difficulty in Identifying Feelings (DIF), Difficulty in Describing Feelings (DDF), and Externally Oriented Thinking (EOT), burnout was measured with the scales emotional exhaustion (EE), depersonalisation (DP), and personal accomplishment (PA) of the Maslach Burnout Test (MBI), hopelessness was measured using the Beck Hopelessness Scale (BHS), and irritability (IRR), depression (DEP), and anxiety (ANX) were evaluated with the Italian version of the Irritability\u201a Depression\u201a Anxiety Scale (IDA). This cross-sectional study recruited a sample of 1445 HCWs from a large urban healthcare facility in Italy from 1 May to 31 June 2021. The comparison between individuals that were positive ( Alexithymia, which is a personality trait, burnout, and hopelessness are interconnected constructs that might have significantly impacted healthcare workers (HCWs) during the COVID-19 pandemic .Alexithymia is a relatively stable personality trait, characterised by the difficulty in identifying, describing, and communicating emotion and in distinguishing emotional experiences from underlying physiological activation ,4. It alHCWs experiencing high stress, trauma, and exhaustion levels during the COVID-19 crisis may find it even more challenging to effectively identify and cope with their emotions ,22. The Burnout, from the English word for \u201cburning out\u201d, indicates a specific work situation mainly affecting people engaged in social work . Nurses,Burnout has been prevalent among HCWs, particularly during the COVID-19 pandemic ,35. The Hopelessness is a psychological construct underlying several psychiatric disorders and refers to the cognitive schemata underlying a negative expectation towards the future . HopelesHopelessness can be a distressing consequence of the prolonged stress experienced by HCWs during the pandemic ,44. The As the combined effects of alexithymia, burnout, and hopelessness can compromise HCWs\u2019 mental well-being, potentially leading to feelings of depression, anxiety, and even suicidal ideation, in the present study, the authors aimed to assess the frequency of and the relationships between alexithymia as a personality trait, burnout, and hopelessness in a large sample of HCWs during the third wave of COVID-19 in Italy. In addition, the clinical predictors of hopelessness, as an indirect measure of potential suicidal ideation, were evaluated in this sample. This cross-sectional study recruited a convenience sample of 1445 HCWs from a large urban healthcare facility in Italy  that provided care to COVID-19 patients during the pandemic. The average age of HCWs was 44.2 \u00b1 12.1 years, and the average working seniority was 17.7 \u00b1 12.1 years; the number of females was 1046 (72.4%), and the number of males was 399 (27.6%). Recruitment occurred throughout the duration of the study. The self-reported data were collected from 1 May to 31 June 2021, which included the peak of the third-wave pandemic that year in Italy. HCWs were invited through work emails to participate voluntarily without exclusion criteria. The project was directly endorsed and commissioned by the Local Health Authority  of Teramo to evaluate HCWs\u2019 well-being during the pandemic, and therefore, no IRB approval was required. Participants provided their consent online before completing the survey.The online questionnaire was composed of third sections. The first section showed in detail the steps and aims of the study and ended with a question asking the respondents whether they agreed to participate. The second section included questions about participants\u2019 age (years), sex , and working seniority.The third section included six rating scales.Alexithymia was evaluated using the Italian version of the 20-item Toronto Alexithymia Scale (TAS-20) . A scoreA patient\u2019s hopelessness status was measured using the Beck Hopelessness Scale (BHS) . Higher The presence of burnout was assessed using the Italian version of the Maslach Burnout Inventory (MBI) . The MBIThe presence of symptoms such as irritability (IRR), depression (DEP), and anxiety (ANX) was evaluated with the Italian version of the Irritability\u201a Depression\u201a Anxiety Scale (IDA) . This st2). Finally, a block-wise linear regression analysis [p values \u2264 0.05 were deemed statistically significant. The differences between subjects that were positive or not positive for alexithymia were tested using analyses of covariance (ANCOVA) with the TAS-20 positivity/negativity as a factor and age, gender, and working seniority as covariates . Effect analysis  was condn = 219) of the 1445 HCWs who scored 61 or more were considered positive for alexithymia.Descriptive statistics of the whole sample and the comparison between subjects, positive or not, for alexithymia are reported in n = 214, 14.8%) or not for alexithymia , controlling for age, gender, and working seniority, revealed that positive subjects showed higher scores for BHS, EE, DP, IRR, DEP, ANX, DIF, DDF, and EOT and lower for PA than the not positive ones (p range: 0.006\u2013<0.001). The effect size calculation (\u03b72) showed that the magnitude of the group effect for all above variables was large.The comparison between individuals that were positive , and the standardised residuals were normally distributed.In the linear regression model , a higheThis was the first study investigating the relationships between alexithymia, burnout, and hopelessness in a large sample of HCWs during the Italian third wave of COVID-19. Overall, our results demonstrated that the third wave of pandemic emergency had an impact on HCWs\u2019 psychological well-being, which was maybe related to the previous waves and the higher workload due to the care of the COVID-19 patients. Moreover, managing the health emergency linked to the spread of COVID-19 required HCWs to make substantial changes in their work concerning organisational, relational, and safety aspects ,54. In sIn our study, the frequency of alexithymia in our sample was 15.2%, which is not higher and somewhat in line with that reported in other studies on the general population, despite the pandemic stress ,13. We hHowever, subjects that were positive for alexithymia showed higher levels of hopelessness, elevated burnout in terms of highly enhanced emotional exhaustion and depersonalisation, and low personal accomplishment, together with more distressing irritable, depressive, and anxiety symptoms. These findings are in line with those of previous studies that have demonstrated that subjects with alexithymia often show higher psychological and psychiatric distress than subjects without alexithymia ,60,61. IThe finding that the DDF scale of TAS-20 was associated with increased hopelessness and burnout is one of the main results of the present study. To date, no studies have investigated the relationship between burnout and hopelessness, and our study findings might indicate that the development of a state of emotional, physical, and mental exhaustion triggered by excessive stress may have weakened the energy of HCWs, which, in turn, decreased efficiency and left them helpless, hopeless, pessimistic, and angry. Therefore, it is possible to hypothesise that a difficulty in describing feelings might leave the HCWs incapable of communicating personal distress and emotional exhaustion, reducing the possibility of asking for help and receiving it, especially in the case of developing psychiatric symptoms.Thus, the results of our study may let us hypothesise that the presence of a difficulty in describing feelings is the background on which higher hopelessness and a reduced sense of competence and efficacy, often associated with an increasing negative view of one\u2019s abilities, may develop and might lower HCWs\u2019 both individual and group resilience ,64. AlexMoreover, alexithymia is a risk factor for experiencing increased levels of chronic psychosocial stress . ChronicIn addition, our results may also support Freyberger\u2019s concept of acute \u201csecondary alexithymia\u201d as a reaction to stressful situations . This ofThis may also explain the higher hopelessness, alexithymia, and burnout seen in HCWs with higher working seniority. In terms of socio-demographic variables, some studies have found that younger HCWs with less work seniority show less burnout and psychological distress, as at the beginning of their careers, they are more motivated ,76, wherOverall, the findings of our study indicate that comprehensive strategies should be implemented to support HCWs\u2019 mental health and mitigate the negative consequences of alexithymia, burnout, and hopelessness . This inThis study has several limitations, so the results should be interpreted cautiously. The first limitation was the usage of self-rating scales with probable biases due to the nature of the self-rating scales themselves, with a social desirability bias especially for TAS-20: social desirability bias is a type of response bias that happens when survey participants give answers in accordance with society\u2019s expectations, despite their own beliefs or experiences. In addition, our study lacks follow-up data, as it was cross-sectional. Also, the sample was recruited during the third wave of the COVID-19 pandemic in Italy, and we lack data on previous waves\u2019 effect on HCWs\u2019 psychological well-being; thus, it is not possible to conclude a cause-and-effect relationship. Finally, the sample was constituted mainly of females, which might limit the generalizability of the results concerning gender. In conclusion, HCWs have faced unprecedented challenges during the COVID-19 pandemic, and alexithymia may be relatively prevalent in HCWs during the fourth wave of the pandemic, significantly increasing emotional and mental health consequences. The impact of alexithymia, burnout, and hopelessness cannot be overlooked. Recognising and addressing these psychological constructs is crucial to safeguarding the well-being of HCWs and ensuring the provision of high-quality patient care. Supportive measures, including emotional awareness training, mental health resources, and fostering a positive work environment, are essential for navigating these challenging times and promoting the long-term sustainability of the healthcare workforce."}
+{"text": "Candida auris are on the rise. In addition, there are new risk factors for fungal disease such as the use of targeted biologics inhibiting important players in antifungal host defense, including IL-17A, IL-17RA, IL-12p40, Tumor necrosis factor \u03b1 (TNF\u03b1). Even though during the last few decades there has been a significant advance in our understanding the mechanistic basis of fungal disease, research in this field has been hampered by difficulties in disease modelling.Invasive fungal diseases are a significant cause of morbidity and mortality worldwide. In the last years the increment of at-risk populations for fungal diseases has been linked with new clinical procedures including immunosuppresive therapy, use of invasive devices, transplantation, but also autoimmunity, diabetes, severe primary infections, cancer, or chronic lung diseases. The diagnosis of invasive fungal diseases is difficult as signs and symptoms are unspecific, thus delaying the start of antifungal therapy. Moreover, resistance to antifungals is emerging, and naturally resistant species such as In this Research Topic we aimed to explore how the use of novel experimental systems can be used to answer important questions about the mechanistic basis of fungal disease. These approaches can help us to better understand the timeline of infection from development to progression, identify critical disease biomarkers that could improve diagnosis, and discover those genes or pathways that could serve as novel therapeutic targets. For these reasons, in this Research Topic we wanted to integrate new methodological approaches developed by early career researchers with a view to improving our understanding in fungal pathogenesis. The application of these technologies to answer different research questions would allow us to improve the diagnosis and treatment of patients with these deadly infections. Here we included research articles and reviews focused on immunology, fungal biology, host-pathogen interactions, fungal pathogenicity mechanisms, and unknown fungal-microbiome connections.Three original research articles, one brief research article, one review and this editorial are included in this Research Topic.Powell et\u00a0al. focused on the role of Tumor necrosis factor \u03b1 (TNF\u03b1) in Coccidioides infection. Coccidiomycosis is an endemic mycosis in the Americas. Clinical management of these patients usually requires long-term antifungal treatment and the use of immunotherapy, as proposed in this article, is of high interest. Nevertheless, the study of Coccidioides infection is complicated by its BSL3 status and the fact that it is difficult to generate infectious spores. The authors used a novel mouse model to dissect the importance TNF\u03b1-mediated response in coccidiomycosis. In their model they suggested that the use of an in vivo model of \u2018static infection state\u201d would recapitulate the phenotype of patients with good resistance to Coccidiodides infections  mice. In this model, A. fumigatus was delivered in coated-beads that facilitate the formation granulomas in infected lungs thus mimicking aspects of the pathohysiology of chronic pulmonary aspergillosis. While HIF-1\u03b1-deficient mice showed smaller granulomas, these mice suffered from uncontrolled fungal growth with enhanced neutrophil cell death. This work provides further important insights in the connection between the hypoxia sensor HIF-1\u03b1 and protective antifungal immunity. Moreover, this model is also a crucial step forward towards the modelling of chronic fungal diseases and opens future avenues for mechanistic studies.The third research article published in the Research topic is focused on aspergillosis. Caenorhabditis elegans infection model. The C. elegans model of neurodegeneration presented in this article displays several advantages compared to other models of disease mainly due to their simple anatomy, transparency, and brief lifespan (Kitisin et\u00a0al. demonstrated that neurodegeneration occurred as a direct consequence of the cryptococcal infections. Thus, C. elegans infection model of cryptococcal-induced neurodegeneration represents a valid alternative to mouse models for evaluation of novel therapeutics to prevent neuronal damage during fungal infections.The last original research article of the topic is focused on modelling of cryptococcal meningitis by using a lifespan . Using tThis Research Topic clearly shows the need to develop new physiological models of fungal diseases and underscores the urgent need for the development of models that better recapitulate pathophysiological mechanisms. There is a need for models that can be used to accurately study infections that are organ specific. The development of infection models to study fungal diseases is limited by the intrinsic capacity of fungi to adapt in the host environments, where they may induce latency, granulomas, static infection states, neurodegeneration, and the huge variety of susceptible hosts. Future research should aim to standardize infection models that better recapitulate the complexity of the host-pathogen interface as shown for a small selection of fungal pathogens by the authors contributing to this Research Topic.SG, TZ, and MG conceived the Research Topic and edited the manuscripts. TZ wrote the first draft of the editorial. All authors read and provided significant inputs into all drafts of the editorial, agreed to be accountable for all aspects of the work and approved the final draft of the editorial for publication."}
+{"text": "However, whether these lncRNAs bear post-translational modifications and how their differential expression is regulated remain largely unknown. In this study, the transcriptome-wide 5-methylcytosine (m5C peaks and 1667 downregulated peaks in the H1N1 infected group. Gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that the differentially modified lncRNAs were associated with protein modification, organelle localization, nuclear export and other biological processes. Furthermore, conjoint analysis of the differentially modified (DM) and differentially expressed (DE) lncRNAs identified 143 \u2018hyper-up\u2019, 81 \u2018hypo-up\u2019, 6 \u2018hypo-down\u2019 and 4 \u2018hyper-down\u2019 lncRNAs. GO and KEGG analyses revealed that these DM and DE lncRNAs were predominantly associated with pathogen recognition and disease pathogenesis pathways, indicating that m5C modifications could play an important role in the regulation of host response to IAV replication by modulating the expression and/or stability of lncRNAs.Our data identified 1317 upregulated m5C modification profile of lncRNAs in A549 cells infected with IAV and demonstrated a significant alteration of m5C modifications on host lncRNAs upon IAV infection. These data could give a reference to future researches on the roles of m5C methylation in virus infection.This study presented the first mThe online version contains supplementary material available at 10.1186/s12864-023-09432-z. Increasing numbers of studies have demonstrated that RNA m5C modification plays important roles in multiple biological processes including RNA processing [5C modifications in low-abundance RNA species such as non-coding RNAs (ncRNAs), and transcriptome-wide identifications of m5C modification in different types of RNAs have been reported [RNAs are subject to a range of covalent modifications at the single nucleotide level, such as pseudouridine, N6-methyladenosine (m6A) , N1-meth6A) [1A) , 5-methy1A) [5C) . So far,ocessing , 5, RNA ocessing , RNA traocessing , and mRNocessing . With threported \u201312.6A, m5C, N4-acetylcytidine (ac4C) and 2\u02b9O-methylated nucleosides (Nm) have been reported to promote viral replication by upregulating viral mRNA stability or translation, or by preventing the recognition of viral RNA through modulation of host RNA-specific innate immunity factors including RIG-I and MDA5 [5C has been found to present at higher levels in retroviral transcripts than in cellular mRNAs, and the modification can regulate RNA splicing and promote the translation of viral mRNAs [e recent study has demonstrated that RNA m5C methylation can control antiviral innate immunity through modulating the m5C methylome of noncoding RNAs (ncRNAs) and their expression, which regulate type I interferons [Evidence is emerging that RNA modifications are involved in the regulation of virus infection. Epitranscriptomic marks such as mand MDA5 . As a real mRNAs . A more erferons .As an important pathogen for seasonal respiratory illness, influenza A viruses (IAV) have caused human pandemic outbreaks such as those occurred in 1918, 1957 and 1968, and they are still continuing to threaten public health , 17. IAV5C is a common epitranscriptomic modification found in RNAs, knowledge surrounding the prevalence and transcriptome-wide distribution of m5C in lncRNA is still very limited, and the roles of m5C modification during IAV infection have not yet been explored. It remains unknown whether m5C modification plays a role in regulating lncRNA expression during IAV infection.Epitranscriptomic regulation has been shown as an important mechanism to control lncRNAs expression and tissue specificity . Althoug5C methylation of lncRNAs in H1N1-infected A549 cells were globally mapped by Methylated RNA immunoprecipitation sequencing (MeRIP-seq), using the uninfected cells as controls. Marked alterations in the amount and distribution of m5C peaks in lncRNAs were detected between the two groups, suggesting that m5C modifications could play important regulatory roles during IAV replication.In this study, the mRNAseq was performed to determine whether the lncRNA expression profile changed upon IAV infection. In total, 12,497 lncRNAs were detected in the IAV infected and uninfected A549 cells, including 1779 novel lncRNAs which were predicted as noncoding transcripts by all the four coding prediction softwares used in this study  and only 32% belonged to intergenic type  and detected their expression levels by RT-qPCR. In the A549 cells infected by 0.1 MOI of IAV at 36 hpi, the qPCR results were consistent with the RNA-Seq results  was performed on H1N1-infected and uninfected A549 cells. We identified 2343 m5C peaks on lncRNA transcripts in A549 cells infected with IAV, and 4022 m5C peaks in uninfected cells , and this enrichment was slightly reduced after infection  m5C methylation in IAV infection, the nearest protein-coding genes paired with the differentially modified lncRNAs were searched out and used for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. GO analysis based on biological processes (BP) revealed that the target-genes of up-methylated lncRNAs (H1N1-infected vs. uninfected cells) were significantly enriched in protein modification by small protein removal, protein deubiquitination, and calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules, while the down-methylated genes were largely enriched in non-membrane-bounded organelle assembly, establishment of organelle localization and nuclear export. The molecular functions (MF) output showed that the genes targeted by the up-methylated lncRNAs were notably involved in cysteine-type deubiquitinase, HMG box domain binding and alcohol dehydrogenase (NAD(P)+) activity. In contrast, the down-methylated lncRNAs were predominantly associated with genes playing roles in transcription coregulator activity, transcription coactivator activity and cadherin binding. The cellular components (CC) data showed that up-methylated lncRNAs mainly targeted genes in SAGA-type complex, MLL1 complex and chromocenter, while the down-methylated lncRNAs were primarily associated with the genes in actin cytoskeleton, cell-substrate junction and focal adhesion . The down-methylated lncRNAs were notably related to genes in Central carbon metabolism and Adherens junction. A number of target genes of hypomethylated lncRNAs were involved in some infection related pathways  Fig.\u00a0B.e the relationship between m5C epitranscriptomic modification and lncRNA expression level. Conjoint analysis of the DM and DE lncRNAs identified 143 hyper-up genes with both m5C modification and expression levels up-regulated, 4 hyper-down lncRNAs with up-regulated modification and decreased expression levels, 81 hypo-up lncRNAs with down-regulated modification and increased expression levels, and 6 hypo-down lncRNAs with down-regulated modification and decreased expression levels   sponges to regulate the expression level of protein-coding genes. To explore the possible regulatory roles of the DM and DE lncRNAs in IAV infection, five lncRNAs including three hyper-up lncRNAs  and two hypo-up lncRNAs  were picked out by retrieving lncbook and miRwalk databases, and their competing endogenous RNA (ceRNA) network were constructed. As shown in Fig.\u00a0It has been reported that IAV infection alters the expression of numerous host lncRNAs, suggesting that this class of noncoding RNAs may play important regulation roles in the virus-host interaction \u201324. PrevIn this study, we examined the expression of lncRNAs in H1N1-infected cells, and detected more lncRNAs on each chromosome in the infected group than the uninfected group, indicating an overall upregulation of lncRNAs expression after H1N1 infection. The phenomenon that the majority of DE lncRNAs have elevated expression levels upon IAV infection had been observed in H1N1 and H3N2 infected cells , 26. Her5C has been reported to act as a modulator for the stability [5C modification of enhancer RNA [5C and m6A modifications of LncRNA NKILA have been found to facilitate cholangiocarcinoma growth and metastasis through the miR-582-3p-YAP1 axis [5C methylation in lncRNAs played an important role in the regulation of type I interferons and antiviral innate immunity [In RNA metabolism, mtability , nucleartability  and prottability  of celluncer RNA  and lncRncer RNA  can incrAP1 axis . Recentlimmunity .5C modification in lncRNAs was affected by H1N1 IAV infection. The results obtained in A549 cells showed that the m5C modification sites and distribution on lncRNAs were globally altered after IAV infection. A total of 1317 upregulated and 1667 downregulated m5C peaks were detected upon H1N1 infection. GO analysis showed that the genes targeted by the m5C differentially modified (m5C-DM) lncRNAs in IAV infection were notably enriched in biological processes of calcium-dependent cell-cell adhesion via plasma membrane cell adhesion molecules, protein deubiquitination, nuclear export and organelle localization. KEGG pathway analyses suggested that these m5C-DM lncRNAs were mainly associated with metabolism  and some infectious pathogens related pathways which were known to be important for virus infections. These data indicated that m5C modification on lncRNAs could play important roles in the host responses to IAV infection.By MeRIP-Seq, we studied how the m5C methylation in the regulation of lncRNAs expression and type I interferon production [5C modifications may be involved in multiple pathways to regulate IAV replication by modulating the expression and/or stability of lncRNAs.By conjoint analysis of the epitranscriptomic profile and expression profile, we identified 143 \u2018hyper-up\u2019, 4 \u2018hyper-down\u2019, 81 \u2018hypo-up\u2019, and 6 \u2018hypo-down\u2019 lncRNAs. GO analysis highlighted the enrichment of target genes of DM and DE lncRNAs in nucleosome assembly, and regulation of type I interferon production pathway which is consistent with the recent report of the involvement of moduction . KEGG pa5C modification with IAV replication. It remains elusive which of these DE and DM lncRNAs identified are common for IAV infection and which of them specifically respond to H1N1 infection in A549 cells. Furthermore, the samples subjected to MeRIP-seq analyses were harvested at 36 hpi. Part of the DE and DM lncRNAs could be indirectly regulated by the cellular factors altered in early infection. Validation of the altered expression and modification of lncRNAs in early infection could be helpful for screening out the key players in IAV infection. Further studies are also needed to answer some key questions on how IAV induces the differential modification level and distribution of m5C, what are the roles of the modifications in the regulation of lncRNA expression and function, and what are the functions of m5C modification of lncRNAs in IAV replication and pathogenesis.Different influenza viruses may influence the host cells in altered pathways or in different degrees. The same virus may also behave differently in varied cells. Our data in this study demonstrated the association of the expression and m5C, and the modification plays positive roles in ribosomal recruitment and RNA splicing to benefit viral gene expression and virus replication [5C modification as well as HIV-1 [6A modifications on its mRNAs and vRNAs have been mapped. The modification has been shown to be able to increase viral RNA expression, and IAV HA m6A mutants show reduced pathogenicity in mice [5C sites on IAV transcripts and determine whether the modifications are involved in virus replication and pathogenesis.Recent studies have revealed that the viral genomic and messenger RNAs of retroviruses including HIV-1 and Murine Leukemia Virus are heavily modified with mlication , 48. Posas HIV-1 . For IAV in mice . It will5C modification upon IAV infection, suggesting a link between lncRNA m5C modification and IAV infection. The data obtained in this study could provide new insights into our understanding of virus-host interactions in influenza virus infection, and prompt further studies to explore the potential of lncRNAs as diagnostic markers and therapeutic targets for the virus infection caused diseases.In conclusion, we performed a transcriptome-wide 5-methylcytosine modification analysis of lncRNAs in A549 cells infected with influenza A virus, and demonstrated the significant alteration of m2. A549 cells were seeded in 100\u00a0mm cell culture dishes. When the confluence reached 80\u201390%, the cells were infected with influenza A virus strain A/WSN/33 at an MOI of 0.1, incubated at 37\u2103 for 2\u00a0h, and then the media was replaced with F-12\u00a0K supplemented with 1% bovine serum albumin , antibiotics and TPCK-treated Trypsin (Sigma). Cell samples were harvested at 36 hpi for subsequent total RNA extraction. All samples are analyzed in triplicate.Human alveolar basal epithelial adenocarcinoma cell line (A549) was purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Influenza virus isolate A/WSN/33 (H1N1) was donated by Dr. Long Liu. A549 cells were cultured in Ham\u2019s F-12\u00a0K  containing 10% fetal bovine serum , 100 U/ml penicillin and 100\u00a0\u00b5g/ml streptomycin (Sangon Biotech) at 37\u2103 in 5% CO260/OD280, and the samples with the value between 1.8 and 2.1 were marked as qualified  following the manufacturer\u2019s instructions. Denaturing agarose gel electrophoresis was used to measure RNA integrity and gDNA contamination  by following the manufacturer\u2019s instructions. Briefly, fragmented RNA was incubated with anti-m5C polyclonal antibody  in IPP buffer for 1\u00a0h at 4\u2103. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher) at 4\u2103 for an additional 2\u00a0h. Then, bound RNA was eluted from the beads with Proteinase K for 30\u00a0min at 55\u2103, and purified by RNA clean&concentrantorTM-5 (ZYMO Research). RNA-seq libraries for the input and IP RNA samples were generated using NEBNext\u00ae Ultra II Directional RNA Library Prep Kit (New England Biolabs). The library quality was evaluated with BioAnalyzer 2100 system (Agilent Technologies) . Briefly, rRNAs were removed from the total RNA with NEBNext\u00ae rRNA Depletion Kit . MeRIP-Seq and RNA-Seq were performed by DIATREBiotech Inc. . Immunoprecipitation of mQuality control of the paired-end reads was performed with Q30, which was followed by trimming of the 3\u2019 adaptor and removal of low-quality reads using Cutadapt software (v1.9.3) . After tWithin the annotated transcripts, lncRNAs were filtered by the classcodes of x (exonic overlap with reference on the opposite strand), u (intergenic transcripts), i (transcripts entirely within intron), j (at least one splice junction is shared with a reference transcript), and o (other same strand overlap with reference exons). Transcripts with length\u2009>\u2009200\u00a0bp and exon number\u2009>\u20092 were maintained. The coding potential of these transcripts was predicted by CNCI (v2) , CPC2 5, CPAT  were identified by MACS2 software [5C peaks with a log2FC\u2009>\u20091  in infected cells were considered to be up-regulated, and those with log2FC < -1 were down-regulated.After transcriptome assembly, annotated lncRNA transcripts were used for msoftware . Differesoftware : m5C pea5C peaks on chromosome was analyzed by R package Circlize [5C peaks on lncRNAs [5C peak regions. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) [https://ngdc.cncb.ac.cn/lncbook/home) [http://mirwalk.umm.uni-heidelberg.de/) [The distribution of lncRNA mCirclize . The norCirclize . Guitar  lncRNAs . The rea lncRNAs . HOMER ss (KEGG)  analysess (KEGG) . The intok/home) , and theerg.de/) . The lnc\u2212\u0394\u0394Ct method. All samples were analyzed in triplicate. The primer sequences used in the qPCR are listed in Table\u00a0Total RNAs from H1N1-infected and uninfected A549 cells were used to synthesize cDNA using the HiFiScript gDNA Removal cDNA Synthesis Kit . Quantitative Real-time PCR (qPCR) was carried out using ChamQ Universal SYBR qPCR Master Mix  according to the manufacturer\u2019s instructions. GAPDH was used as a normalization control. The relative expression level of each lncRNA was calculated with the 2Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4"}
+{"text": "The glenohumeral joint dislocation can be associated with major nerve injury. The reported prevalence and risk factors for major nerve injury are variable and this injury can have a severe and life-long impact on the patient. The objectives of this study were to analyse the prevalence of major nerve injury following shoulder dislocation and examine risk factors. Management and outcomes of nerve injury were explored.A 1\u00a0year retrospective cohort study of 243 consecutive adults who presented with a shoulder dislocation was performed. Data were collected on patient demographics, timings of investigations, treatment, follow-up, and nerve injury prevalence and management. The primary outcome measure was prevalence of nerve injury. Risk factors for this were analysed using appropriate tests with Stata SE15.1.p\u2009=\u20090.004) and older age (p\u2009=\u20090.02) were significantly associated with major nerve injury. Sex, time to successful reduction and force of injury were not associated with major nerve injury in this cohort. Patients with nerve injury made functional recovery to varying degrees. Recurrent shoulder dislocation was common accounting for 133/243 (55%) attendances.Of 243 patients with shoulder dislocation, 14 (6%) had neurological deficit. Primary dislocation (Shoulder dislocation requires careful assessment and timely management in the ED. A 6% rate of nerve injury following shoulder dislocation was at the lower border of reported rates (5\u201355%), and primary dislocation and older age were identified as risk factors for nerve injury. We emphasise the importance of referring patients with suspected major nerve injury to specialist services. The shoulder is the most frequently dislocated major joint, with an incidence of 40.4 per 100,000 and 15.5 per 100,000 in men and women respectively  with sevOur primary objectives were to analyse the incidence of nerve injury following shoulder dislocation and identify risk factors for this, in patients presenting to the emergency department (ED) of a single major trauma centre. Risk factors of interest included those relating to standard of care, injury related factors and intrinsic patient factors. Our secondary objectives were to explore the management and outcomes of patients sustaining nerve injury following shoulder dislocation.This was a retrospective cohort study of consecutive patients who presented to the ED with suspected shoulder dislocation, between 1st March 2016 and 28th February 2017. Local approval for the work was given, formal ethical review was not required as per our institution\u2019s guidelines.The study included adults (over 18\u00a0years of age) at presentation with a shoulder dislocation that required reduction. Patients were identified using clinical coding for \u2018dislocated shoulder\u2019. Exclusion criteria included; subluxations that did not require reduction, dislocations that had relocated before arrival, incorrect coding and no case notes available.All data was sourced from electronic health records . We captured the time of injury; time of arrival to ED; time of first radiograph; time of analgesia; pain score (out of 10) at baseline; time from arrival to successful reduction; force of injury and neurovascular status of affected limb. Successful reduction time was taken as the documented time of reduction attempt if there was a post-reduction radiograph demonstrating a relocated shoulder. If time of reduction attempt was not documented the time of successful post-reduction radiograph was used.Force of injury was defined as either high energy or low energy based upon our department major trauma triage guidance. High energy mechanisms were defined as: pedestrian versus car incidents; all falls from greater than 1\u00a0m or 5 stairs (including found at bottom of stairs); high speed road traffic accidents\u2009>\u200960 miles per hour; ejections from vehicle; all vehicle roll-overs; death in same vehicle; all road traffic accidents involving cyclists or motorcyclists; all incidents involving horses; multiple areas of injury with concerns that the patient has polytrauma. All other mechanisms were defined as low energy. A successful first reduction (yes/no) was defined as an anatomically reduced shoulder on radiograph after one documented reduction attempt. The assessment of neurovascular status in the department would comprise of evaluation of the sensory/motor function and the perfusion of the upper limb.Data were analysed using StataSE v15.1 . Wilcoxon\u2009=\u200990), incorrect coding (n\u2009=\u200917), inaccessible data (n\u2009=\u20096). The demographic data are shown in Table There were 357 patients in the initial list coded with a diagnosis of a dislocated shoulder. Of these patients 243 met the inclusion criteria. Reasons for exclusion were: subluxation only . The incidence was higher in males and on the right. One patient had 3 episodes of bilateral dislocations due to seizures. Low-energy injuries were more common in this cohort. For patients presenting with recurrent dislocation 64 attended once over the year, 10 attended twice, one attended three times, two attended four times, one attended five times and one patient attended 19 times. The patient attending 19 times had poorly controlled seizures which caused them to dislocate their shoulders.p\u2009<\u20090.001. Those with recurrent dislocation were on average 18\u00a0years younger. Low-energy injury was more frequent with recurrent dislocation 118/133 (89%) compared to primary dislocation 85/110 .Patients with recurrent dislocation were younger  compared to primary dislocations , p\u2009=\u2009\u2009<\u20090.001. Patients with recurrent dislocation were on average 15\u00a0years younger. There was no association between the force of the injury and recurrent dislocations (primary dislocation 85/110 (77%) low energy versus 78/93 (84%) recurrent dislocations, p\u2009=\u20090.24).Following removal of multiple attendances (40 attendances dropped) patients with recurrent dislocation were still significantly younger  compared to primary dislocation , 218/243 (90%) patients were assessed for neurovascular status. 14/218 patients (6%) had a neurological deficit, these patients are detailed in Table p\u2009=\u20090.02) whereby the odds of sustaining a major nerve injury increased by 20% with each decade of life . Patient sex, and time from arrival to reduction were not significantly associated with major nerve injury. 9/14 (64%) patients with major nerve injury had their dislocation reduced within 2\u00a0hours. Two patients waited over 2\u00a0hours to have their dislocated shoulder reduced and three patients waited over 3\u00a0hours. The odds of sustaining a major nerve injury were sixfold higher in patients who sustained their first dislocation (OR 5.94 [95% CI 1.50\u201323.46] p\u2009=\u20090.01), compared to those with recurrent dislocations. Force of injury was not associated with major nerve injury, and there was a slightly higher rate compared to those without . Following removal of multiple attendances (35 attendances dropped) older age was still significantly associated with major nerve injury (p\u2009=\u20090.05), as was first time dislocation (p\u2009=\u20090.02).Risk factors for major nerve injury were examined Table . Older ap\u2009=\u20090.70). 169 patients (70%) had a shoulder radiograph within 60\u00a0min of arrival. 141 patients (60%) had a first reduction attempted within 2\u00a0hours of arrival, and 192 (80%) had first attempted reduction within 3\u00a0hours. The median time from arrival in the ED to successful reduction was 97\u00a0min (range 15\u2013406). 225 patients (93%) had a post-reduction radiograph performed. Four patients self-discharged prior to post-reduction radiograph but it is not clear why the fourteen others did not have this performed. In total 233 patients (96%) were booked for follow up: 197 (85%) in fracture clinic, 14 (6%) in other orthopaedic clinics e.g. already under upper limb surgeons, 8 (3%) with both fracture clinic and major nerve clinic follow up, and 15 (6%) had other arrangements. Three patients self-discharged.115/140 (82%) patients describing moderate or severe pain (pain score 4\u201310/10) were offered analgesia within 30\u00a0min of arrival and 134/140 (96%) had been offered by 60\u00a0min. Time to successful reduction was not associated with time to pain relief . This has clinical and medicolegal consequences  as neuroOf the 14 patients suspected of having neurological deficit following dislocation, nine were referred to a specialist major nerve injury clinic. Although the majority of patients were managed nonoperatively, input from the specialist therapy team is recommended . We founOlder age was identified as a significant risk factor, as has been demonstrated by other authors . The litIn terms of management of nerve injury following shoulder dislocation we advise that all suspected nerve injuries are referred to a specialist major nerve service, ideally from the emergency department. Low energy injuries can usually be managed conservatively in the first instance with physiotherapy, but may require nerve conduction studies if adequate recovery is not demonstrated at three weeks. If required, nerve exploration and reconstruction can be undertaken within 3\u00a0months and no later than 6\u00a0months. High energy injuries carry a higher suspicion of serious nerve injury hence should have a lower threshold for early nerve exploration (days to weeks).The limitations of this study were that it was retrospective with some incomplete data and the number of patients sustaining major nerve injury is small. There were some bilateral cases and repeat attendances meaning that there is dependency in our data which has not been accounted for. The time taken for the first reduction attempt was variable and delayed in some cases, which may affect the applicability of this study. Please contact authors if data is required.Shoulder dislocation requires immediate assessment and timely management in the ED. Nerve injury was identified in 6% of patients presenting with shoulder dislocation which is consistent with current literature. We also report the associations between older age and primary dislocation with major nerve injury following shoulder dislocation. The importance of performing and documenting a comprehensive neurovascular assessment for shoulder dislocation due to the risk of neurological injury is emphasised, as well as the need to refer patients with nerve injury to specialist services."}
+{"text": "We sought to compare risk factors contributing to unintentional, homicide, and suicide firearm deaths in children. We conducted a retrospective review of the National Fatality Review Case Reporting System. We included all firearm deaths among children aged 0\u201318 years occurring from 2007 to 2016. Descriptive analyses were performed on demographic, psychosocial, and firearm characteristics and their relationship to unintentional, homicide, and suicide deaths. Regression analyses were used to compare factors contributing to unintentional vs. intentional deaths. There were 6148 firearm deaths during the study period. The mean age was 14 years (SD \u00b1 4 years), of which 81% were male and 41% were non-Hispanic White. The most common manners of death were homicide (57%), suicide (36%), and unintentional (7%). Over one-third of firearms were stored unlocked. Homicide deaths had a higher likelihood of occurring outside of the home setting  compared with unintentional deaths. Suicide deaths had a higher likelihood of occurring in homes with firearms that were stored locked  compared with unintentional deaths. Each manner of firearm death presents a unique set of psychosocial circumstances and challenges for preventive strategies. Unsafe firearm storage practices remain a central theme in contributing to the increased risk of youth firearm deaths. Firearm injury affecting youth is a largely preventable public health crisis in the United States. It represents the leading cause of death in children with the highest case-fatality rate among pediatric trauma-related injuries . FirearmThe US is an outlier when compared to other high-income countries when comparing firearm deaths. Among World Health Organization data for high-income countries, 96.7% of all children aged 0\u20134 years who died by firearm in 2015 were from the US . In one The American Academy of Pediatrics has stated that the most effective means to prevent any type of firearm injury among children is to reduce the presence of guns in homes and communities . DespiteInformation is limited regarding risk factors for firearm deaths in children, especially information related to individual, familial, and psychosocial factors ,18,19,20The purpose of this study is to compare circumstances contributing to unintentional, suicidal, and homicidal firearm deaths in children. In particular, we sought to understand how risk factors related to unintentional deaths differ from intentional homicide and suicide deaths. Unintentional firearm deaths are less common than suicide or homicide deaths but are often viewed as preventable ,19,27. TWe performed a secondary analysis of previously collected data from the National Fatality Review Case Reporting System (NFR-CRS). We obtained de-identified data on all firearm-related deaths among children aged 0\u201318 years that occurred during the 10-year period of 2007\u20132016. We included all firearm-related deaths where the manner of death was homicide, suicide, or unintentional. Deaths with manner documented as natural, undetermined, pending, or unknown were excluded. Suicide deaths under the age of 7 years were excluded due to concern for misclassification, the rarity of suicide, and the lack of routine universal screening for mental health risk factors in this age group . We alsoThere are over 2600 variables available in the NFR-CRS. The variables for this study were first selected based on relevance to our study population using a comprehensive literature review ,26. We tData for this analysis were obtained from the NFR-CRS, a web-based system that records and standardizes information from Child Death Review (CDR) team meetings . The CDRDescriptive analyses based on counts (proportion), mean (SD), or median (IQR) were first performed on the decedents\u2019 risk factors grouped by the manner of death. These were followed by two separate logistic regression analyses conducted to identify predictors that differed between each intentional group  and the unintentional comparison group, expressing the strength of association as odds ratios (ORs) and 95% confidence intervals (95% CIs). Covariates had varying degrees of missingness due to unknown responses or underreporting from data submitted by individual CDR teams. A multiple imputation (MI) analysis was performed to estimate missing values based on covariate profiles under the missing at-random assumption within each group using the MI package in Stata 16 . Due to During our 10-year study period, there were a total of 6148 firearm deaths in children, and 6024 met the inclusion criteria for this study . DemograHandguns (64%) were the most common firearm used, followed by shotguns (11%) and hunting rifles (6%). Large proportions (57\u201377%) of licensing and safety variables were missing, but still, a higher proportion of unintentional deaths (21%) involved unlicensed firearms compared to homicide (14%) and suicide (12%) deaths. When reported, most firearms were without safety features, stored unlocked in all groups (35%), with the highest prevalence among the unintentional group (70%). Firearms stored loaded and with ammunition were especially common in unintentional deaths followed by suicides.Demographic characteristics in homicide and suicide deaths vs. unintentional deaths are shown in In subsequent analyses, results adjusted for demographic differences were presented alongside unadjusted comparisons using multiple imputed and raw data. In evaluating the relationships, the adjusted results based on imputed data were given priority. In In The NFR-CRS results affirm our understanding of the individual and familial-level factors contributing to firearm deaths in children, especially that certain psychosocial, and environmental circumstances predispose to each manner of firearm death. Homicide deaths are more likely to occur in an urban setting, at public locations, and among racial/ethnic minorities. In contrast, firearms in homes play a central role in unintentional and suicide deaths, especially firearms stored unlocked in unintentional firearm deaths. These results underscore the enormous risk of having firearms in households with children.Although the NFR-CRS offers only a snapshot of deaths in the study period, it contains fairly representative data when compared to the Web-based Injury Statistics and Query Reporting System (WISQARS). The WISQARS had 20 045 firearm deaths (vs. 6024 in the NFR-CRS) in all 50 states during the study period, with similar compositions of homicide (62% vs. 57% in NFR-CRS), suicide (32% vs. 36%), and unintentional deaths (5% vs. 7%). Similarly, homicides were most common in NH-Black children (60% vs. 56% in NFR-CRS), while suicides occurred most often in NH-White children (78% vs. 75%) [Consistent with prior studies, unintentional firearm deaths in children are more common in younger White males and occur primarily in the home setting ,27,33,34Our results are consistent with prior studies reporting that the majority of homicidal deaths occur in an urban setting among Black males with an average age of 14 years ,33,35. USuicide victims were far more likely to have died from firearms that belonged to the decedent or a family member. Approximately one-quarter of decedents in suicide deaths had preceding mental health concerns. This further elucidates the importance of safe firearm storage to be universally applied, as only a subset of suicide decedents had reported mental health diagnoses at their CDR team assessment. Lethal means screening and counseling are important tasks in the healthcare setting, but knowledge gaps still remain regarding the best approach for lethal means counseling and efficacy ,26.A comprehensive preventive strategy is essential for reducing the medical and public health burden of firearm injuries in the US. Many preventive strategies have focused on universal screening and education-based interventions in healthcare settings. The effectiveness of this \u201cone size fits all\u201d approach has been debated, especially because of its limited impact on reducing firearm injuries ,39,40,41There are several potential limitations that may impact our study. Not all states participate in the NFR-CRS, not all participating states review all child deaths, and some states do not make their data available for research purposes. Consequently, mortality rates cannot be calculated using these omitted data. Some NFR-CRS variables have a high proportion of missing data. To account for this, we estimated missing values using multiple imputation. The lack of substantial differences between imputed and non-imputed data is consistent with the missing at-random assumption and supports the adequacy of the data for drawing unbiased effect estimates. Key variables with very large proportions of missing data were not imputed and were excluded from further analyses. Data from 34 states met the inclusion criteria for this study, and they may not be fully representative of all firearm fatalities among children in the US. Despite these limitations, the breadth of information and level of detail available in the NFR-CRS for all manners of death is not available in any other mortality data. This provides a unique opportunity to identify potential risk and protective factors important for future study of firearm fatalities among youth and may inform development of prevention strategies.The NFR-CRS highlights the complexities affecting firearm deaths in children. Each manner of firearm death is accompanied by unique circumstances that need to be understood and addressed with preventive efforts. Homicide deaths were more likely among racial ethnic/racial minorities in urban environments, highlighting the importance of further research on the effectiveness of community-based violence interventions. After adjusting for age, race/ethnicity, year of death, and urban/rural setting, the risk factor profiles in young firearm decedents were found to differ between intentional (homicide and suicide) and unintentional outcomes in ways that potentially help identify persons at risk. Firearm homicides are more likely than unintentional deaths to involve documented victims of maltreatment that may not be supported by open CPS cases. Suicide and unintentional deaths tend to have similar risk factor profiles; both were more likely to occur in the home environment predominantly with firearms that originate in the home. Unlocked firearms were far more likely among unintentional deaths, emphasizing the need for increased primary prevention and motivation for caregivers to improve home firearm safety. The absence of a risk difference between the mechanism underlying firearm death does not rule out the importance of a potential risk factor between victims and comparable non-victims. In spite of many challenges, it is important to take every opportunity to identify risk factors, evaluate the impact of targeted preventive strategies, and allocate resources to the most vulnerable households and communities in order to mitigate the burden of this public health crisis."
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