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+{"text": "Rhizopus oryzae glucoamylase (RoGA) consists of three domains: an amino (N)-terminal raw starch-binding domain (SBD), a glycosylated linker domain, and a carboxy (C)-terminal catalytic domain. The 36-amino-acid linker region (residues 132\u2013167) connects the two functional domains, but its structural and functional roles are unclear.RoGA and its involvement in protein expression, a number of RoGA variants containing deletions and mutations were constructed and expressed in Saccharomyces cerevisiae. Deletion analyses demonstrate that the linker region, especially within residues 161 to 167, is required for protein expression. In addition, site-directed mutagenesis and deglycosylation studies reveal that the linker region of RoGA contains both N- and O-linked carbohydrate moieties, and the N-linked oligosaccharides play a major role in the formation of active enzyme. Although the linker segment itself appears to have no ordered secondary structural conformation, the flexible region indeed contributes to the stabilization of functional N- and C-terminal domains.To characterize the linker sequences of RoGA.Our data provide direct evidence that the length, composition, and glycosylation of the interdomain linker play a central role in the structure and function of  Aspergillus niger  and Rhizopus oryzae (RoGA) because of their stability and high activity m.r.w., calculated by the equation: [\u03b8]m.r.w. = (100\u03b8obs)/(nlc), where \u03b8obs is the observed ellipticity in degrees, n is the number of amino acids, l is the length of the light path in centimeters, and c is the molar concentration of the protein.Circular dichroism measurements were carried out with an AVIV model 202 spectropolarimeter . Far-UV wavelength scans were recorded from 200 to 260 nm with a bandwidth of 1.0 nm, using a 0.1-cm path length cuvette at 25\u00b0C. Each spectrum was an average of three consecutive scans and was corrected by subtracting the buffer spectrum. Thermal denaturation experiments were performed by increasing the temperature from 25 to 96\u00b0C, allowing temperature equilibration for 1.5 min before recording each spectrum. All of the data are expressed in terms of mean residue ellipticity, [An GA, Aspergillus niger glucoamylase; Ro GA, Rhizopus oryzae glucoamylase; CBM, carbohydrate-binding module; SBD, starch-binding domainSCL designed and generated most of the mutants, carried out most functional assays, and drafted the manuscript; WTL expressed and purified recombinant SBDs. SHL involved initial cloning and enzymatic assay designs; WIC participated in the structural determination employing spectroscopic methodologies; BKH assisted recombinant yeast generation and growth. IPL participated in manuscript preparation. CCS participated in experimental design and coordination. MDTC conceived and supervised this project and manuscript writing. All authors read and approved the final manuscript."}
+{"text": "Network reconstruction methods that rely on covariance of expression of transcription regulators and their targets ignore the fact that transcription of regulators and their targets can be controlled differently and/or independently. Such oversight would result in many erroneous predictions. However, accurate prediction of gene regulatory interactions can be made possible through modeling and estimation of transcriptional activity of groups of co-regulated genes.Escherichia coli (E. coli). The network is updated via a covariance model describing the activity of gene sets controlled by common regulators. The proposed model-selection algorithm was used to annotate the likeliest regulatory interactions in E. coli on the basis of two independent sets of expression data, each containing many microarray experiments under a variety of conditions. The key regulatory predictions have been verified by an experiment and literature survey. In addition, the estimated activity profiles of transcription factors were used to describe their responses to environmental and genetic perturbations as well as drug treatments.Incomplete regulatory connectivity and expression data are used here to construct a consensus network of transcriptional regulation in Information about transcriptional activity of documented co-regulated genes (a core regulon) should be sufficient for discovering new target genes, whose transcriptional activities significantly co-vary with the activity of the core regulon members. Our ability to derive a highly significant consensus network by applying the regulon-based approach to two very different data sets demonstrated the efficiency of this strategy. We believe that this approach can be used to reconstruct gene regulatory networks of other organisms for which partial sets of known interactions are available. One of the goals of systems biology is to elucidate functionally relevant regulatory interactions,2. SinceTo overcome these limitations more complex statistical and mathematical models have been proposed, which are briefly summarized below and in Table Yet another approach to inferring gene regulatory networks is through non-greedy decomposition of gene expression data matrices to uncover hidden, often overlapping, regulatory signals and transcriptional connectivity patterns. Since the data does not have to be a time series, one can collect data for as many different experiments as possible and combine them to increase the sample size and prevent the problem of under-determination. Principal component analysis (PCA)  or singuIn this paper we introduce a new and highly efficient approach to the problem of gene network inference through a simple model selection algorithm. The algorithm uses gene expression data and documented transcriptional connections to predict a more complete structure of the transcriptional network. The method is based on covariance models of the co-regulated gene sets. We demonstrate that this approach can uncover previously uncharacterized regulatory interactions and simultaneously estimate the activity profiles of regulators from the corresponding covariance matrices. Comparison with the relevance network and GGM algorithms on two different data sets demonstrated the advantage of using the \"gene\"-\"regulon\" associations (the present method) over the \"gene\"-\"regulator\" associations (relevance networks and GGM). The proposed method outperformed both algorithms on both data sets at a very high significance margin with a recall value of more than 62% and precision value of 64%. We confirmed some of the predicted interactions by experiment and literature mining.E. coli. The conditions covered a spectrum of environmental and genetic perturbations and drug treatments. The environmental perturbations, in addition to those described in [Microarray gene expression data for more than 100 arrays representing 46 biologically distinct conditions have been used to reconstruct the underlying large scale transcriptional regulatory network of ribed in  (Data seMD3) web site . This set contained expression levels of E. coli genes across 524 arrays which resolved into 189 different experimental conditions. It should be noted here that not only do these two data sets cover very different genetic or environmental perturbations, but they also have been collected on two different microarray platforms: cDNA microarrays and Affymatrix Genechips.A second data set used in our study was published in  and was [The connectivity matrix obtained from RegulonDB . This coK genes controlled by a TF are treated as K realizations of an independent random variable with the same distribution and, therefore, the weighted sample covariance matrix estimation method was applied to approximate the TF's model covariance matrix. Higher weight is given to those gene samples that are exclusively controlled by the TF. The bootstrap procedure is incorporated to increase the estimation accuracy of covariance model for those TFs with the low number of known targets. Then the algorithm computes the Gene-Regulon association score for each gene-regulon pair using the likelihood function defined in the Method Section. Finally, the activity profiles of transcription factors are estimated using the eigenvalue decomposition procedure. The regulon-based methodology assumes that the expressions of target genes vary with the activity of their regulator, which does not have to be determined solely by its transcript levels but can be a combination of latent factors including abundance, modification status, interaction with low molecular weight effectors or other proteins.For each transcription factor, the core regulon, consisting of the set of known targets, is identified from the known connectivity matrix described above. This information is used to learn a covariance model for each transcription factor. The covariance matrices are estimated from the expression data of genes assigned to regulons. The gene expression measurements of the group of To assess the relative performance of our algorithm, we compared our algorithm with the relevance network method developed in , and witSince we were interested in transcriptional regulatory interactions (interactions between transcription factors and their target genes), we built a network by comparing scores for all possible pairs of transcription factor-gene targets. To make a fair comparison, in each method for each gene we ranked the regulators based on their association scores with that gene. A regulator which has the maximum association score with a gene was assigned to that gene. The second regulator was assigned to the gene if the corresponding association score was greater than the minimum of the association scores of the genes assigned to that regulator in the first round. This procedure was repeated and assignments were made, if warranted by the association score, for the lower ranking regulators as well. This procedure is different from those that use a global threshold parameter to select the edges. Assignment of regulators based on a global threshold for all TFs results in a very limited number of predictions, although with a good precision. Due to the large scale of the data, it is reasonable to assume that there should be at least one regulator that can explain the gene expression data of each gene. Although, this association may not be discovered through the dependency between the expression level of the gene and the mRNA level of a gene coding for the transcription factor, it may be discovered using gene-regulon based association, and this is what we would like to demonstrate.We compared the prediction results of these three algorithms over the set of known interactions in RegulonDB database , which wThe results for two data sets and three algorithms is presented in Table The second reason for outperforming the relevance network algorithm is the capacity of our method to capture condition-specific activity and the co-variance of the gene expression profiles across different conditions. On the other hand, the relevance network algorithm treats gene expression levels across different conditions as ensembles of single random variables to estimate the probability distribution for each gene, and therefore it can not account for condition-specific activity of the genes when the sample size is not large enough. See [Additional file We applied the proposed method to a large-scale gene expression data set to evaluate its capacity to recover known regulatory interactions and predict new ones. We ignored any TF assignments with a very small number (< 5) of known interactions. We assigned to each gene a regulator based on the maximum probability of association calculated using the proposed algorithm presented in the methods section. When we limited the number of associations for each gene to one, the algorithm was able to recover the correct association for 86% of the genes with known interaction, i.e., 1044 genes out of 1210 genes were associated with their known regulators. When two regulators were assigned to genes, an additional 542 known interactions were recovered, which included known interactions for 26 genes that were not in the set of 1044. That increased the percentage of genes with correct associations to 88%, and this implies that for 74% of genes, i.e. for 516 out of 696 genes having two or more regulators, both predicted regulators were correct.hisJ and artJ, which were not among known interactions in the RegulonDB database, but have been recently reported in the literature [In addition to recovering known interactions, the algorithm discovered new, un-annotated interactions. Some of the discovered interactions could be independently confirmed. For example, the algorithm predicted two targets of ArgR, terature . For thiompT, dppA, eco, pntA, pntB, csiE, sdaB sdaC, yhjE and ygdH, most of which were also verified using a qPCR experiment , on average 13 TFs were active per condition in our data set (11 \u2013 in the Affymetrix data set). The distribution of the number of active TFs across the conditions is shown in Fig. Not only did we find that more than one TF appeared to be active in any given condition, but also that many TFs were likely to be mediating transcriptional responses in multiple conditions Fig. . Given tThe correlation analysis of TFs activity profiles revealed that the activity profiles of almost half of the transcription factors considered in this study are correlated with one another. Some global regulators such as CRP, IHF, FNR were correlated with more than 10 TFs. However, some local regulators, such as OmpR, PhoP, and EnvY, also showed a high degree of correlation with other TFs. Figure S1 [see Additional file However several transcription factors, including LexA, GcvA, SoxR, DsdC and FadR, did not show high degree of correlation with other TFs, even though they were relatively responsive in a high number of conditions in the study.Based on the two expression data sets, covering regulatory states for almost all genes in the genome, each gene (operon) was assigned a transcription factor(s) that was likely to control the expression of that gene. To this end, the present algorithm was applied to both data sets. For each data set, every gene was assigned three top-ranking regulators with the highest probabilities of association, calculated using equation 3). If a gene had the same regulator(s) predicted from both sets, the regulatory association between the gene and regulator(s) was deemed true and cataloged for the purpose of network refinement. This resulted in a list of 1719 genes associated with at least one regulator [see Additional file . If a gedinI and dinP are known targets of LexA, but not among the known interactions set used in this study. Also, yafNOP, yebB, yebG, yigN, and yjjB-dnaTC-yjjA were predicted to be LexA targets. yafNOP is a neighboring operon of dinB with a weak but significant score for a LexA binding site in its promoter region. Regulatory regions of yebG, yigN and yebB contain high scoring LexA binding sites. The possibility that dnaT and dnaC, two genes involved in DNA replication, are under LexA control is intriguing, and warrants further experimentation.We incorporated the operon information to further refine the set of regulatory interactions. The consensus regulator for an operon was chosen as a common regulator of genes in the same operon, as predicted on the basis of both data sets. This resulted in a list of potential regulator(s) for each operon [see Additional file Overall, even though the two data compendia appeared to be substantially different as far as dominant activity profiles are concerned, transcriptional profiles of as many as 1407 genes and 773 multigene operons could be explained at least in part by the activity of the same regulator(s) in both data sets. This result implies that, provided a sufficiently diverse collection of experimental conditions, the method will converge on true transcriptional regulators of any given gene in a genome, including regulators themselves [see Additional files Genome-wide transcriptional data allow for systematic analysis of regulatory patterns of gene expression. These patterns are at least in part determined by interactions between transcription factors and their cognate target genes. Accurate prediction of such interactions, which is essential for understanding phenotypic outcomes of genetic and environmental perturbations, depends on the quality of models capturing essential regulatory features and on their underlying assumptions. One such feature is that the transcriptional activity of co-regulated genes should sufficiently absorb in itself the activity of their common regulator. Moreover, the information about transcriptional activity of the known co-regulated genes (a core regulon) should also be sufficient for discovering new target genes, whose transcriptional activity significantly co-vary with the activity of the core regulon members.de novo interactivity matrix, which can be used in combination with gene expression data to construct a reliable gene regulatory network by means of these supervised learning methods.We introduced an effective approach to predict interactions between regulators and genes through a simple model selection algorithm. The algorithm takes advantage of both gene expression data and the knowledge of known gene-TF associations to simultaneously discover new interactions between regulators and genes and to estimate the activity profiles of the regulators. The proposed approach, unlike other methods, associates the expression of genes in a regulon with the activity of transcription factors rather than with the expression levels of genes encoding for the transcription factors. We demonstrated that incorporation of information about the activity profiles of transcription factors allows for a reliable identification of many known as well as previously uncharacterized regulatory interactions, which could not be achieved by methods solely relying on gene expression associations. We should mention that the power of a regulon-based association framework presented in this paper and of the supervised inference method  relies oWe consider the gene regulation process as an input-output model where the transcription rate (output product) is controlled through the activity level of the group of specialized transcription factors. We assume, given the activity of regulators, that this process is approximately linear, although the dynamic behavior of this biological process can be much more complex. In the linear modele represents a vector of gene expression measurements across m conditions, S is a m \u00d7 k matrix of the k regulators' activities and c is a sparse vector that represents the interaction between the gene and regulators. We assume S and c are both unknown and \u03f5 is an m elements vector of random noise with zero mean and covariance matrix of \u03c32I.c, sparse. Furthermore, the direct measurement of the regulators' activity is a challenging and expensive, if not an impossible, task. Therefore, one does not have access to S, the activity profiles of regulators. The objective is to estimate the activity profiles of the regulators that control transcription (initiation) of groups of genes. At the same time, we also wish to provide for each gene the set of regulators that can explain the observed gene expression data.The fact that the expression level of genes is controlled by only a few regulatory nodes makes the resulting network, and therefore f be a set consisting of the expression vectors of genes known to be controlled by a single regulator f. Then,Here, we present an efficient algorithm, which takes advantage of known interaction among genes and regulators. Let \u03a9sf, the activity profile of the regulator f, is unknown and cf is the interaction coefficient, which is assumed to be a random variable with distribution \u03c5, \u03b32). The likelihood function for sf [where n for sf  is,wheresf, maximizes p(eg|sf). On the other hand, to compute p(eg|sf) the regulatory signals need not be known explicitly and the knowledge of the sample mean (\u03bcf) and the covariance matrix (\u03a3f) corresponding to each set \u03a9f is sufficient for model selection. Therefore, one only needs to estimate these sample mean and covariance matrices from the data. The covariance matrix \u03a3f, which is the covariance matrix of gene expression data in \u03a9f, can be estimated using sample covariance matrix. Assume Ef to be the expression measurements of genes in \u03a9f, then one can estimate the weighted covariance matrix corresponding to regulator f by,Among the set of all regulators, the correct regulator, Nf is the number of samples in \u03a9f, \u03bci and \u03bcj are ith and jth entries of sample mean, \u03bcf and wk's are weights which sum to one.where f's as sets of genes controlled only by one regulator, if sf represents the true model, then the covariance matrix \u03a3f can be represented by its first rank approximation using eigenvalue decomposition. Therefore, ignoring the noise terms, the inverse of the covariance matrix can be efficiently computed through its first rank approximation, which not only speeds up the algorithm but also reduces the effects of the noise.Since we considered \u03a9\u03bb1 and u1 are respectively the principal eigenvalue and the principal eigenvector of the covariance matrix. Given data sets of known TF-gene interactions one can form the sets of \u03a9f's and use equations 4,5 and 3, respectively, to compute p(eg|sf) for all TFs and assign to each gene g the regulator f that provides the maximum value.where f) can be viewed as the activity profile of a regulator f. Notice that the activity profiles of regulators are principal eigenvectors of different covariance matrices, which necessarily need not be, and, indeed they are not, orthogonal to each other. Therefore, the estimated activity profiles are different from those estimated by algorithms such as Principal component analysis (PCA) [The knowledge of regulator-gene interactions represents a low-resolution view of molecular interactions inside a cell. It does not provide any details about how and when these interactions occur. Therefore, as a complement to this information, in some biological studies, the question might be which regulators and how they respond under different environmental or genetic perturbations. One way to tackle this problem is to study the activity levels of different transcription factors across different conditions. The principal eigenvector  of the sample covariance matrix (\u03a3is (PCA)  or singuis (PCA) ,24 and iis (PCA)  which deHZ conceived the method, designed the study, performed the analysis and wrote the manuscript. DS carried out the DNA microarray experiments for amino acid data set. PS obtained the chromatin-IP data for Lrp. MK participated in the design of the study and wrote the manuscript. AK coordinated the study, analyzed microarray data, and wrote the manuscript. All authors read and approved the final manuscript.Supplementary Information. Additional information about performance evaluation, algorithms comparison, and transcription factor sub-networks.Click here for fileSupplementary Figure 1. Network of transcription factors with correlated profiles. The existence of an edge between two TFs indicates that the correlation between their activity profiles is above a threshold value of 0.70. Such similarities may indicate a certain degree of regulatory redundancy, i.e. different regulators controlling subsets of overlapping genes. Indeed, when we examined to what extent the correlations between the profiles are indicative of TFs regulating common genes, we observed that transcription factor pairs with high correlation regulate common genes with higher probability than TF pairs with low correlations. 55% of TF pairs with correlation above 0.70 appeared to have common targets, compared to 20% of TF pairs with correlation less than 0.70.Click here for fileTF-Gene Interactions. A set of genes with common regulators predicted from both data sets.Click here for fileTF-Operon Refined Interactions. A refined catalog of transcriptional interactions.Click here for fileSupplementary Figure 2. Regulatory network of transcription factors. The consensus regulatory interactions predicted using both data sets. This subnetwork comprises of 101 transcription factors (nodes) with 118 predicted interactions (edges) among them. All interactions are directed from a TF-regulator toward a TF-target. 76 (66%) predicted interactions (red edges) were previously known and include 36 known auto-regulators. The remaining 42 predicted interactions (blue edges) are new. In addition, 13 regulators identified as targets did not have any previously identified regulators.Click here for file"}
+{"text": "Therapeutic hypothermia (TH) has become standard management following out-of-hospital cardiac arrest (OHCA). Recent evidence suggests TH increases the risk of pneumonia. We retrospectively assessed infective indicators after OHCA and evaluated the effect of antibiotics on survival.We identified all patients admitted to the ICU of a regional primary angioplasty hospital following OHCA from May 2007 to December 2010. We recorded ICNARC predicted mortality scores, blood and respiratory (protected catheter aspiration) culture results, white blood cell count (WBC) and C-reactive protein (CRP), hospital outcome and ICU length of stay. All chest radiographs (CXRs) were reviewed by a respiratory consultant (JW). Any antibacterial therapy was recorded.9/l). Fifty-six of 144 patients  received antibiotics during the first 7 days of their ICU stay . The hospital mortality rate for these patients 53.6% (40.7 to 66.0) was significantly less than those not receiving antibiotics 75.0% (65.0 to 82.9)  with absolute risk reduction of 0.214 (0.055 to 0.365) and NNT of 5 (3 to 18). There was no difference in age (59.9 \u00b1 4.2 vs. 62.9 \u00b1 3.5) or ICNARC predicted mortality (75.1 \u00b1 5.2 vs. 78.4 \u00b1 4.2) between the groups.A total of 144 patients were admitted to the ICU following OHCA. Mean age was 61.7 years (95% CI 59.0 to 64.4). The mortality rate was 66.67% (58.62 to 73.84) with mean ICNARC predicted mortality of 77.11% (73.84 to 80.39). Of 144 patients, 138  had at least one positive marker of infection within 72 hours. Sixty-four had microbiology samples analysed, 34 of which were positive . Of 88 patients who had a CXR, 26  had consolidation. Ninety-six of 115 patients  had a CRP >100 mg/l  within 72 hours and 82 of 115  had an abnormal WBC (<4.0 or >11.0 \u00d7 10The post-arrest management of OHCA is commonly complicated by infections, the diagnosis of which is delayed by a universal increase in inflammatory markers, body temperature control, delay in the processing of samples and poor quality radiography. We have shown a significant reduction in mortality in patients receiving antibiotics compared with patients who do not, despite there being no difference in age or predicted mortality between the groups. This could be due to treatment of an aspiration pneumonia, an anti-inflammatory effect or that some patients did not survive long enough to receive antibiotics. It suggests that a formal clinical trial is warranted."}
+{"text": "Dryopteris crassirhizoma has been used as a traditional herbal medicine for treating various inflammatory and infectious diseases such as tapeworm infestation and mumps. In the present study, we investigated the bone protective effect of water extract of the rhizome of Dryopteris crassirhizoma (WEDC). We found that WEDC inhibits osteoclast differentiation via directly acting on osteoclast precursors. In osteoclast precursors, WEDC inhibited receptor activator of nuclear factor-\u03baB ligand- (RANKL-) induced expression of c-Fos and nuclear factor of activated T cells cytoplasmic 1, a key downstream target of c-Fos during osteoclast differentiation. We found that WEDC inhibits RNAKL-induced activation of extracellular-regulated kinase and NF-\u03baB that mediates c-Fos expression and osteoclast differentiation. In addition to the inhibitory effect of osteoclast differentiation, WEDC markedly suppressed bon-resorbing activity of mature osteoclasts, which was accompanied by disruption of actin ring structure. Furthermore, administration of WEDC suppressed RANKL-induced trabecular bone loss in mice. Collectively, our results demonstrate that WEDC inhibits not only osteoclast differentiation by inhibiting RANK signaling pathways in osteoclast precursors but also bone resorption by disrupting actin ring in mature osteoclasts, thereby contributing to its protective effect on bone loss. The rhizome of  Bone homeostasis is maintained by the coordinated actions of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. An imbalance in favor of bone resorption, most often due to excess osteoclastic activity, leads to bone loss in pathological conditions such as osteoporosis, lytic bone metastases, and rheumatoid arthritis . \u03baB ligand (RANKL) is an essential cytokine that stimulates entire processes for the development of bone-resorbing osteoclasts [\u03baB and mitogen-activated protein kinases (MAPKs) including extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 through recruitment of the adapter molecules such as TNF receptor-associated factor 6 (TRAF6) [\u03baB p50/p52, c-Fos, or NFATc1 results in blockage of osteoclastogenesis [Osteoclasts are multinucleated bone-resorbing cells that originate from hematopoietic mononuclear precursors. Receptor activator of nuclear factor-eoclasts . The effeoclasts . Stromaleoclasts . Thus, R (TRAF6) . These s (TRAF6) \u20138. Genetogenesis \u201311. Dryopteris crassirhizoma (WEDC) efficiently inhibits osteoclast differentiation. D. crassirhizoma is a perennial herbaceous plant widely distributed in Asia, and its rhizome is commonly used as an effective vermicide in traditional Chinese medicine. It has also been prescribed for treating respiratory tract infections, uterine bleeding, mumps, and feverish illnesses [D. crassirhizoma has antioxidant, anticancer, anti-inflammatory, and antibacterial activities [D. crassirhizoma on bone metabolism. In the present study, we investigated the inhibitory effect and action mechanism of WEDC on osteoclast differentiation and function. The in vivo efficacy of WEDC was also examined in a murine model of bone loss.In the screening of traditional Korean herbal medicines for antiosteoclastogenic activity, we found that water extract of the rhizome of llnesses . Previoutivities \u201316. Howe\u03b1-MEM and FBS were purchased from Thermo Fisher Scientific Inc. . Phalloidin-TRITC, 1\u03b1,25-dihydroxyvitamin D3 (VitD3), and p-nitrophenyl phosphate were purchased from Sigma-Aldrich . Recombinant human M-CSF was kindly provided by Dr. Yongwon Choi . Recombinant human soluble RANKL was prepared as described in a previous report [\u03baB\u03b1 (Ser32), and I\u03baB\u03b1 were purchased from Cell Singling Technology . Antibodies against NFATc1, c-Fos, \u03b2-actin, and GAPDH were from Santa Cruz Biotechnology . s report . AntibodD. crassirhizoma was purchased from Yeongcheon Oriental Herbal Market . A voucher specimen (no. W197) was deposited in the herbal bank of KM-Based Herbal Drug Research Group, Korea Institute of Oriental Medicine. The rhizome of D. crassirhizoma (50\u2009g) was extracted with 500\u2009mL of boiling water for 3\u2009h. The extract was filtered through a testing sieve (150\u2009\u03bcm) and lyophilized in a freeze dryer. To prepare WEDC, the lyophilized powder (yield: 12.78%) was resuspended in distilled water, centrifuged at 10,000\u2009\u00d7g for 5\u2009min, and filtered through a 0.2\u2009\u03bcm sterile filter. The rhizome of \u03b1-MEM complete medium containing 10% FBS, antibiotics (100\u2009U/mL penicillin and 100\u2009\u03bcg/mL streptomycin), and M-CSF (60\u2009ng/mL). To generate osteoclasts from BMM cultures, BMMs (1 \u00d7 104 cells/well) were incubated with M-CSF (60\u2009ng/mL) and RANKL (100\u2009ng/mL) for 4 days in 96-well plates. Cell viability of BMMs was measured with Cell Counting Kit-8 assay , after culturing BMMs (1 \u00d7 104 cells/well in a 96-well plate) in the presence of M-CSF (60\u2009ng/mL) for 2 days. Mouse calvarial osteoblasts were obtained from calvariae of newborn mice as described previously [5 cells/well) and osteoblasts (2 \u00d7 104 cells/well) were cocultured with 10\u2009nM VitD3 for 6 days in 48-well tissue culture plates. All cultures were replenished with fresh medium on day 3.Bone-marrow-derived macrophages (BMMs) were prepared from mouse bone marrow cells as described previously  and culteviously . For ostp-nitrophenyl phosphate as substrate according to the manufacturer's instructions in a TRAP assay buffer . TRAP staining was performed according to the protocol described in BD Biosciences Technical Bulletin no. 445. TRAP-positive multinucleated cells (TRAP(+)MNCs) having more than three nuclei were counted as osteoclasts.Cells were fixed in 10% neutral buffered formalin and permeabilized with 0.1% Triton X-100. Total TRAP activity was measured spectrophotometrically using 5 cells/well in a 6-well plate) were preincubated with WEDC for 3\u2009h and then stimulated with VitD3 for 24\u2009h. BMMs (4 \u00d7 105 cells/well in a 6-well plate) were incubated with WEDC for 3\u2009h and then stimulated with RANKL for the indicated times. Total RNA was isolated using RNA-spin total RNA Extraction Kit , and cDNA was synthesized from 1\u2009\u03bcg of total RNA using AccuPower RT-PreMix (Bioneer). SYBR green-based QPCR amplification was performed using cDNA diluted to 1\u2009:\u20093, 10\u2009pmol of primers, and AccuPower GreenStar QPCR Master Mix (Bioneer) in the Applied Biosystems 7500 Real-Time PCR System . The primer sequences used were described previously [Osteoblasts (3 \u00d7 10eviously . The rel\u03bcg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a polyvinylidene fluoride membrane, and then immunoblotted with the indicated antibodies.Cells were washed twice with ice-cold PBS and lysed in ice-cold lysis buffer  supplemented with protease inhibitor and phosphatase inhibitor cocktail tablets . Cell lysates were centrifuged at 10,000\u2009\u00d7g for 15\u2009min at 4\u00b0C. Protein concentration of total cell lysates was determined by suing BCA protein assay kit (Thermo Fisher Scientific Inc.). Equal amounts of proteins (30\u20093 (10\u2009nM) on collagen gels for 6 days. The generated osteoclasts were seeded on an Osteo Assay Surface plate , allowed to settle for 2\u2009h, and then incubated with the indicated concentrations of WEDC for 24\u2009h. Cells were stained for TRAP to detect osteoclasts and photographed. Resorbed pits by osteoclasts were photographed and analyzed by using Image J software, after removing cells with sodium hypochlorite bleach.Mature osteoclasts were generated by coculturing mouse bone marrow cells and primary osteoblasts with VitD\u03bcg/mL in PBS) to stain F-actin. The peripheral actin ring-like structures in osteoclasts were photographed using a fluorescence microscope (Olympus IX53 inverted microscope).BMMs were incubated with M-CSF and RANKL for 4 days to generate osteoclasts as described above. The cultures were treated with vehicle or the indicated concentrations of WEDC for 30\u2009min, fixed, permeabilized, and then incubated with phalloidin-TRITC  were orally administrated with distilled water or WEDC (0.25\u2009g/kg of body weight twice daily) for 5 days. RANKL (1\u2009mg/kg of body weight) or PBS was intraperitoneally injected on days 3 and 4. The mice were sacrificed on day 6, and the right femurs were dissected, cleaned of soft tissue, and fixed in 10% neutral buffered formalin. Microcomputed tomography (micro-CT) was performed with the SMX-90CT system . Scans then were integrated into 3D voxel images. All bone images were reconstructed by the VG Studio MAX 1.2.1 program . The regenerated trabecular bone volume/tissue volume, number, thickness, and separation were calculated with TRI/3D-BON . All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The experimental protocols were approved by the Institutional Animal Care and Use Committee at Korea Institute of Oriental Medicine.n \u2265 3). Two-group comparisons were performed with Student's t-tests, while multiple-group comparisons were performed with analysis of variance followed by Dunnett's test. A P value <0.05 was considered statistically significant.All values are presented as mean \u00b1 SD , a concentration that markedly inhibited osteoclast differentiation in the coculture system, did not affect RANKL and OPG mRNA expression in basal and VitD3-stimulated osteoblasts . WEDC slightly inhibited RANKL-induced ERK activation but not JNK or p38 activation. It also attenuated RANKL-induced p65 phosphorylation and I\u03baB\u03b1 phosphorylation and degradation  complex that stimulates I\u03baB\u03b1 phosphorylation and degradation, allowing nuclear translocation of NF-\u03baB heterodimer containing the p50 and p65 subunits [\u03baB\u03b1, posttranslational p65 modifications such as phosphorylation, acetylation, and ubiquitination represent another mechanism regulating NF-\u03baB transcriptional activity [\u03baB\u03b1 phosphorylation and degradation. Therefore, our findings suggest that WEDC inhibits osteoclast differentiation, at least in part, by suppressing RANKL-induced activation of ERK and the classical NF-\u03baB signaling pathway, without interfering with the RANK-TRAF6 pathway.Previous studies have shown that activation of MAPKs  and NF-ntiation \u20138. It waof TRAF6 . Howeversubunits . In addiactivity . It has activity , 22. In \u03baB signalling pathway is involved in bone-resorbing activity of osteoclasts as well as osteoclast differentiation. It has been shown that blocking NF-\u03baB pathway by overexpressing a dominant-negative form of IKK beta suppresses bone-resorbing activity of osteoclasts, whereas NF-\u03baB activation by expression of a constitutively active form of IKK beta upregulates it [\u03baB pathway by the IKK beta mutants affected osteoclast survival [\u03baB pathway, was shown to inhibit bone resorption by disrupting actin ring [\u03baB pathway may contribute to its antiresorptive function.In addition to the inhibitory effect on osteoclast differentiation, WEDC also decreased resorbed pit area by mature osteoclasts without affecting the number of osteoclasts, indicating that WEDC inhibits bone-resorbing activity of osteoclasts. Given the crucial role of actin ring structure in bone-resorbing activity of osteoclasts , our finlates it . Neithersurvival . In additin ring . Therefoin vitro results, administration of WEDC protected bone loss induced by RANKL. Since injection of exogenous RANKL rapidly induces trabecular bone loss through stimulating osteoclast differentiation and function [in vivo remains to be elucidated. Given the crucial role of excessive RANKL activity in pathological bone destruction, our findings strongly suggest that WEDC may be useful in preventing or treating various bone destructive diseases. Consistent with the function , the proD. crassirhizoma [D. crassirhizoma, including antioxidant, antibacterial, and antitumor activities [Phloroglucinols, triterpenes, flavonoid glycosides, and phenolic compounds have been isolated from the rhizome of irhizoma \u201329. Of ttivities , 25, 30.in vivo. These findings suggest that WEDC may be useful in preventing or treating bone diseases associated with excessive bone loss.We have demonstrated that WEDC inhibits osteoclast differentiation and function by inhibiting RANK signaling pathways in osteoclast precursors and by disrupting actin ring in mature osteoclasts, respectively. Furthermore, WEDC suppressed RANKL-induced bone loss"}
+{"text": "The intracellular protein calretinin (CR) is frequently used as a marker of specific  cortical interneuronal population  with a CR-ires-Cre mouse line, which expresses the enzyme Cre-recombinase in a fashion similar to endogenous CR expression. In the offspring from this cross, the cellular expression of the tdTom label was indirectly activated by the expression of CR. In these animals, the orientation selectivity, size tuning, and temporal and spatial frequency tuning of tdTom+ cells were estimated After comparing the results with those acquired from the general tdTom negative neuronal population, the authors concluded that in none of the studied visual response properties (VRPs) the tdTom+ population is significantly different from the tdTom\u2013 population.On the first sight, it would suggest that the CR+ (inter)neurons do not differ in their VRPs from the general neuronal population, composed of the pyramidal neurons and other (CR negative) interneuronal types.However, when the expression of tdTom label was directly compared with the expression of CR (via immunohistochemistry and in situ hybridization), it could be shown that only 20% of tdTom+ cells expressed also calretinin. By contrast, 96% of CR+ cells expressed also tdTom. This showed that actually many of tdTom+ neurons expressed CR only transiently during their development. And indeed, the fact that 60% of tdTom+ cells also expressed SatB2  underlines the conclusion that the majority of tdTom+ cells are pyramidal neurons which expressed CR transiently during their development. A further comparison with other interneuronal markers  showed a low colocalization between CR and SOM and no colocalization between CR and PV. Hence, the exclusive expression of CR or PV (but never both of them) in adult cortical neurons well-known across all studied species including mouse  complicates the interpretation of the results with regard to the VRPs of these cells. The study of Camillo et al. shows that the neurons with a common \u201cCR history\u201d do not differ in their VRPs from the general neuronal population. In earlier studies, significant differences of some of the VRPs between excitatory neurons and GABAergic interneurons have been documented in mouse visual cortex. In some of those studies, a discrimination between individual types of inhibitory neurons was not (Sohya et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares 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": "This issue's invited Editorial is provided by Todd Pawlicki, AAPM Secretary and Chair of Task Group No. 281, Governance Assessment Communications Plan. This is an opinion article and as such does not necessarily reflect the views and position of the JACMP EIC leadership team nor those of the JACMP Board of Editors. It is being provided to inform the medical physics community of the work and sentiments of TG\u2010281 and to assist the AAPM membership in its decision respecting the upcoming vote on this topic. \u2013Michael Mills, JACMP EICThe American Association of Physicists in Medicine (AAPM) was founded by physicists on November 17, 1958 in Chicago prior to the annual meeting of the Radiological Society of North America (RSNA). The AAPM was incorporated on November 10, 1965 with 16 members making up the Board of Directors (the Board). The Science, Professional, and Education Councils were created in 1972, 1973, and 1974, respectively. The Administrative Council was added later in 2010. Along the way, the AAPM has operated two highly successful journals, created many impactful technical documents, and has spearheaded other professional organizations such as the Commission on Accreditation of Medical Physics Education Programs (CAMPEP) and the Society of Directors of Academic Medical Physics Programs (SDAMPP). The membership has grown to over 8,500 and the Board has grown to 49 members (of which 38 are voting members). Over the past 59\u00a0yr, the AAPM has established itself as an internationally leading organization for the application of physics to medicine.Given all of its successes and impact, the AAPM has not been without its refinements and course corrections. The American College of Medical Physics (ACMP) was started by the AAPM in the early 1980s only to be absorbed by AAPM in 2012. Likewise, the effort for licensure did not achieve its aim of requiring licensure for clinical medical physicists in all 50 States. The Annual Meeting has morphed over the years first to include a science track and later the education track followed by the professional track. Much later, the AAPM inherited the Spring Clinical Meeting from the ACMP partly because it better satisfied the practical needs of clinical physics members\u2019 that were not being met by the annual meeting alone. So goes the life for an organization of dedicated and engaged members such as the AAPM; you win some, you lose some but are still able to maintain a positive impact on healthcare both at home and abroad.It is prudent to describe the current governance structure of the AAPM. There are five officers of the AAPM\u2014the President\u2010elect, President, Chair of the Board, Treasurer, and Secretary. This group along with the Executive Director constitutes the Executive Committee (EXCOM). EXCOM runs the day\u2010to\u2010day activities of the AAPM as well as performs Board\u2010related functions such as strategic decision making, fiduciary responsibilities, resource allocation, etc., in the intervals between Board meetings as provided by the AAPM Rules. There are 12 Board Members\u2010at\u2010large, 21 AAPM Chapter\u2010elected Board members, and 6 nonvoting members of the Board: Council Chairs, our Representative to the AIP Board, and the Executive Director. The President\u2010elect, Treasurer, Secretary, Members\u2010at\u2010large, and Chapter\u2010elected members are all elected by the General Membership or a subset of the General Membership . There are four Councils: Administrative, Education, Professional, and Science. The Chairs of these Councils are appointed positions. There is also a Strategic Planning Committee that is chaired by the Chair of the Board and has nine other appointed members.With the complex healthcare landscape and new opportunities ahead, the AAPM has a responsibility to its members for continuous improvement. For the first time in many years, the AAPM has completed a critical assessment of itself and the ways in which it could do even better. About two and half years ago, the Ad Hoc Committee on Governance Assessment (AHCGA) was convened. This group, along with the Board of Directors and Strategic Planning Committee, has been assessing the current governance structure of the AAPM. The Board engaged a consulting group that specializes in nonprofit organizational governance, completed a membership survey (the first one in over 10\u00a0yr), and conducted several surveys of current AAPM leadership. As a result of this effort, several recommended changes to AAPM's governance structure are being proposed. These changes are tethered to the \u201cDNA\u201d of the organization and in concert with its ethos. For example, AAPM leadership should be made up of the various member stakeholders and constituencies with General Membership input through the elected positions. The purpose of this editorial is to describe the proposed changes in order to inform and inspire member readers as they cast their votes for this change.Recognizing that the AAPM's core strength is its members, the AAPM must continue to be led by its major member stakeholder groups of clinical, science, chapters, educators, and members in both imaging and therapy subspecialties. As such, a new Council structure is being proposed to consist of the following Councils: Clinical Practice, Science, Regional Organization, Education, and Member Services.The Clinical Practice Council will be focusing on stewardship of the profession of clinical medical physics with aspects of accreditation, credentialing, and practice guidelines constituting the purview of this Council. Communication with key collaborators will be an important task and include vendor relations, regulatory and legislative affairs, and patient communications as well as media response to clinical issues.The Science Council will continue to function in much the same ways as it currently functions. It will have several responsibilities including: (a) oversee scientifically based policies and position papers, administer most of the scientific task groups and working groups through the Therapy Physics Committee and Imaging Physics Committee, (b) address research issues via the Research Committee, (c) provide assessment of technology through the Technology Assessment Committee, and (d) be responsible for press inquiries on scientific matters. A key aspect of its work will be to address the governmental aspects of research funding.The Regional Organization Council is perhaps the most novel construct of the proposed structure and is being proposed in recognition that the profession has not realized optimal value from a collaboration of the AAPM with its Chapters. There are common needs of all chapters, regardless of geography, which can be addressed by identifying and focusing on them. Therefore, this Council will be tasked with ensuring synergy and coordination of Chapter activities with the broader AAPM efforts. Consequently, important areas to be addressed are education, vendor support of chapters, local regulatory issues, communication between the AAPM and Chapters as well as cultivation of future AAPM leaders. The Regional Organization Council will also work to identify Chapter needs and secure resources to help them achieve their specific goals.The Education Council will maintain most of its current structure and charges but will now oversee both the International Educational Activities Committee and International Affairs Committee for better coordination of global outreach efforts.The Member Services Council will carry important functions by focusing on the services and tools that will help members succeed in their profession. This Council will be responsible for publishing and the e\u2010presence of the AAPM including management of the association's journals. It will also handle AAPM meetings, awards and honors, and ensuring that AAPM's history is documented. Other important member needs will be addressed through the Membership Committee and Ethics Committee.The proposal further recommends rightsizing the Board to 13 members consisting of the major member stakeholders. The new Board configuration calls for the following composition: the Chair of each of the Councils (5), the President\u2010elect, President, Chair of the Board, and Immediate Past Chair of the Board (4), the Treasurer (1), the Secretary (1), the Executive Director (1), and an optional non\u2010AAPM member (1). The purpose of allowing a nonmember to participate on the Board is to ensure expertise that is not typically obtainable through member appointments, e.g., hospital administrator, legal, marketing, or sales expert, or perhaps someone with experience in nonprofit revenue generation that does not increase the membership dues. It was felt that a Board seat would help get the most qualified person possible as well as establish a long\u2010term relationship for the benefit of AAPM and the profession rather than what one would get from a consultant. The non\u2010AAPM member Board position is not mandatory and will only be filled as the Board deems necessary.As way of reference, two of our sister societies have similar governance structures as the newly proposed structure. On the imaging side, the RSNA with 54,000 members is a society comprised of radiologists, physicists, and other medical professionals that has an eight\u2010member Board that includes six Directors, the President, and the President\u2010elect. The Directors are liaisons for the cabinets of Education, Information Technology and the Annual Meeting, International Affairs, Publications and Communications, and Science. The President handles external relations and the President\u2010elect also serves as the Secretary\u2010Treasurer. On the therapy side, the American Society for Radiation Oncology (ASTRO) with over 10,000 members composed of physicians, nurses, biologists, physicists, therapists, dosimetrists, and other health care professionals is governed by a 15\u2010member Board. The Board consists of the Chair, President, President\u2010elect, Secretary\u2010Treasurer (one position), Immediate Past Chair, and the Chair and Vice\u2010chair for each of their Councils. Their five Councils are Clinical Affairs and Quality, Education, Government Relations, Health Policy, and Science.The AAPM's governance proposal also includes the creation of a new operations group of 11 members \u2014 the Operations Committee \u2014 that will handle the day\u2010to\u2010day operations of the association, precluding the need for the AAPM's Executive Committee (EXCOM). The Council Vice\u2010chairs will make up the Operations Committee along with the President\u2010elect, President, Treasurer, Secretary, Government and Regulatory Affairs Committee Chair, and Executive Director (nonvoting).The General Membership will elect future Board members as Vice\u2010chairs of the Councils, who will also first serve on the Operations Committee for 2\u00a0yr. Vice\u2010chairs will then succeed to Council Chairs and serve a two\u2010year term on the Board. The four\u2010member presidential chain  will each serve one\u2010year terms. The Treasurer and the Secretary will serve up to two consecutive 2\u2010yr terms. Determination of candidates for the General Membership vote will be the responsibility of the Nominations Committee.The Nominations Committee will have seven members. It will be chaired by the Immediate Past Chair of the Board. There will be one member from the Regional Organizations Council and four other members consisting of one person from academic therapy physics, one from community practice therapy physics, one from academic imaging physics, and one from community practice imaging physics. All will be staggered 2\u2010yr terms and be elected by a General Membership vote. In this way, the membership has input into the nominating process so that the AAPM leadership succession is open and transparent. The current Chair of the Board will be a nonvoting member. The purpose of the Nominations Committee is to make nominations for Council Vice\u2010chairs, President\u2010elect, Secretary, Treasurer, and the Nominations Committee.Lastly, a Governance Committee, chaired by the Secretary, will be created and tasked with several key functions not currently performed within the AAPM but crucial to good organizational governance. It will be tasked with routinely and critically assessing the effectiveness of the AAPM's governance, ensuring diversity of Board members, as well as developing and ensuring the necessary knowledge and governance skills of Board members.In closing, it is important to remember that when making a change like this, \u201cperfect\u201d is the enemy of \u201cbetter\u201d. Not every issue will be automatically addressed with the proposed changes\u00a0and not every change will suddenly produce fantastic results. Over the years however, one constant for the AAPM has\u00a0been change. In keeping with that tradition, the above\u2010described governance changes are just the next step in a natural evolution of the AAPM. The proposed changes will be presented for a General Membership vote shortly after the Annual Meeting in Denver. TG\u2010281 encourages you to cast your vote on August 23, 2017 and play a vital role in shaping the future of your organization.AAPM Task Group No. 281 Governance Assessment Communications Plan:"}
+{"text": "Tumor development and therapeutic resistance are linked with tumor-associated macrophage (TAM) and myeloid-derived suppressor cell (MDSC) infiltration in tumors via chemokine axis. Chemokine expression, which determines the pro or anti-inflammatory status of myeloid cells, are partly regulated by the nuclear factor-kappa B (NF-\u03baB) pathway. Here, we identified that conditional deletion of canonical NF-\u03baB signaling (p65) in myeloid cells inhibited syngeneic glioblastoma (GBM) through decreased CD45 infiltration in tumors, as characterized by decreased TAMs (CD206+) and MDSCs (Gr1+ CD11b+), increased dendritic cells (CD86+) and cytotoxic T cells (CD8+) in the p65 knockout (KO) mice. Proinflammatory cytokines  and myeloid differentiation factor (Endoglin) were increased in myeloid cells from p65 KO tumor, which demonstrated an influence on CD8+T cell proliferation. In contrast, p65KO athymic chimeric mice with human GBM, failed to inhibit tumor growth, confirming the contribution of T cells in an immune competent model. The analysis of human datasets and GBM tumors revealed higher expression of p65 in GBM-associated CD68+ macrophages compared to neighboring stroma. Thus, canonical NF-\u03baB signaling has an anti-inflammatory role and is required for macrophage polarization, immune suppression, and GBM growth. Combining an NF-\u03baB inhibitor with standard therapy could improve antitumor immunity in GBM. Hypoxia and neovascularization are signature histopathologic features of GBM2, which is most lethal during the first year after initial diagnosis, despite surgical resection and other standard therapies3. Recent reports suggest that tumor growth depends on the tumor microenvironment (TME)4. Peripheral macrophages and microglia are the most abundant non-cancerous cell types in GBM, in some cases accounting for up to 30% of the total tumor composition6. Tumor-associated hypoxia is known to upregulate hypoxia inducible factor 1-\u03b1 (HIF1-\u03b1), transcribe stromal cell-derived factor 1\u03b1 (SDF-1\u03b1), and promote secretion of proangiogenic factors to recruit CXCR4+ bone marrow-derived cells (BMDCs) in the tumor milieu10. The myeloid populations of BMDCs, such as tumor-associated macrophages (TAMs) and immune regulatory myeloid-derived suppressor cells (MDSCs), are critical in tumor development12. TAMs in the TME are skewed towards an M2 polarized state and are a central target in cancer therapy13. Several chemokines, such as macrophage colony stimulating factor-1 (m-CSF/CSF1) and monocyte chemotactic protein-1 (MCP1/CCL2) are known to contribute to the recruitment of heterogeneous myeloid cells to the tumors due to the presence of CSF1 receptor (CSF1R)16.Glioblastoma (GBM), a grade IV astrocytoma as classified by World Health Organization, is a highly malignant, vascular, and invasive subtype18. Chemokines are critical in regulating cancer-associated transport, activation, and proliferation of several cell types, including myeloid, lymphoid, endothelial and epithelial cells20. Previously, we identified that chemokine signaling, especially through CXCL7, plays a key role in GBM growth and antiangiogenic therapy resistance. Targeting CSF1R+ myeloid cells significantly decreased CXCL7 and thus the GBM growth12. Interestingly, chemokines, including CXCL7, are secreted by the host peripheral macrophages and are regulated through the NF-\u03baB signaling in murine models17. In human TAMs, CXCL8 or IL8 expression is mediated through NF-\u03baB driven transcription in response to m-CSF and MCP121. Moreover, it has been widely recognized that chemokines are one of the major targets of canonical NF-\u03baB signaling.Chemokines and pro-inflammatory peptides are often expressed in response to the induction of expression of nuclear factor-\u03baB (NF-\u03baB) by cytokines or other stimuli in cancer22. NF-\u03baB follows p50 and p65 (RelA) mediated canonical as well as p52 and RelB mediated non-canonical pathways25. NF-\u03baB cross-talks with different kinases, such as GSK3-\u03b2, p38, or PI3K, which modulate the NF-\u03baB transcriptional activity or affect upstream signaling pathways26. NF-\u03baB cooperates with multiple transcription factors in pathways such as STAT3 and p53, which either directly interact with NF-\u03baB subunits or affects NF-\u03baB target genes in the nucleus. Depending on the context, such as in different tumor types, NF-\u03baB signaling could be tumor promoting or anti-tumorigenic in cancer cells and their microenvironment27. It has recently been shown that NF-\u03baB signaling can drive GBM cancer stem cells28, but surprisingly, no data is available in the GBM microenvironment, and it is not understood whether the canonical NF-\u03baB pathway has a proinflammatory or anti-inflammatory role in GBM tumor recruited myeloid cell populations.NF-\u03baB is considered as a master regulator of inflammation mechanisms, is increasingly recognized as a crucial player in many steps of cancer initiation and progression, and thus serves as a critical link between inflammation and cancerThe present study is focused on studying myeloid cell-associated canonical NF-\u03baB signaling with a special interest in GBM models. We identified that deleting myeloid cell associated NF-\u03baB signaling resulted in M2 to M1 polarization and enhancement of CD8+T cell-mediated antitumor immunity in an immune competent mouse model. Further, data were validated in an immunocompromised athymic nude chimera model, which showed tumor growth advantages in the absence of a T cell component. Here, we report for the very first time that GBM growth is influenced by myeloid cell-associated NF-\u03baB signaling in the host through anti-inflammatory phenotypes, and is a potential target for GBM therapy. We anticipate that combining standard temozolomide therapy with a pharmacological NF-\u03baB inhibitor could improve the outcome of GBM treatments in the clinic.Lyz2tm1(cre)Ifo/J) male mice were crossed with p65flox/flox female mice on the C57BL6 background, resulting in conditional p65 deletion in cells of the myeloid compartment. p65fl/fl/LysMCre (KO), (n\u2009=\u20097) and LysMCre control (n\u2009=\u20096) mice were used to develop syngeneic GL261 GBM tumors following orthotopic tumor cell implantation at day 1, as shown previously by our laboratory29. Magnetic resonance imaging (MRI) was used to monitor tumor growth on day 22 following implantations (end-point). MRI data analysis identified significantly decreased GBM tumor growth in p65KO mice compared to controls  Cre  spheroid formation assays. No difference in spheroid size was observed in a cell culture experiment in the presence of 50% conditioned medium derived from 24\u2009hours\u2019 culture of wild-type, control or p65 KO BMDMs. (Supplementary data\u00a0GL261 GBM tumor bearing p65KO (n\u2009=\u20093) and control mice (n\u2009=\u20093) were analyzed for cellular differences in the tumor microenvironment. p65KO mice implanted with GBM had statistically significant cellular reductions in CD45+ leukocyte Fig.\u00a0, macrophsorted CD11b+ cells from the GL261 TME were subjected for protein lysate preparation followed by cytokine protein arrays. The customized array involved 44 factors with Th1 and Th2 cytokines, growth factors, angiogenic factors, and chemokines  or p65 control (n\u2009=\u20094) mice. Different ratios of T cell: CD11b cell cocultures were performed, ranging from 1:1 to 1:1/32 Fig.\u00a0. Interes6 cells34. Once mice achieved 70% engraftment (chimera), U251 (n\u2009=\u20096) or PDX GBM811 cells (n\u2009=\u20096) were orthotopically implanted and followed-up by MRI at the 3-week or 8-week protocol, respectively  and normoxia (21% O2) conditions. We identified that hypoxia-induced p65 mRNA expression in human macrophages compared to macrophages cultured in normoxia. The p65 overexpression under hypoxia was associated with decreased pro-inflammatory and cell differentiation mediators such as PPAR\u03b3, MIP1\u03b1, MCP1, CSF1, Endoglin, STAT1, STAT5, and IRF5  brain data, with 10 normal adjacent and 542 GBM brain samples, suggested there is significantly increased RELA (p65) expression  in GBM (2.25 fold) as compared to normal brain Fig.\u00a0. AnalysiRF5 Fig.\u00a0. This waRF5 Fig.\u00a0. High p638. NF-\u03baB pathway through Notch 1 signaling activates microglia following hypoxia exposure39. The canonical NF-\u03baB pathway is required for the human myeloid dendritic cell development and function40, however, NF-\u03baB signaling also regulates M2 polarization41. Recently, NF-\u03baB signaling was found to be a mediator of MDSC expansion in an aging model42. In addition, noncanonical NF-\u03baB signaling was reported to mediate STAT3-stimulated IDO upregulation, an immune suppressive mediator in MDSCs43. The present study identified that tumor-associated myeloid cells deficient in NF-\u03baB signaling display anti-inflammatory properties, which was evident by increased inflammatory mediators following conditional deletion of p65 in myeloid cells in our GBM model.Conflicting data exists in the literature, with the majority from the immunology field, suggesting the production of pro-inflammatory or immune suppressive mediators in the presence of active canonical NF-\u03baB signaling in myeloid cells. NF-\u03baB signaling has been known to regulate macrophage-driven inflammation in hematological and autoimmune diseases44. In an ovarian tumor model, transplantation of macrophages with inhibited IKK2 led to a reduction in tumor burden through the switch from M2 to M1 macrophage phenotype45. In a hepatocellular carcinoma model, targeting an NF-\u03baB signaling mediator, an I\u03baB-super repressor, in the liver macrophages (Kupffer cells) led to a decreased tumor incidence46. NF-\u03baB signaling, particularly the p65 subunit, has been shown to modulate M2 polarization in a hepatoma model, where tumor cells produced TLR2-related ligands, which are capable of activating p65 nuclear translocation in TAMs47. After turning on related gene expression, nuclear p65 is exported into the cytoplasm for autophagosomal degradation which limits NF\u03baB activity and drives TAM to M2 macrophage polarization47. In the present study, we anticipated that deletion of NF-\u03baB signaling in myeloid cells may limit the above mechanisms of M2 polarization, which was evident by the decreased M2 (CD206) macrophages and increased M1 (CD86) macrophages in GBM tumor-bearing p65 KO mice compared to control mice. Interestingly, NOS2+ macrophages (M1) through NO production were found to be involved in T cell infiltration and tumor rejection48. Similarly, we also noticed that increased M2 to M1 polarization and CD8+ infiltration in tumor-bearing p65 KO mice as compared to control mice. Interestingly, one study has identified the anti-tumor role of macrophage-associated NF-\u03baB signaling in mammary tumor-mediated lung metastasis model49. Moreover, most studies suggest a tumor-promoting effect of NF-\u03baB in myeloid cells, including TAMs. Despite these data pointing to a pro-tumor role for NF-\u03baB in macrophages, genes regulated by NF-\u03baB could also lead to an anti-tumor phenotype, suggesting that effects may be more complex. Altogether, the extent of NF-\u03baB signaling driven mechanisms in critical immune cells such as cancer-associated heterogeneous myeloid cell populations are poorly understood and controversial in solid cancers50.First, we observed that p65 KO mice had reduced syngeneic GBM growth. Thus, canonical NF-\u03baB signaling in myeloid cells is required for the GBM growth. Our tumor growth data corroborates with previous reports: In a colitis-associated cancer model, deletion of a canonical NF-\u03baB inhibitor of nuclear factor kappa-B kinase (IKK2) in the myeloid lineage led to a reduction in tumor incidence and size51. The macrophage-derived cytokines IL-1\u03b2 and TNF-\u03b1, are potent activators of NF-\u03baB. In turn, their expression is controlled by NF-\u03baB, thus creating a positive feedback loop. Interestingly, physical and oxidative stress mediators, such as NOS2 or COX-2, may induce NF\u03baB pathway in myeloid cells53. We found that p65 deleted myeloid cells produced an increased amount of IFN-\u03b3, TNF-\u03b1, IGF1, endoglin, MCP-1 (CCL2) and MIP-1\u03b1 (CCL3). Interestingly, these factors belong to HMGB1 signaling pathways identified in a web-based pathway analysis. HMGB1 has been shown to activate pro-inflammatory signals in the literature56 and promote autophagy of tumor-associated myeloid cells such as MDSCs57. This could explain the decreased MDSCs and pro-tumor TAMs in p65KO group compared to p65 controls. Further, T cell proliferation, especially CD8+T cells, was increased following T cell coculture with p65KO myeloid cells compared to control tumor myeloid cells. Co-culture supernatant with p65KO myeloid cells was characterized to have increased IFN-\u03b3, TNF-\u03b1 and IL-1\u03b2 compared to control myeloid cells. It is anticipated that IFN-\u03b3, TNF-\u03b1 and IL-1\u03b2 cytokines were produced by the p65 deleted myeloid cells in the coculture, which supported the T cell survival and proliferation. The culture of T cells with IFN-\u03b3, TNF-\u03b1, and IL1-\u03b2 cytokine showed similar T cell proliferation patterns. These data indicate that proinflammatory cytokines IFN-\u03b3, TNF-\u03b1, and IL1-\u03b2 have a proliferative effect specifically on the CD8+T cell population and an antiproliferative effect on the CD4+T cell population. This study result corroborates with our previous co-culture study findings in manuscript  overexpression in canonical NF-\u03baB signaling inhibited proinflammatory IL-12 cytokine expression in normal macrophages. TAMs isolated from p50 knockout mice showed normal production of M1 cytokines and were associated with the decreased tumor growth. Thus, p50 overexpression accounts for the inability of TAMs to mount an effective M1 antitumor response capable of inhibiting tumor growth63. Nevertheless, these data indicate that canonical NF-\u03baB signaling has anti-inflammatory effects, which favor GBM tumor growth. However, the expression of proinflammatory cytokines in p65 deleted myeloid cells is extremely complex and requires future investigation in the context of T cell function. Moreover, studies support that NF-\u03baB signaling through context-dependent functions may play a pivotal role in governing heterogeneous myeloid cell function during inflammation and cancer development. Please note that microenvironment with intact immune system play a critical role here because anti-tumor effects were only seen in syngeneic tumor model (GL261) in immunocompetent animals. Other human tumor models in immune compromised mice with p65flox/flox control or p65 KO chimera did not decrease in tumor growth. We anticipate that p50 dimers might have inhibited the expression proinflammatory cytokines  through inhibiting the p50-p65 complex in the nucleus in control mice. However, p50 dimers would not be able to suppress the proinflammatory cytokine promoter due to absence of p65 (p50-p65 complex) in myeloid cells in p65 KO mice65. Please see Fig.\u00a0Previous reports indicated the involvement of NF-\u03baB in T-cell proliferation and activation33. Our in vitro data showed no change in invasive potential or sphere-forming potential following incubation of conditioned medium of normal BMDM, p65 KO or control BM cells cocultured with GL261 or HF2303 tumor cells. Therefore, the decrease in CD44+ CSCs in invasive GL261 tumors in p65 KO mice compared to controls could be the result of the anti-tumor effect of CD8+T cells in syngeneic GBM model. This is further evidenced by the human PDX GBM 811 or U251 tumors in chimeric nude models, which showed comparable CD44+ CSCs in TME in the absence of host T cell component. Moreover, the present study supports that CSC phenotypes in the immunocompetent hosts are mediated through the NF\u03baB signaling in macrophages and cytotoxic CD8+T cells. However, a functional stemness assay such as limiting dilution transplantation between GBM tumors in control and p65KO mice, which is out of the scope of the present study, may provide a better understanding of the role of myeloid NFkB in regulating stemness in GBM models.Previously, it was identified that macrophages induce invasiveness of cancer cells and CSCs in breast cancer modelet al., reported that human cell line models such as U251 and U87 are more immunogenic compared to GL261 model66. Therefore, using a low immunogenic cell line may not reflect the clinical scenario in our study. Unfortunately, GL261 is the only plausible syngeneic murine cell line available, as it recapitulates human glioblastoma characteristics such as hypervascularity, hypoxia, and hyper-invasive phenotypes67. GL261 also depicts a mutation status similar to human tumors and is very well characterized for cancer therapeutic studies68.We investigated that myeloid cell-associated and p65 mediated NFkB signaling is critical in immunogenic tumors such as a murine GL261 tumor. Please note that human tumors are higher in immunogenicity, for example, a study by Jacobs In summary, we identified that myeloid NF\u03baB signaling is heterogeneous in the human GBM-associated stroma. The present study identified the anti-inflammatory role of myeloid NF\u03baB signaling in promoting GBM tumor growth through immune suppression mechanisms. Therefore, targeting/inhibiting myeloid-specific NF\u03baB signaling in GBM could inhibit the immune suppressive TAMs and improve the anti-tumor immunity Fig.\u00a0. We antiad libitum. Body weight was measured twice weekly as an indicator of overall animal health. All efforts were made to ameliorate the suffering of animals. CO2 with secondary method was used to euthanize animals for tissue collection at the end of the study.All the experiments were performed according to the National Institutes of Health (NIH) guidelines and regulations. The Institutional Animal Care and Use Committee (IACUC) and Institutional Review Board (IRB) of Augusta University  approved all the experimental protocols. All animals were kept under regular barrier conditions at room temperature (21 to 25\u2009\u00b0C) with exposure to light for 12\u2009hours and dark for 12\u2009hours. Food and water were offered fl/fl/LysMCre animals, p65fl/fl mice69 were bred to B6.129P2-Lyz2tm1(cre)Ifo/J (Jackson Lab) animals until homozygous animals were obtained. Genotypes were confirmed via PCR-based genotyping and p65 knockdown was confirmed via western blotting of various cell types.To obtain the p652 at 37\u2009\u00b0C in a humidified incubator. Patient-derived GBM cells (HF2303) was obtained from Dr. Tom Mikkelsen\u2019 s lab at Henry Ford Hospital and was grown in neurosphere medium (NM), composed of DMEM/F-12 supplemented with N2 (Gibco), 0.5\u2009mg/ml BSA (Sigma), 25\u2009\u03bcg/ml gentamicin (Gibco), 0.5% antibiotic/antimycotic (Invitrogen), 20\u2009ng/ml basic fibroblast growth factor, and 20\u2009ng/ml EGF (Peprotech). Cells were maintained in culture for up to passage 10 (low passage). Patient-derived PDX GBM cells (GBM811) was obtained from North Western University and was propagated in immunocompromised NOD-SCID mice. The tumor was disintegrated into a cell suspension at the time of intracranial tumor implantation29.GL261 syngeneic (C57BL/6 mouse derived) GBM cell line was obtained from Dr. Ted Johnson (Augusta University) and was authenticated in 2016. The human glioma U251 cell line was obtained from Dr. Steve Brown of Henry Ford Health System. The cell line was authenticated in July 2014 using the STR profiling method. The cell line, U251 was grown in high glucose (4.5\u2009g/L) Dulbecco\u2019s modified eagle\u2019s medium (DMEM) and GL261 in RPMI , supplemented with 10% fetal bovine serum (FBS), 2\u2009mM glutamine and 100\u2009U/ml penicillin and streptomycin at 5% CO3, U251: 2.4\u2009\u00d7\u2009105, and PDX GBM811: 5\u2009\u00d7\u2009104) in a volume of 3\u2009\u00b5l was lowered to a depth of 2.5 mm and then raised to a depth of 2 mm. During and after the injection, the careful note was made of any reflux from the injection site. After completing the injection, we waited 2\u20133\u2009minutes before withdrawing in a stepwise manner. The surgical hole was sealed with bone wax. Finally, the skull was swabbed with betadine before suturing the skin.Animals were anesthetized with 100\u2009mg/kg ketamine and 15\u2009mg/kg xylazine i.p. The surgical zone was swabbed with betadine solution, the eyes coated with Lacri-lube, and the animals were immobilized in a small animal stereotactic device . After draping, a 1-cm incision was made 2 mm to the right of the midline 1 mm retro-orbitally; the skull was exposed with cotton-tip applicators, and a 23\u2009G needle tip was used to drill a hole 2 mm to the right of the bregma, taking care not to penetrate the dura. A 10\u2009\u00b5L Hamilton syringe with a 26G-needle containing tumor cells  were sublethally irradiated (6\u2009Gy)34. After 24\u2009hours, recipient mice were injected intravenously with BM cells (10\u201315\u2009\u00d7\u2009106 cells) collected from donor transgenic mice (control or p65 KO mice). All mononuclear cells were separated from red blood cells using lymphocyte cell separation media .Human U251 and PDX GBM811 were orthotopically implanted in chimeric mice developed through our published protocol34. An appropriate state of anesthesia was obtained with isoflurane . After positioning using a triplanar FLASH sequence, MR studies were performed using pre-contrast T1, T2-weighted and post-contrast T1-weighted MRI scans with the following parameters: (1) Standard T1-weighted multislice sequence , # of averages\u2009=\u20094); (2) T2-mapping sequence  sequence, TE\u2009=\u200910, 20, 30, 40, 50, 60 msec, TR\u2009=\u20093000 msec, 256\u2009\u00d7\u2009256 matrix, 13\u201315 slices, 1 mm thick slice, 32 mm field of view (FOV), # of averages\u2009=\u20092). Post contrast T1WI was used to determine the volume of tumors in vehicle and p65 deleted mice by drawing irregular ROI to encircle the whole tumor in each image section-containing tumor using ImageJ software, and the area was then multiplied by the thickness of image slice to determine the volume (cm3). Two investigators blinded to the animal groups determined tumor volume.All MRI experiments were conducted using a 7 Tesla 12\u2009cm (clear bore) magnet interfaced to a varian console with actively shielded gradients of 49 gauss/cm and 100\u00b5s rise times or a horizontal 7 Tesla BioSpec MRI spectrometer  equipped with a 12\u2009cm self-shielded gradient set (45 gauss/cm max). Detailed MRI procedures were adopted from our several previous publicationsFreshly isolated tumor, spleen and BM samples from each group were passed through a 40\u00b5m-cell strainer to make single cells. Cells were labeled with antibodies (Bio Legend) against immune cell antigens such as CD45, CD4, CD8, Gr1, CD11b, F4/80, CD68, CD206 , and CD86  in the tumor as well as in spleen and BM. CSC antigens such as CD133 and CD44 were analyzed on tumor cells (CD45\u2212). Immunogenicity or presence of MHC1 (H2Ld-H2Db) on GL261 tumor cell line was analyzed using specific antibody (BioLegend). Flow cytometry data were acquired using Accuri C6 machine (BD Biosciences) and analyzed by BD Accuri C6 software.After MRI at day 22, control mice (n\u2009=\u20093) and p65 KO mice (n\u2009=\u20093) were euthanized and brains were collected for paraffin fixed tissue sections and later stained to determine histology through standard H&E, expression of CD11b (Abcam) and p65  through immunofluorescence, expression of proliferation marker Ki67 (Dako) and apoptosis marker cleaved caspase 3  through immunohistochemistry using standard methods. Mice staining data were validated using human GBM tumors (n\u2009=\u200910) collected from Augusta University Biorepository under an IRB approved protocol. The data were acquired using an automated all-in-one microscope .www.qiagenbioinformatics.com)12. Freshly isolated total CD3+T cells isolated from normal spleen and lymph nodes were labeled with CFSE dye per the manufacturer\u2019s protocol , and were stimulated with 10\u2009\u03bcg/mL plate-bound anti-CD3, 2.5\u2009\u03bcg/mL soluble anti-CD28, and cocultured with CD11b+ cells. Non-stimulated T cells and CD11b monoculture were used as a control for the assay. T cell proliferation was monitored 72\u2009hours later by flow cytometry70. To identify the direct effect of proinflammatory cytokines such as IFN-\u03b3, TNF-\u03b1, and IL1-\u03b2 on T cell proliferation, sorted T cells were cultured similarly. Stimulated T cells were treated with IFN-\u03b3 (100 U/ml), TNF-\u03b1 (100 ng/ml), or IL1-\u03b2 (100 ng/ml), or a combination of the three. Following co-culture, T cells were labeled with CD4 and CD8, and CD11b cells were labeled with CD86 mature DC marker to compare the cell proliferation at all the conditions. Co-culture supernatant was tested for cytokine expression using membrane-based protein array, as described above.CD11b positive myeloid cells from tumors from p65 control and p65KO mice were harvested using positive selection method (Miltenyi Biotec). Purity for each population ranged from 90\u201399%, as detected by flow cytometry. Sorted CD11b cells were used for total protein isolation and were processed for customized mouse cytokine array (44 factors) (Ray Biotech). Membranes were imaged using Las-3000 imaging machine . All signals (expression intensity) emitted from the membrane were normalized to the positive control spots of the corresponding membrane using ImageJ software. A web-based Ingenuity Pathway Analysis (IPA) interfaced for the pathway and network analysis (Bone marrow cells from p65 KO mice (n\u2009=\u20092) were collected through the standard protocol. CD11b positive myeloid cells and CD11b negative cells were harvested using positive selection method (Miltenyi Biotech). Brain tissue from P2 pups was dissociated using the Neuronal Dissociation Kit (Miltenyi #130\u2013092\u2013628), and microglia were isolated using CD11b positive selection (Miltenyi #130-093-636). Cells and tissues were processed for protein isolation using Pierce RIPA buffer . Protein concentrations were estimated with Pierce, BCA protein assay kit , and separated by standard Tris/Glycine/SDS gel electrophoresis. Membranes were incubated with primary antibodies against p65 , and \u03b2-actin  followed by horseradish peroxidase-conjugated secondary antibody . The blots were developed using a Pierce Super Signal West Pico Chemiluminescent Substrate kit . Western blot images were acquired by Las-3000 imaging machine .4 GL261 or HF2303 (patient GBM derived cells) tumor cells were plated on matrigel in 96 wells plate. The tumor cells were cultured with the 50% conditioned medium derived from the culture of wild-type BMDMs, control BMDMs or p65 KO BMDMs. Images were acquired right after adding conditioned medium (day 0), on day 2 and day 5 of culture with 5x magnification using Amscope inverted microscope equipped with a digital camera (Amscope).Assay was performed using a standard protocol. Briefly, a total of 1\u2009\u00d7\u200910www.oncomine.org) and GEO dataset (GSE4630)35.The mRNA expression data from brain cancer patient\u2019s datasets were analyzed using Oncomine data   or Student t-test. Differences were considered statistically significant at p-value\u2009<\u20090.05.Supplementary information"}
+{"text": "Oryzias latipes) is a popular animal model used in vertebrate genetic analysis. Recently, an efficient (~\u200930%) knock-in system via non-homologous end joining (NHEJ) was established in zebrafish using the CRISPR/Cas9 system. If the same technique were applicable in medaka, it would greatly expand the usefulness of this model organism. The question of the applicability of CRISPR/Cas9 in medaka, however, has yet to be addressed.Medaka (We report the highly efficient generation of knock-in transgenic medaka via non-homologous end joining (NHEJ). Donor plasmid containing a heat-shock promoter and a reporter gene was co-injected with a short guide RNA (sgRNA) targeted for genome digestion, an sgRNA targeted for donor plasmid digestion, and Cas9 mRNA. Broad transgene expression in the expression domain of a target gene was observed in approximately 25% of injected embryos. By raising these animals, we established stable knock-in transgenic fish with several different constructs for five genetic loci, obtaining transgenic founders at efficiencies of >\u200950% for all five loci. Further, we show that the method is useful for obtaining mutant alleles. In the experiments where transgene integrations were targeted between the transcription start site and the initiation methionine, the resultant transgenic fish became mutant alleles.With its simplicity, design flexibility, and high efficiency, we propose that CRISPR/Cas9-mediated knock-in via NHEJ will become a standard method for the generation of transgenic and mutant medaka.The online version of this article (10.1186/s40851-017-0086-3) contains supplementary material, which is available to authorized users. Oryzias latipes) is a small freshwater teleost species. Similar to zebrafish (Danio rerio),\u00a0medaka is a popular animal model for vertebrate genetic analysis and offers many advantages, including the availability of highly polymorphic inbred strains that can be effectively used for genetic mapping ..hsp70.1 Zebrafish hspa8 promoter, approximately 2.6\u00a0kb in length , 10, wasTemplate DNA for sgRNA synthesize was PCR-amplified from pDR274  with thevacht and nr5a1 genes, we tested two sgRNAs. The sgRNA that yielded best results in F0 expression assay was chosen to generate stable transgenic fish  purified donor DNA. Each embryo was injected with a solution containing ~\u20099\u00a0ng/\u03bcl of sgRNA for digesting Tbait, ~\u200918\u00a0ng/\u03bcl of sgRNA for digesting genome DNA, ~\u2009200\u00a0ng/\u03bcl of Cas9 mRNA, and ~\u20099\u00a0ng/\u03bcl of donor plasmid. Injection volume was adjusted such that approximately 30\u201375% of injected embryos were dead within one week after injection  and a 3\u2032 primer that was specific to the donor plasmid . To examine whether the tandem-array insertion in the same direction occurred, a PCR reaction was performed with the two primers within the donor plasmid.sox5 and pax7a transgenic fish, nucleotide sequences of PCR products were determined to examine the joint regions of the insertions.For the Images were taken using an MVX10 microscope , an MZ APO stereomicroscope , and an LSM700 confocal laser-scanning microscope .vacht  gene, which is known to be expressed in cholinergic neurons , was chosen as an initial target. The donor plasmid (Tbait-hs-lRl-GFP) contains a bait sequence  upstreacht Fig. .Fig. 1StWe tested two sgRNAs  in the Tg[vacht-hs:lRl-GFP] fish described above was aimed such that RFP expression could be converted to GFP expression by the application of Cre. To conveniently change RFP transgenic fish to GFP transgenic fish, we generated transgenic medaka that ubiquitously express Cre-mCherry-NLS fusion protein  in earlyTg[zhsp8:Cre-mCherry-NLS] transgenic medaka was generated by a Tol2-based transgenic method Fig.\u00a0. Transgenr5a1  gene, and examined if the same technique worked for this gene. The nr5a1 gene is known to be expressed in cells in the hypothalamus (hp), interrenal gland (ir), and gonad (g)  , interrenal gland (ir), and gonad (g) Figs. . The anivacht and nr5a1 genes, we did not aim to disrupt gene functions. The insertion sites were set upstream of the genes, not in the exons. In zebrafish studies, the NHEJ-mediated knock-in technique has also shown to be effective for obtaining mutant alleles by inserting the transgene into exons  transgenic zebrafish. The same strategy could be used to generate transgenic medaka that express reporter/driver genes that are not fluorescent themselves.We report that the NHEJ-mediated knock-in system is highly efficient in medaka, and is very useful for establishing mutant alleles. With its simplicity and high efficiency, we propose that the method described may become a standard technique for the generation of transgenic and mutant medaka.Additional file 1: Table S1.Genomic location of potential off-target sites for sgT (sgRNA for Tbait) (DOCX 14 kb)Additional file 2: Table S2.Status of the insertions in the transgenic fish generated in this study (DOCX 16 kb)Additional file 3: Figure S1.GFP expression in Tg[pax7a-hs:GFP] and Tg[sox5-hs:GFP].  Tg[pax7a-hs:GFP].  Tg[sox5-hs:GFP].  Dorsal views of the head region magnified.  Lateral views of the anterior trunk region magnified. The signal in the tectum is especially strong in Tg[pax7a-hs:GFP] (A). Presumable pigment cell progenitors of xanthophore and leucophore (B) and xanthophore (D) on the body surface are positive for GFP in Tg[pax7a-hs:GFP] and Tg[sox5-hs:GFP], respectively. (JPEG 1442 kb)Additional file 4: Figure S2.sox5 transgenic fish with the forward integration (#1, #3, and #4 strains; Additional file sox5 transgenic fish with the reverse integration (#2 strains; Additional file sox5 transgenic fish with the forward integration (#1, #2, #3, and #7 strains; Additional file sox5 transgenic fish with the reverse integration (#6 strain; Additional file pax7a transgenic fish with the forward integration (#1, #3, #4, and #6 strains; Additional file pax7a transgenic fish with the reverse integration (#2 strains; Additional file pax7a transgenic fish with the forward integration (#1and #6 strains; Additional file pax7a transgenic fish with the reverse integration (#2, #3, #4, and #5 strains; Additional file Nucleotide sequences of the joint region of the insertions for the sox5 and pax7a transgenic fish The PCR products that span the genomic DNA and the donor DNA (see Additional file"}
+{"text": "Porphyromonas gingivalis has been extensively associated with both the onset and progression of periodontitis. We previously isolated and characterized two P. gingivalis strains, one from a patient exhibiting severe chronic periodontitis (CP3) and another from a periodontally healthy individual (H3). We previously showed that CP3 and H3 exhibit differences in virulence since H3 showed a lower resistance to cationic peptides compared with CP3, and a lower ability to induce proliferation in gingival epithelial cells. Here, we aimed to determine whether differences in virulence between these two strains are associated with the presence or absence of specific genes encoding virulence factors. We sequenced the whole genomes of both P. gingivalis CP3 and H3 and conducted a comparative analysis regarding P. gingivalis virulence genetic determinants. To do so, we performed a homology search of predicted protein sequences in CP3 and H3 genomes against the most characterized virulence genes for P. gingivalis available in the literature. In addition, we performed a genomic comparison of CP3 and H3 with all the 62 genomes of P. gingivalis found in NCBI's RefSeq database. This approach allowed us to determine the evolutionary relationships of CP3 and H3 with other virulent and avirulent strains; and additionally, to detect variability in presence/absence of virulence genes among P. gingivalis genomes. Our results show genetic variability in the hemagglutinin genes. While CP3 possesses one copy of hagA and two of hagC, H3 has no hagA and only one copy of hagC. Experimentally, this finding is related to lower in vitro hemmaglutination ability of H3 compared to CP3. Moreover, while CP3 encodes a gene for a major fimbrium subunit FimA type 4 (CP3_00160), H3 possess a FimA type 1 (H3_01400). Such genetic differences are in agreement with both lower biofilm formation ability and less intracellular invasion to oral epithelial cells exhibited by H3, compared with the virulent strain CP3. Therefore, here we provide new results on the genome sequences, comparative genomics analyses, and phenotypic analyses of two P. gingivalis strains. The genomics comparison of these two strains with the other 62 genomes included in the analysis provided relevant results regarding genetic determinants and their association with P. gingivalis virulence. Porphyromonas gingivalis is a Gram-negative anaerobic bacterium that significantly contributes to the onset and progression of periodontitis  and periodontitis subjects  show differences in their virulence. For example, strains from periodontitis patients exhibit a higher resistance to antimicrobial peptides    by the bacterium through erythrocyte binding  obtained from a Chilean periodontitis patient exhibiting severe clinical signs of inflammation and tissue damage, as well as of an attenuated strain (H3) obtained from a periodontally-healthy individual. In addition, we performed a comparison between CP3 and H3 with all the available genomes of in vitro even when compared to the highly virulent strain W50; H3 showed low virulent properties in vitro  and probing depth (PD) . While CP. gingivalis reference strains ATCC 33277, W50 (ATCC 53978), and clinical isolates CP3 and H3, were grown as described previously  = 0.6\u20130.8. For invasion assays, gingival epithelial cells (OKF6/TERT2) were infected at a multiplicity of infection (MOI) of 100 for 90 min at 37\u00b0C and 5% CO2. After infection, cells were washed with PBS and incubated 2 h with fresh media supplemented with 300 ug/mL gentamicin and 200 \u03bcg/mL metronidazole. Then, cells were rinsed with PBS and incubated with 1% saponin, followed by serial dilution of the resulting supernatant, and plating on blood agar plates supplemented with hemin and menadione. After 5\u20137 days, colony-forming units (CFU) were quantified. Adhesion assays were performed as described above, except that cells were infected at 4\u00b0C. Results were compared by applying ANOVA testing and Dunnett's multiple comparison to analyze statistical differences.P. gingivalis clinical isolates CP3 and H3 in tubes containing lysis/stabilization buffer. Samples were lysed using bead-beating, and DNA was extracted by a guanidine thiocyanate silica column-based purification method . All downloaded P. gingivalis genomes were reannotated using Prokka as with CP3 and H3 genomes.In order to perform a comparative genomic analysis of CP3 and H3 with others P. gingivalis dataset , using the pyani Python3 module  for results' visualization. kSNP3.0 was used for SNP identification in the 64 P. gingivalis genomes and the core polymorphic positions were used for the phylogenetic reconstruction through Maximum Likelihood inference using RAxML v8.2.9. The obtained tree was plotted using FigTree v1.4.4 .Average nucleotide identity (ANI) was calculated for all 64-genome P. gingivalis was performed through the Roary v3.7.0 pipeline . Also, virulence genes were identified in the 64-genome dataset using pan-genome results to evaluate presence/absence and protein sequence variation of these genes. According to shared and unique genes between CP3 and H3 genomes, a Venn diagram was plotted using the VennDiagram R package (https://cran.r-project.org/package=VennDiagram). The inferred phylogenetic tree was annotated based on the presence/absence of the virulence genes that are part of the accessory genome, using the pheatmap R package and FigTree.The pan-genome analysis of the 64-genome P. gingivalis cultures grown to exponential phase were obtained, diluted, and inoculated to each well of a 96-well flat bottom plate in supplemented BHI medium . Then, plates were incubated anaerobically at 37\u00b0C for 72 h. After the incubation time, the supernatant was removed, wells were washed twice by immersion in distilled water and the plate was left to air dry for 1 h. Biofilm staining was performed by adding 100 \u03bcL of 0.1% safranin and incubating for 15 min, followed by two washes by immersion in distilled water. Finally, the dye retained in the biofilm was eluted incubating with 100 \u03bcL of 95% ethanol for 5 min, the elution was transferred to a new 96-well plate and the absorbance was measured at 490 nm. Results were compared by applying ANOVA testing and Dunnett's multiple comparison to analyze statistical differences.P. gingivalis strains  were grown to an exponential phase culture, which was adjusted to DO600 = 2.0 (3 \u00d7 108 CFU/mL). Then, two hundred \u03bcL of each suspension was added to one well of a 96-well round-bottom plate. After that, each suspension  was serially diluted, by taking 100 \u03bcL and mixed with 100 \u03bcL PBS (1:2 dilution). This step was repeated until 1:64 dilution. Finally, each well was mixed with an equal volume of 2% sheep erythrocytes at 37\u00b0C for 3 h. Experiments were repeated three times.One mL of defibrinated horse blood was centrifuged at 2,100 rpm for 5 min. The resulting pellet (red blood cells) was washed 3 times and then diluted in PBS to 2% solution. In parallel, hagC and 16S rRNA genes, 1,000 ng of RNA were used to synthetize cDNA and amplify by quantitative real-time polymerase chain reaction (qRT-PCR) using appropriate primers  and the Brilliant II SYBR qPCR master mix  in a Mx3000p qPCR system . The cycling program was as follows: 95\u00b0C for 10 min, followed by 45 cycles of 95\u00b0C for 10 s, 58\u00b0C for 30 s, and 72\u00b0C for 20 s. Finally, a melting curve was obtained by incubating at 95\u00b0C for 15 s, 60\u00b0C for 1 min, and 95\u00b0C for 15 s, to detect non-specific product formation and false positive amplification. As an endogenous control, we determined the16S rRNA gene expression levels. Data were statistically analyzed using PRISM software . Comparisons between strains were made using by applying ANOVA testing and Dunnett's multiple comparison to analyze statistical differences.Total cytoplasmic RNA was isolated from CP3, W50, and H3 strains using the TRIZOL method as described previously  and SGAZ00000000 (H3), under BioProject PRJNA521311. The version described in this paper is version SGBA01000000 (CP3) and SGAZ01000000 (H3).This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession de novo assembly of the 2.25 and 2.31 Mb-length genomes of CP3 and H3, respectively. CP3 and H3 genomes are composed by 118 and 165 contigs, with a N50 value of 37,600 and 39,176 bp, GC content of 48.47 and 48.35%, and predicted protein count of 1,896 and 1,955, respectively. These values are similar with average statistics values for the 62 P. gingivalis available genomes at NCBI's RefSeq database . Both assembled genomes exhibit high completeness, having a 95.3% of bacterial orthologs present in the OrthoDB database. The average coverage of CP3 and H3 genomes is 300x and 28x, respectively. Genome features of both strains are displayed in A total of 3.43 million sequences were sequenced and an average of 8% of the raw sequence reads were filtered out. Quality-controlled reads of the CP3 L1 and L2 libraries, and H3 library were used to perform the P. gingivalis strains, an ANI analysis using the 64-genome P. gingivalis dataset  was performed , 12 are part of the 64-genome dataset accessory genome and were selected to evaluate their distribution along the phylogeny  and exhibits exactly the same pattern of virulence genes than CP3, though they are not closely related  in the literature, and a screening of these genes in CP3 and H3 genomes through a bidirectional blast was conducted , H3 have only four . Three of these genes belong to the CP3 and H3 core genome (>95% identity) and encode non-characterized hemagglutinins . The other three hag copies found in CP3 genome and one of the H3 gene copies belong to their accessory genome (<95% identity). Moreover, there are differences in the type of hemagglutinins found in the accessory genome: while hagA is only present in CP3 (CP3_01901) and no in H3, CP3 possesses two copies of hagC , while H3 possesses only one (H3_01383)  .fimA, which encodes different major fimbriae types. Interestingly, while type IV fimA is present in CP3 and absent in H3, type I fimA is present in H3 and absent in CP3 , H3 only agglutinates when 3.0 \u00d7 108 CFUs where added. The hemagglutination titer of red blood cells showed by the virulent reference strain W50 was similar compared to CP3 and higher compared to H3  and H3 possesses fimbria type I  possesses a significantly higher ability to form biofilms that H3 (encoding type I fimA) (Since there are differences in the  I fimA) . InteresP. gingivalis has been largely associated with periodontitis, showing both one of the highest prevalence and abundance among periodontitis-associated species , and the other from a periodontally healthy subject (H3)  and ~34 to 35% of \u201cunique\u201d genes  ], revealed the presence of heteroduplex types; some of them significantly associated with periodontitis and others equally detected in health or disease  induce different cellular responses (Missailidis et al., Regarding FimA genotypes, type I P. gingivalis virulence factors, not only FimA has been related to epithelial cell adhesion/invasion and biofilm formation. HagA promotes adhesion to epithelial cells, and antibodies against its hemagglutinin domain protects against oral infection in a murine model (Frazer et al., in vitro model. The hagB gene of P. gingivalis, which is 98.6% identical to hagC, encodes HagB that mediates adhesion to host cells (Progulske-Fox et al., hagC found in H3 has a major effect on its ability of adhere to and invade epithelial cells. Similarly, HagA has been associated with co-aggregation between P. gingivalis and Treponema denticola, which is another periodontitis-associated bacteria (Ito et al., Interestingly, among P. gingivalis, they have been related to exopolysaccharide synthesis in other bacterial species. In Bacillus subtilis, the locus epsHIJK is necessary for the synthesis poly-N-acetylglucosamine, which is a key component of the biofilm (Roux et al., epsJ genes are annotated as putative glycosyltransferases, showing similarity to icaA in Staphylococcus aureus and pgaC in Escherichia coli. EpsD and EpsJ have also been associated to exopolysaccharides synthesis in Lactococcus lactis (van Kranenburg et al., P. gingivalis virulence, we cannot discard that these genetic differences between CP3 and H3 could participate in their biofilm formation ability (P. gingivalis genomes, this study provides relevant results regarding genetic determinants and their association with P. gingivalis virulence.Unlike previous reports comparing clinical isolates obtained from patients showing different periodontal disease severity, no differences in capsular antigen genotypes were found between CP3 and H3 strains (van Winkelhoff et al.,  ability . AltogetThe datasets generated for this study can be found in NCBI PRJNA521311.P. gingivalis strains. JP-D contributed to conception and critically reviewed the manuscript. EC-N contributed to the design of the genomic analyses, wrote, and reviewed the manuscript. DB conceived and designed the research, wrote, and reviewed the manuscript. All authors contributed to the final version.KM performed the comparative genomics analyses, wrote, and reviewed the manuscript. AH analyzed the data, wrote and reviewed the manuscript. CS carried out hemagglutination, adhesion/invasion assays, and RT-qPCR experiments. IB carried out biofilm assays. MO carried out hemagglutination assays. CM performed sequencing of 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": "Peripartum cardiomyopathy (PPCM) is a rare cause of heart failure that develops during the last month of pregnancy or within first months of delivery. We report the case of a 40-year-old woman diagnosed with severely symptomatic PPCM characterized by left ventricular ejection fraction (LVEF) of 10% and significant dyssynchrony secondary to a left bundle branch block (LBBB). Early cardiac resynchronization therapy (CRT) was used to achieve remarkable functional and LVEF recovery. This case suggests that early CRT must be considered for patients suffering from severely symptomatic PPCM despite optimal medical therapy for whom advanced heart failure therapies are proposed. Peripartum cardiomyopathy (PPCM) is a rare and idiopathic cause of heart failure that develops during the last month of pregnancy or within first months of delivery. As any heart failure with reduced left ventricular ejection fraction (LVEF), a left ventricular assist device (LVAD) or a heart transplant must be considered in severely symptomatic patients despite optimal medical therapy. Less than 5% of patients suffering from PPCM will have an enlarged QRS width but this characteristic must not be overlooked as some could benefit from cardiac resynchronization therapy (CRT) .A 40-year-old woman  was evaluated at our specialized center for rapidly progressive symptoms of heart failure in the first week postpartum. The patient\u2019s medical background and first three pregnancies were unremarkable. The last pregnancy was uneventful except for a caesarian section for nuchal cord during a term spontaneous labor. She was prescribed antibiotics on day five postpartum for new onset dyspnea and dry coughing. She rapidly deteriorated with dyspnea at rest, orthopnea, and paroxysmal nocturnal dyspnea. Echocardiography showed a left ventricular ejection fraction (LVEF) of 10%, severe left ventricular dilatation with left ventricular end-systolic diameter (LVESD) of 58 mm and moderate functional mitral regurgitation. The electrocardiogram showed sinus rhythm and new onset left bundle branch block (LBBB) with a QRS width of 158 ms. Dobutamine perfusion was initiated for low cardiac output signs along with intravenous Furosemide. The coronary angiogram was normal and the Swan\u2013Ganz catheterization showed a cardiac index of 1.71 L/min on inotropic support with normal pulmonary artery and capillary wedge pressures. The cardiac MRI showed no sign of inflammation or fibrosis. At this point, the differential diagnosis included peripartum cardiomyopathy and idiopathic dilated cardiomyopathy. However, the latter seemed less likely because of the absence of previous symptoms and the acute onset of symptoms in the early postpartum period, which is characteristic of peripartum cardiomyopathy. Valsartan 40 mg PO twice a day and Eplerenone 25 mg PO daily was initiated. Slow weaning of Dobutamine was completed after five days and small dose Metoprolol was eventually added. Despite uptitration of therapy, the patient did not improve. Bromocriptine was discussed with the patient considering some evidence to support its use but was not initiated because of the initial preference of the patient to breastfeed. However, the inherent difficulties of the long hospitalization led the patient to abandon breastfeeding [After the intervention, the patient improved to a NYHA functional class II. Control echocardiography showed resolution of mitral regurgitation and a mild increase in LVEF of 15%. The patient was followed at our specialized heart failure outpatient clinic and the titration of the optimal medical therapy was done including Sacubitril-Valsartan up to 49\u201351 mg PO twice a day, Bisoprolol 10 mg PO daily, and Aldactone 25 mg PO daily. The echocardiography follow-up showed increase in LVEF to 35% and 50% after seven and thirteen months respectively .Treatment of PPCM is mainly based on guidelines directed optimal medical therapy but studies with specific treatments, like Bromocriptine and Pentoxifylline, suggest some benefits . AlthougIn the IPAC study, no patient with both baseline LVEF < 30% and an LVEDD \u2265 60 mm had complete recovery at 12 months . With a In conclusion, in presence of LBBB with severe systolic dysfunction, early CRT should be considered to accelerate functional recovery in patients with PPCM that are evaluated for advanced support therapies considering that the other options are far more invasive and morbid."}
+{"text": "P=0.033 and P=0.063). And it was demonstrated that PMRT were independently associated with increased OS according to univariate and multivariate analyses. However, no significant differences in BCSS or OS were observed between the groups stratified by the number of positive lymph nodes. In conclusion, PMRT significantly improved OS for TNBC patients with T1-2 and 1-3 lymph nodes involved. Additional prospective studies are needed to provide a stronger evidence base for choosing patients for PMRT.There is consensus on the routine use of postmastectomy radiotherapy (PMRT) in patients with four or more positive axillary lymph nodes. However, the benefits of PMRT in patients with T1-2 and 1-3 involved lymph nodes still remain controversial. Data from the Surveillance, Epidemiology, and End Results Program (SEER) of the United States between 2010 and 2012 were used to analyze the outcomes of 675 triple-negative breast cancer (TNBC) patients with T1-2 and 1-3 lymph nodes involved. Those patients were subdivided into radiotherapy (RT) (312) and no-RT groups (363). After a median follow-up time of 37 months, Kaplan-Meier analysis showed that PMRT significantly improved overall survival (OS) but not breast cancer-specific survival (BCSS) in the total cohort of 675 patients ( Breast cancer is a highly heterogeneous disease, which is reflected in different clinical, pathologic, and molecular features. Currently, breast cancers are classified into four molecular subtypes including, luminal A, luminal B, human epidermal growth factor receptor 2 (HER2) overexpressed, and triple-negative breast cancer (TNBC) subtypes according to estrogen receptor (ER), progesterone receptor (PR), and HER2 status by gene expression profiling , 2. The Adjuvant radiotherapy plays a pivotal role in the treatment of breast cancer, since it can reduce the risk of loco-regional recurrence and overall mortality , 6. And Given the poor prognosis and aggressive characteristics of TNBC patients and the controversy surrounding PMRT as appropriate for patients with small tumors with 1\u20133 positive nodes, our study is designed to investigate the effect of postmastectomy radiotherapy on OS and breast cancer-specific survival (BCSS) among TNBC patients with T1-2 and 1-3 positive lymph nodes by utilizing population-based SEER data, and to further determine which patients are most likely to benefit from PMRT.A summary of demographic and clinical characteristics is shown in P=0.033 and P=0.063). In addition, no significant differences in OS was observed between the groups stratified by the number of positive lymph nodes; P=0.073, P=0.455, P=0.144 for 1 LN, 2 LNs, and 3 LNs. there was no significant difference in BCSS when patients were stratified by the number of LN; P=0.181, P=0.452, and P=0.176 for 1 LN, 2 LNs and 3 LNs.We used the Kaplan-Meier plot and log-rank tests to compare BCSS and OS between different subgroups according to lymph node their status and the results are illustrated in P=0.009 for OS and HR, 1.484 with a 95%CI, 1.035 to 2.127 and P=0.032 for BCSS. Compared with tumors sized 2 cm to 5 cm (T2), patients with \u22642 cm (T1) tumors had a better OS and BCSS with univariate analyses; HR, 0.617 with a 95%CI, 0.416 to 0.917 and P=0.017 for OS and HR, 0.548 with a 95%CI, 0.353 to 0.852 and P=0.008 for BCSS. This result retained significance in multivariate analyses of OS and BCSS  and 20-year rates of breast cancer mortality . But theIn clinical practice, considering the delayed complications from radiation including cardiac toxicity, lymphedema, skin fibrosis, and so on, some surgeons and radiation oncologist do not recommend PMRT to patients with 1-3 positive axillary lymph nodes. But TNBC is a special subtype of breast cancer. Patients with TNBC are likely to suffer poorer survival and are prone to early locoregional recurrence and distant metastasis. Previous studies have demonstrated that different molecular subtypes had different response to PMRT. Wen et al.  found thAlthough no statistical difference was observed for BCSS among the total cohort and by stratified analysis, a benefit trend was reflected from Kaplan-Meier analysis which was consistent with the trend in OS, thus these observations should be interpreted with caution. We speculated that the small percentage of patients who have died from breast cancer and the small sample size of patients who died in subgroup analysis could be one potential explanation for the finite benefits from PMRT. And hopefully will extending the follow-up time make our results more satisfactory.There are several limitations of our study. Firstly, we were compelled to focus on short follow-up time because the information regarding HER-2 expression in the SEER database was not available until 2010. But for the TNBC subtype, an early peak of recurrence occurs within the first 2-3 years after diagnosis. Secondly, the SEER database is absent from information about LRR. However, information about chemotherapy is offered by the latest version of the SEER database. And we endeavored to mitigate several confounding variables, such as endocrine and trastuzumab treatment by selecting TNBC as the object of our study rather than other subtypes.In conclusion, our study shares some similar results with the recent literature that there is a significant improvement on OS after PMRT among TNBC patients with T1-2 and 1-3 positive axillary lymph nodes. In accordance with current consensuses and guidelines, we suggest that PMRT should be strongly considered in T1-2N1 breast cancer patients particularly in TNBC, an aggressive and poor prognosis disease. Additional prospective randomized studies are needed to provide a stronger evidence base for selecting patients for PMRT.The data in the SEER database do not require informed patient consent because cancer is a disease reported by every state of the United States. But a Data-Use Agreement for the SEER 1973\u20132014 Research Data File has been completed.in situ disease, and no record of radiotherapy. We calculated follow-up durations from January 1, 2010 to December 31, 2014 to ensure adequate follow-up duration.SEER*Stat version 8.3.4 was used to generate a case listing. We identified 675 patients screened according to the following inclusion criteria: female; year of diagnosis from 2010 to 2012; age of diagnosis between 20 years and 80 years; breast cancer as the first and only malignant cancer diagnosis; pathologically confirmed invasive ductal carcinoma-not otherwise specified (ICD-O-3 8500/3), unilateral cancer; TNBC subtype ; histological grades I-III; AJCC T stagesI-II; known tumor size category; known lymph node status; record of chemotherapy therapy, mastectomy received. We excluded patients with inflammatory breast cancer, P-values were two-sided and P<0.05 were considered statistically significant. All statistical analyses were carried out using SPSS version 22.0 software .Patients were divided into RT group and no-RT group categorized by radiotherapy status. A Chi-squared test was used to compare the demographic and clinical characteristics of the included cases between the two groups. The Kaplan-Meier analysis was performed to generate survival curves, and the log-rank test was used to compare the unadjusted BCSS and OS rates of patients with different lymph node status. BCSS was measured from the date of diagnosis to the date of breast cancer death. OS was defined as the time from the date of diagnosis to the date of death due to any cause (including breast cancer) or the last follow-up. Adjusted hazard ratios (HRs) with 95% confidence interval (CI) were calculated using a Cox proportional hazard regression model to estimate the outcome-related factors. All"}
+{"text": "Growing evidence reported that vitamin D deficiency is a common finding in obesity. Vitamin D status also seems to be sex-related, although little is known regarding this association. Therefore, the aim of this study was to investigate the sex-related differences of serum 25OH vitamin D (25OHD) concentrations across body mass index (BMI) classes and, if there were any differences, whether they could be explained by sex-related differences in body composition. We enrolled 500 subjects . Body composition was assessed by bioelectrical impedance analysis (BIA) phase-sensitive system. Serum 25OHD concentration was quantified by a direct, competitive chemiluminescence immunoassay. Vitamin D deficiency was defined as a serum 25OHD concentrations < 20 ng/mL (50 nmol/L). Stratifying the sample population according to sex and BMI categories, 25OHD concentrations were significantly higher in males compared to females in all BMI classes and decreased along with the increase of BMI values. Females with vitamin D deficiency had higher fat mass (FM) % compared to males with vitamin D deficiency. The 25OHD concentrations inversely correlated with FM % in both sexes. In a multiple regression analysis model, sex, FM %, and BMI were predictive factors of 25OHD concentration. In conclusion, our study suggests that 25OHD concentrations were lower in females than males across all BMI categories. Given the tight correlation between 25OHD concentrations and FM %, it can be hypothesized that the lower 25OHD concentrations in females than males can be explained by the fact that females have a higher amount of fat than males.  Howeverngements . Furtherngements . Johnsonngements  reportedngements .Thus, the objectives of our study were (1) to investigate the sex differences in vitamin D status across BMI classes and, (2) if any differences exist, whether it could be explained by sex-related differences in body composition. The experimental design of the study was an observational prospective cross-sectional study.n. 173/16). Every enrolled subject provided informed consent after a thorough explanation of the protocol.The study was performed at the Department of Clinical Medicine and Surgery, Endocrinology Unit, University Federico II, Naples , from 13 October 2016 to 25 March 2019. The study was carried out taking into account the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. The protocol was formally approved by the Ethical Committee of the University of Naples \u201cFederico II\u201d Medical School . We enrolled female subjects that were non-pregnant and non-lactating. A full medical history, including drug use, was collected. Hypocaloric diet in the last three months (5 subjects);Chronic diseases that could interfere fluid homeostasis, such as liver or renal chronic diseases, cancer, acute or chronic inflammatory diseases (13 subjects);Altered concentration of serum creatinine, serum calcium, or albumin;Presence of T2DM  \u2265 6.5% (\u226548 mmol/mol) on two occasions, or both at the same time. Participants on antidiabetic medication were considered to have T2DM (18 subjects);Uncontrolled thyroid or parathyroid disease (3 subjects);Current therapy with calcium, vitamin D supplementation or osteoporosis therapies, anti-inflammatory drugs, statin, and other hypolipidemic agents (22 subjects);Alcohol dependence diagnosed based on the Diagnostic and Statistical Manual of Mental Disorders (DSM)-V diagnostic criteria (2 subjects);Pacemakers or defibrillators which could potentially interfere with BIA assessment;2 (8 subjects).Patients having a BMI lower than 18.5 kg/mIn order to increase the homogeneity of the subject samples, we included only adults of both gender with the following criteria of exclusion :Hypocalo2), kg/m2) was calculated after measuring weight and height. A wall-mounted stadiometer  was used to measure height while a calibrated balance beam scale  was used to assess weight. The degrees of obesity were established according to the World Health Organization\u2019s (WHO) criteria: BMI: 18.5\u201324.9 kg/m2, normal weight; BMI: 25.0\u201329.9 kg/m2, overweight; BMI: 30.0\u201334.9 kg/m2, grade I obesity; BMI: 35.0\u201339.9 kg/m2, grade II obesity; BMI \u2265 40.0 kg/m2, grade III obesity [Measurements were performed in the morning, after an overnight fast. The anthropometric assessment was performed following standard criteria by the same nutritionist. The subjects were recommended to dress in light clothes and to remove shoes during the assessment ,16,17. TThe BIA phase-sensitive system  was used by experienced observers to assess body composition  as previWe stored the blood samples, collected in the morning after an overnight 8 h fast, at \u221280 \u00b0C until they were processed. Serum 25OHD concentrations were measured with chemiluminescence . The analytical measurement range of detection was 4\u2013150 ng/mL, whereas the intra-assay coefficients of variation (CVs) were 5.4%, 2.8%, and 4.7%, and the inter-assay CVs were 10.1%, 4.8%, and 7.9% for low, medium, and high points of the standard curve, respectively, as previously reported ,24,25.Vitamin D deficiency was defined as a serum 25OHD concentrations < 20 ng/mL (50 nmol/L), insufficiency between 21 and 29 ng/mL (from 52.5 to 72.5 nmol/L), and normal concentrations \u2265 30 ng/mL (75 nmol/L)  as previThe biases of our study were the following: (1) the data were not adjusted for seasonal variation and this was due to the fact that the subjects were enrolled in the very same season; (2) we did not adjust the data for sun exposure; however, we enrolled people living in the same Metropolitan area and with the same similar lifestyle habits regarding sun exposure; (3) we did not correct the data for vitamin D dietary intake; however, we excluded people taking vitamin D supplements.www.clincalc.com) based on the results from Carnevale et al. [The sample size was determined using the software ClinCalc tool  test was used to determine the significance of differences in frequency distribution among BMI classes  and 25OHD categories . Pearson r correlation coefficients were used in order to investigate correlations among 25OHD concentration with age, anthropometric measurements and body composition parameters after adjusting for BMI and FM. In addition, a multiple linear regression analysis model (stepwise method), expressed as R2, Beta (\u03b2) and t, with 25OHD concentration as dependent variables were used to estimate the predictive value of gender, BMI, and FM % on 25OHD concentration. Data were collected and analyzed using the MedCalc\u00ae package . The data distribution was evaluated by the Kolmogorov\u2013Smirnov test and the abnormal data were normalized by logarithm. Skewed variables were back-transformed for presentation in tables and figures. Baseline descriptive statistics, including means and standard deviations for continuous characteristics and frequencies and percentages for categorical characteristics, were calculated. Similarly, descriptive statistics for all primary and secondary outcome measures at all time points in the study were also calculated. The differences between males and females in terms of age, anthropometric characteristics, 25OHD concentration, and body composition parameters were analyzed by Student\u2019s paired The study population consisted of 500 participants: 250 males and 250 females. Age, anthropometric characteristics, and vitamin D of the study population are reported according to sex in Although both males and females had comparable values of BMI, 25OHD concentrations were significantly higher in males than females, although both of them had vitamin D deficiency. The percentage of subjects with vitamin D insufficiency were significantly lower in males compared to females, while the percentage of subjects with vitamin D sufficiency was higher in males compared to females. Although there was no significant differences regarding the percentage of subjects with vitamin D deficiency between males and females, we found a trend toward a higher percentage of subjects with vitamin D deficiency in females.In p < 0.001). Moreover, we found a trend toward higher FM % in females compared to males in both vitamin D insufficiency and sufficiency categories.The correlations among 25OHD concentrations, age, BMI, and body composition parameters assessed by BIA are reported in p < 0.001), followed by BMI and sex. Results are reported in To compare the relative predictive power of sex, FM %, and BMI on 25OHD concentration, we performed a multiple linear regression analysis using a model that included as independent variables sex, BMI, and FM %, and 25OHD as a dependent variable. Using this model, FM % was entered at the first step , FM %, TBW (Lt), and ICW. A positive correlation between R (\u03a9) and 25OHD concentrations were found only in males.As shown by the results of the multivariate analysis, sex, BMI and FM were predictive factors of 25OHD concentrations. As already reported, FM represents a reservoir of 25OHD and its metabolites ,27 thus The strengths of our study include the prospective collection of data in a large, homogenous population of individuals with different BMI categories and the assessment of body composition that allow us to partially explained the sex difference in 25OHD concentration. We did not adjust data for seasonal variations, but in order to minimize this bias, we enrolled all the subjects in the same season. Another limit of our study was the lack of assessment of sun exposure. However, all the subjects live in the same metropolitan area and, thus, have similar lifestyle habits regarding sun exposure. Although dual X-ray absorptiometry is considered the gold standard to measure body composition, we used BIA to assess body composition given the good relative agreement of BIA with dual X-ray absorptiometry results ,42. The In conclusion, females had significantly lower 25OHD concentrations than males among the different class of BMI. This sex difference was mainly explained by the higher FM % in women than men. If these findings are also confirmed in other seasons, they may create a background for the revision of vitamin D supplementation dosages. Indeed, future guidelines for monitoring 25OHD concentrations should take into account not only life stages but also sex. Further, a careful assessment of body composition in order to quantify FM % may be warranted in order to adjust vitamin D supplementation dosages. Randomized controlled trials exploring the dose\u2013response effect of vitamin D supplementation according to gender are highly warranted."}
+{"text": "One of the greatest challenges currently facing those studying Mendelian disease is identifying the pathogenic variant from the long list produced by a next-generation sequencing test. We investigate the predictive ability of homozygosity mapping for identifying the regions likely to contain the causative variant.We use 179 homozygous pathogenic variants from three independent cohorts to investigate the predictive power of homozygosity mapping.We demonstrate that homozygous pathogenic variants in our cohorts are disproportionately likely to be found within one of the largest regions of homozygosity: 80% of pathogenic variants are found in a homozygous region that is in the ten largest regions in a sample. The maximal predictive power is achieved in patients with <8% homozygosity and variants >3\u2009Mb from a telomere; this gives an area under the curve (AUC) of 0.735 and results in 92% of the causative variants being in one of the ten largest homozygous regions.This predictive power can be used to prioritize the list of candidate variants in gene discovery studies. When classifying a homozygous variant the size and rank of the region of homozygosity in which the candidate variant is located can also be considered as supporting evidence for pathogenicity. It is now possible to screen thousands of genes in a single test. However, this generates an extensive list of variants. One of the greatest challenges currently facing those studying Mendelian disease is identifying the pathogenic variant amongst the myriad of other variants.3 Identifying target genes within shared regions of homozygosity is a critical step in consanguineous families with recessive disorders.4 Regions of homozygosity are created when identical-by-descent haplotypes are inherited from parents. A homozygosity map can be generated directly from next-generation sequencing data, identifying regions likely to contain the causative variant.5Searching for shared regions of homozygosity between affected individuals has been used to identify genes causing recessive Mendelian diseases.6 Thus we would expect variants that cause recessive Mendelian disease to be contained in the largest regions of homozygosity.The number and size of homozygous regions within an individual\u2019s genome is influenced by ancestral population effects and recent consanguineous events. It is important to differentiate the two cases as disease-causing variants are likely to be in regions of recent homozygosity; variants in ancestral regions of homozygosity have been exposed to selection in a homozygous state for sufficient time for selection to act on them. Ancestral regions of homozygosity are likely to be smaller, less than a megabase, whereas homozygous regions that are the result of recent consanguinity tend to be multiple megabases in length.To test the hypothesis that homozygous pathogenic variants are more likely to be found in the largest regions of homozygosity in a sample, we used a data set of 99 consanguineous patients with previously identified homozygous pathogenic variants. We then replicated our findings in two further cohorts, with 17 and 63 patients respectively.9 99 consanguineous patients were diagnosed as having a homozygous pathogenic variant.Our discovery cohort consisted of patients referred to the molecular genetics department at the Royal Devon and Exeter Hospital for genetic testing for neonatal diabetes (NDM) or hyperinsulinemic hypoglycemia (HH). Samples were sequenced on a targeted gene panel test for monogenic diabetes and HH.8 with a pathogenic or likely pathogenic homozygous variant identified using trio exome sequencing and shared via DECIPHER.10We replicated our findings in two further cohorts: first, consanguineous patients with severe pediatric disorders where exome sequencing identified 17 homozygous pathogenic variants; and second, 63 consanguineous children from the Deciphering Developmental Disorders (DDD) study11 Levels of homozygosity were similar between cohorts: discovery cohort mean 8.7% (SD 4.5%), severe pediatric disorders cohort 8.8% (6.6%), DDD cohort 9.2% (4.5%).Patients were defined as consanguineous if more than 1.5% of their genome was covered by homozygous regions >3\u2009Mb. This is the expected percentage of homozygosity for offspring of second cousin marriages.Informed consent was obtained at referral. See 13 For the two replication cohorts, regions of homozygosity were calculated from VCF files using SavvyVcfHomozygosity.13 The pathogenic variants in our samples were discovered independently of the regions of homozygosity mapping; they were not used to guide variant discovery.For our discovery cohort, regions of homozygosity were detected directly from the targeted sequencing data using SavvyHomozygosity, which uses off-target reads.In our discovery cohort we found that the largest regions of homozygosity in each sample were more likely to contain the pathogenic variant. In fact, the rank (receiver operator characteristic [ROC] area under the curve [AUC] 0.666), size (AUC 0.627), and relative size (size of homozygous region divided by size of the largest region in the sample) (AUC 0.668) all have predictive power . We have sufficient power to detect this effect: a minimum of 51 samples is required to detect the proportion with 80% power and P\u2009=\u20090.05. This pattern is demonstrated by the ROC curve in Supplementary Figure\u00a0Figure\u00a0We replicated our findings in two independent cohorts. The rank, size, and relative size of the homozygous regions all have predictive power in both replication cohorts . If we only include samples with <8% homozygosity and exclude variants within 3\u2009Mb of a telomere the AUC increases to 0.735 and 92% of causative variants are in one of the ten largest homozygous regions . Additionally genes near telomeres were more likely to have causative variants that were not in the ten largest regions  levels of homozygosity and we suggest that the biological principle should be generally applicable across individuals and populations\u2014that the causative homozygous variant will tend to be in a larger homozygous region, because these are the result of recent consanguineous events. This metric should be applicable for all consanguineous patients\u2014consanguinity (homozygosity >1.5%) can be determined from sequencing data and does not need to be known a priori.The samples for this study are from multiple global populations, which could be a confounding factor as different populations are known to have different patterns of homozygosity.The predictive power of homozygous regions should be agnostic to the method used to call the regions; however, certain areas of the genome are harder to sequence and thus contain more false heterozygous variants, which have the potential to artificially break up large regions of homozygosity. This can be reduced by using only variants that are in Hardy\u2013Weinberg equilibrium and allowing some heterozygous variants within homozygous regions.14  to widely used tools such as SIFT (AUC 0.631\u20130.848) and PolyPhen (AUC 0.596\u20130.859)We can apply our results to prioritize the list of candidate variants in gene discovery studies. For example, 80% of pathogenic variants are found in a homozygous region that is in the ten largest regions but only 61% of homozygous bases fulfill the same criteria. Using such a prioritization enriches the remaining regions for pathogenic variants. This is of particular value for gene discovery within consanguineous cohorts without multiple affected members in a single family to narrow down target regions.In conclusion, the size, rank, and relative size of the homozygous region a variant is found in provides evidence of its likely pathogenicity. 92 percent of pathogenic variants are found in the ten largest regions of homozygosity (excluding samples >8% homozygosity and variants within 3\u2009Mb of a telomere). We suggest this criterion could be used in the context of the ACMG guidelines as a potential source of supporting evidence for variant pathogenicity.Supplementary Figure 1Supplementary Figure 2Supplementary InformationSupplementary Table"}
+{"text": "Zea mays L.) in the smallholder farms of eastern India are outcomes of a complex interplay of climatic variations, soil fertility gradients, socio-economic factors, and differential management intensities. Several machine learning approaches were used in this study to investigate the relative influences of multiple biophysical, socio-economic, and crop management features in determining maize yield variability using several machine learning approaches. Soil fertility status was assessed in 180 farms and paired with the surveyed data on maize yield, socio-economic conditions, and agronomic management. The C&RT relative variable importance plot identified farm size, total labor, soil factors, seed rate, fertilizer, and organic manure as influential factors. Among the three approaches compared for classifying maize yield, the artificial neural network (ANN) yielded the least (25%) misclassification on validation samples. The random forest partial dependence plots revealed a positive association between farm size and maize productivity. Nonlinear support vector machine boundary analysis for the eight top important variables revealed complex interactions underpinning maize yield response. Notably, farm size and total labor synergistically increased maize yield. Future research integrating these algorithms with empirical crop growth models and crop simulation models for ex-ante yield estimations could result in further improvement.Yield gaps of maize ( Bridging the large yield gaps in smallholder farms of Asia and Africa, with significant regional and interpersonal variations, is necessary to reduce global food insecurity  for classification purpose. The ANN was run in the WEKA data mining package. We optimized parameters of ANN via \u2018CVParameterSelection\u2019 module. Note that RF, SVM, and ANN were applied on the whole dataset and further applied on a split of data (135 training 75% and 44 test 25%).In the present study, we first used a C&RT algorithm known for predicting quantitative or classifying categorical targets by recursively dividing the dataset . The C&R2), cross-validation RMSE (RMSEcv), residual prediction deviation (RPD), and bias were used for judging model predictability.After establishing the influencing variables by the abovementioned classification algorithms, the RF regression was used to predict the Maize yield using the whole dataset with full cross-validation. The coefficient of determination (R-1) surpassed the overall productivity of Bankura (3.41 t ha-1) by 11.14%, no significant yield difference was observed between them. Pooled total maize yield varied from 0.11\u20138.25 t ha-1 with Q1, Q2, Q3, and Q4 ranging from 0.11\u20131.86, 1.86\u20134.0, 4.0\u20134.81, and 4.81\u20138.25 t ha-1, respectively. Considerable variation in soil properties was apparent between districts. Malda had finer-textured soils with higher OC (21% higher), EC (61.54% higher), and pH (18.33%) (-1) with larger interquartile range (37.5% larger) was found in Malda. A similar trend was obtained for available P.Although the overall productivity of Malda (3.79 t ha(18.33%) . On the -1), available N (77\u2013308 kg ha-1), available P (4.9\u201321.7 kg ha-1), available K (24.0\u2013432.9 kg ha-1), clay (10.8\u201344.8%), sand (17.2\u201383.2%), and silt (4\u201342%). Organic carbon was significantly correlated with all parameters except available N, P, and S.The statistical moments of all measured soil variables for pooled data are shown in 1) , clay minerals , alkyl asymmetric\u2013symmetric doublet (2\u03c51), carboxylic acids (3\u03c51), smectite (\u03c51+\u03b4a or \u03c51+\u03b4b) or illite (\u03c51+\u03b4), respectively  or  or 74] oThe RF perfectly classified the yield data with 0% misclassification both on the whole dataset and the 75% training set (n = 135) . ConversUsing the whole dataset, the RF relative variable importance analysis based on the Gini criterion exhibited an interesting trend. The leading influential variables were all the numeric variables that complemented the C&RT important predictors , althoug2 value of 0.94 (RMSE = 846 kg ha-1). The RF regression variable importance plot  was sufficiently sensitive to capture spectral features of soil OC and clay minerals. Assigning precise wavebands for individual soil parameters was difficult due to the complex nature of soil matrix. Consequently, determining the relationship between the size of the PC score and the loading was not straightforward. For simplicity, we used negative spectral scores for each SPC1 and SPC2 as comprehensive indicators of both soil OC and clay content. In contrast, PC1 had significantly larger positive loadings on clay (0.972) and OC (0.543) while PC2 had significantly larger positive loading on available P (0.482) than rest of the variables with minor positive and negative loadings. The larger the absolute value of loading weight, the greater the contribution of the corresponding input variable to the output. Thus, clay and OC were the most influential variables in PC1 while available P was most influential in PC2. Large positive values of PC1 represented large values of clay and OC, while large positive values of PC2 were associated with high soil available P. To aid interpretation, we used PC1 to denote the combined effect of clay and OC while PC2 denoted available soil P.-1 , which is close to the recommendations of the state department of agriculture. It is well known that optimum plant stand is key to achieving resource use efficiency and higher productivity in maize, and this is critical in situations where farm resources are scarce and optimum nutrient management is not assured [In the C&RT analysis, the appearance of seeding rate as the primary splitting node stems from the fact that most of the farmers in Malda sow maize seeds with specific row arrangements (line sowing method) while the farmers of Bankura prefer broadcasting with a higher seeding rate. Data suggested optimum seeding rates in the range of 17.63\u201327.78 kg ha assured . Nutrien assured , 58, 59, assured , 60. How assured , 62.The variable \u2018agro-ecological region\u2019, represented by two districts, was not selected by C&RT as an explanatory variable, suggesting that site effects were explained instead by the biophysical and management variables. Notably, it was observed that seeding rate, organic manure, and total labour showed more than one threshold value that reappeared as splitting criteria, signifying their multi-modal distribution in the dataset. These variables did not have a monotonous relationship with maize yield and had optimal quantitative ranges associated with higher maize yields (in combination with ranges of other variables). This was expected since maize yield variability, like that of many other crops, is governed by complex interactions of climatic, socio-economic, and crop management practices , 63, 64.According to Tittonell et al. , soil feYield variability was also attributable to differences in farm size and productivity. Efficiency of farm size increases with the number of family members of working age and with the household\u2019s working capital or resource endowment . Farm siA misclassification rate between 47 and 0% is large but realistic, based on the complex interactions among several biophysical, management, and socio-economic factors affecting maize yield. Such interactions are common in smallholder systems ,77, whicIn the RF proximity plot , farms hAlthough RF regression was able to satisfactorily predict maize yield, we refrained from over-interpreting the model prediction accuracy. Our main objective was to test the capability of a new methodological framework to help explain different factors and their interactions that affect maize yield. Our objective was not to develop a laboratory-grade predictive model. Moreover, Jame and Cutforth  argued tIn the RF partial dependence plots , the posThe yield gap of maize in eastern India is a complex interplay of climatic variations, soil fertility gradients, differential management intensities and farmer socioeconomics. With an increasing shift to maize-based cropping systems in eastern India replacing the conventional rice-based system, understanding maize yield determinants has become critical for creating effective interventions. This study has drawn upon a host of complex interacting yield determining factors, using machine learning approaches like PSR, C&RT, RF, SVM, and ANN to identify important biophysical, socio-economic, and crop management factors for explaining maize yield. The C&RT relative variable importance plot identified farm size, total labor, soil factors, seed rate, fertilizer, and organic manure as influential factors. Among three classification approaches compared for classifying maize yield classes, ANN produced the smallest misclassification rate on the test set and outperformed RF and SVM. In the RF classification scheme, all the numeric variables appeared as the leading influential variables to classify maize yield. Moreover, the RF partial dependence plots exhibited a positive relationship between farm size and maize productivity. A nonlinear SVM boundary for the leading eight influential variables revealed complex interactions between influential factors in determining maize yield response. These algorithms may be used both in future empirical studies and in developing efficient crop simulation models for ex-ante yield estimations of field crops.S1 File(DOC)Click here for additional data file.S2 File(DOC)Click here for additional data file."}
+{"text": "Understanding the aetiology of the diverse recovery patterns in bilingual aphasia is a theoretical challenge with implications for treatment. Loss of control over intact language networks provides a parsimonious starting point that can be tested using in-silico lesions. We simulated a complex recovery pattern  to test the hypothesis\u2014from an established hierarchical control model\u2014that loss of control was mediated by constraints on neuromodulatory resources. We used active (Bayesian) inference to simulate a selective loss of sensory precision; i.e., confidence in the causes of sensations. This in-silico lesion altered the precision of beliefs about task relevant states, including appropriate actions, and reproduced exactly the recovery pattern of interest. As sensory precision has been linked to acetylcholine release, these simulations endorse the conjecture that loss of neuromodulatory control can explain this atypical recovery pattern. We discuss the relevance of this finding for other recovery patterns. Lesions induced by stroke or by head injury in speakers of more than one language elicit a variety of recovery patterns, e.g., ,2. We laNeuropsychological case reports of language recovery in bilingual speakers document instances best characterised as indicative of intact language networks with impaired control. For instance, S.J., a Friulian-Italian speaker, had intact clausal processing in both languages but in conversation was unable to avoid switching inappropriately into Friulian, for example, when speaking to an Italian-only speaker . ProblemConsistent with this separation of language networks and their control\u2014but particularly challenging from an explanatory point of view\u2014are case reports of individuals with a pattern of alternate antagonism and paradoxical translation. Paradis et al.  reportedGreen  offered To simulate the recovery patterns of alternate antagonism and paradoxical translation, we introduce in-silico lesions to a synthetic subject engaged in active inference, described in detail in Active inference provides a first principles, Bayesian, account of how agents actively engage with a given environment ,15,16. CThis Bayesian approach is formally distinct from previous computational approaches to aphasia ,18,19,20Under active inference, we are concerned with what agents must believe to render their behaviour optimal, instead of why it appears pathological. This allows us to characterise patients with brain damage as operating under ideal Bayesian assumptions but with a poor  generative model ,23. In oThe in-silico lesions used in this work are motivated by the neuromodulatory control account described above. These lesions alter the confidence (or precision) in prior beliefs about the causes of sensory observations. These changes in precision can be linked to (a loss of) neuromodulatory control, and to acetylcholine in particular ,26,27. IOur simulations were carried out using an active (Bayesian) inference model of three tasks: picture naming, word repetition and translation. In this section, we first provide a concise overview of active inference and then present the generative model used for the simulations. Please see Active inference brings together perception and action under a formal, first principles, Bayesian framework ,15,16. IF)  or maximF) ,30. HereUnder active inference, sentient creatures not only have capacity to infer the state of the world but can also influence the future  by taking actions. These actions are selected from trajectories of all plausible actions  ,32 in acP(\u03c4o|\u03c4s):The expected free energy can be derived from the free energy by taking an expectation under predicted outcomes in the future From this free energy formulation, we can optimise expectations about hidden states, policies and precision through inference and optimise the model parameters through learning. This involves the  message passing of sufficient statistics of posterior beliefs among neuronal populations. Here, the sufficient statistics are just the expected probability of being in a particular state or pursuing a particular policy . Variational message passing can be formulated as a gradient descent on variational free energy, using a mean-field approximation ,34. ThisThe process theory underwriting active inference is based on a partially observable Markov decision process (POMDP). This can be defined as a generative model which, in its simplest form, has discrete outcomes that are caused by discrete hidden states, and is described extensively in previous work ,37. A ge\u03c0.In this generative model, outcomes  depend upon hidden states and hidden states depend upon policies. Outcomes and hidden states are generated by two sets of categorical probability distributions parametrised by Outcomes are generated by selecting appropriate policies using a softmax function of their expected free energies. Therefore, policies are more probable, a priori, if they minimise the expected free energy, which depends upon prior preferences about outcomes. From this, sequences of hidden states are generated using state transitions specified by the selected policy. These hidden states generate outcomes. The hidden states can also influence the expected free energy. Here, model parameters are equipped with a prior  distribution.This model specification may sound rather technical and complicated; however, it is the simplest and most generic kind of generative model for deeply structured state transitions of the sort required to generate behaviours such as speech. The form of this generative model can be considered the \u2018structure\u2019 in structure\u2013function relationships. This structure is thought to underwrite functional brain architectures that realise active inference; e.g., . This peer time) .Our aim was to illustrate how alternate antagonism with paradoxical translation could be mediated by a loss of sensory precision, and an implicit loss of neuromodulatory control. For this purpose, we modelled three language tasks using a minimal generative model: (1) picture naming, where the subject is presented with a visual stimulus and must verbally identify it , (2) worThe generative model has one level with five (latent or hidden) state factors: context, target language, heard language, concept and epoch, along with five outcome modalities: task, language, audition, visual and feedback . The conThe likelihood, A, is represented by the lines connecting states to outcomes in The transition matrices, B, are represented by lines modelling transitions among states within each factor in The model was allowed to choose from a set of 2 different one-step policies (sequences of actions); both are a different permutation of how (controlled) state transitions might play out. The prior beliefs about the initial states were initialised to 1 for all repeated and target word levels, epoch 1 for the epoch factor and zero otherwise. The model had no capacity to learn between each simulation. Additionally, certain aspects of this generative model and the optimisation process  can be mapped onto the functional anatomy in the brain, e.g., states  probability distributions: a precise distribution which has confident beliefs centred around the true value, and an imprecise distribution which implies flatter beliefs about the true value . In termFor our simulations, perturbing precision control was implemented using discrete updates to the precision hyperparameter, \u03c9, over A until saturation. This mimics precision control\u2014regulated by higher levels of the model hierarchy\u2014where belief updates influence parameter uncertainty. Neurobiologically, changes in the precision of beliefs about outcomes given states of the world (A) is altered by acetylcholine ,26,27. ABoth our model simulations\u2014lesioned and control\u2014were exposed to the same paradigm procedure each day . This inIn this section, we describe the simulation of alternate antagonism and paradoxical translation recovery patterns in bilingual aphasia based on the patient cases reported in  and summWe simulated these recovery patterns using an active inference under a generative model of the three tasks: picture naming, word repetition and translating a heard word . We distSimulation of the recovery pattern was accomplished with discrete changes in sensory precision \u03c9; i.e., 0.1, 0.25, 0.5, 1, that alternated between the heard language affected. These changes limit the subject\u2019s ability to select the appropriate language. Over time, these failures of inference resolve\u2014as precision increased\u2014to rebalance control over processing in both languages. The responses of a lesioned subject \u2014and a coAs shown in To make this concrete, the impaired belief updating is shown in www.fil.ion.ucl.ac.uk/spm/. Specifically, the code (and simulated data) necessary to reproduce the simulations and figures can be found here: www.github.com/ucbtns/aapt.The data presented above were simulated using a generic optimisation scheme implemented using standard functions. These are available under the SPM academic software: Bilingual patients reveal a variety of patterns of language recovery. Our goal was to explore the putative neurocomputational basis of one complex pattern  with the hope of identifying the factors that might elicit other patterns of recovery. We build on a symbolic hierarchical control model ,13 that The tenet of our approach is that behaviour has to be understood from the perspective of agents acting in their worlds. This tenet is embodied in the active inference (Bayesian) approach to understanding behavioural deficits following on neurological damage. Central to the approach is the notion that action in the world is underwritten by the agent\u2019s model of how the world generates her sensations. The objective\u2014in active inference\u2014is to adjust the generative model, so it provides the best explanation of sensory observations, thereby minimising surprise and reducing uncertainty about the present and future states of the world.To simulate the recovery pattern we lesioned an in-silico active inference agent and probed her responses to three key tasks: picture naming , word repetition  and translating a heard a word . Our in-silico lesion reduced the model\u2019s ability to select an appropriate action . It did so by altering confidence, that is, precision, in prior beliefs about the causes of a sensory observation such as hearing a word in L1. Before this sensory precision stabilised, the synthetic subject exhibited a pattern of alternate antagonism, combined with paradoxical translation with repetition remaining essentially intact. Our simulations accordingly provide a proof in principle that aberrant precision control can explain a complex pattern of recovery seen in neuropsychology. The simulation is agnostic with respect to details of neural implementation. However, in line with  that losNeuromodulators exert widespread influence on neural networks in the brain. Recent research  establisThe findings we report concern just one pattern of recovery\u2014alternate antagonism and paradoxical translation . HoweverOur simulations involved explicit manipulation of precision\u2014mimicking fluctuating precision control\u2014regulated by some higher levels of the model hierarchy. We acknowledge that a more dynamical account is possible in a more expressive generative model. For this, we could go in one of two ways\u2014either include continuous lower level parameters, responsible for speech production including the fundamental pitch (F0),  or discrChanges in precision are sufficient to account for a complex pattern of recovery but our simulation does not establish that they are necessary. Other manipulations within the generative model may be equally successful in explaining similar recovery patterns. For example, one could manipulate priors over target language state transitions. That is, how do an agent\u2019s posterior beliefs about the current target language shape their beliefs about the future. Such manipulations would directly affect language control and might be associated with noradrenaline release, cf. .Regardless of the outcomes of further research, the simulations we report establish the importance of understanding how language networks are controlled in order to understand recovery patterns in bilingual speakers. Furthermore, they may be relevant to language recovery in speakers of a single language, where network control is increasingly acknowledged as crucial for understanding language recovery .Our active (Bayesian) inference approach offers a different (complementary) perspective to other computational approaches, which is particularly useful for understanding behavioural deficits arising from neurological damage. This approach defines computational pathologies, underwritten by abnormal prior beliefs, that are a Bayes-optimal response to damage. We demonstrated this by lesioning an in-silico active inference generative model and established that variations in confidence in prior beliefs (precision) about the causes of a sensory observations (such as hearing a word) were sufficient to explain the recovery pattern of alternate antagonism and paradoxical translation. Neurobiologically, changes in precision have been linked to the neuromodulator acetylcholine. As such, our data support the notion that constraints on a neuromodulatory resource mediate a loss of language control, whereas stabilising that resource yields normal performance."}
+{"text": "There are 3 million refugees living in the United States today whose health and wellbeing may be diminished by not being able to understand and use health information. Little is known about these barriers to health in multiethnic refugee communities.This present study examined (1) the relationship between English proficiency, health literacy, length of time in the US, and health status; and (2) differences in poor health status caused by limited English proficiency and low health literacy individually and in combination to better understand which barriers might be addressed by improving refugee health.N = 136) age 18 to 65 years were recruited using health clinics and refugee resettlement agencies. Survey questions included demographics, health status, health literacy, English language proficiency, social determinants of health, and barriers to getting health care. Interpreters were used as necessary. We used a cross-sectional study with purposeful sampling.Refugees (r = 0.77) between health literacy and English proficiency; they were moderately correlated with health status . Length of time in the US only modestly correlated with health status (r = 0.16). Health literacy and English proficiency taken individually were strong predictors of health status  but not significant. Their interaction, however, was significant and accounted for most of the effect .There is a high correlation :e230\u2013e236.][English proficiency and health literacy individually and in combination facilitate poor health and present health-related barriers for refugees. Length of time in the US for refugees may not correlate with health status despite studies that suggest a change in health over time for the larger immigrant population. The combined effects of limited English proficiency and low health literacy can create significant barriers to good health outcomes in refugee populations. Length of time in the US for refugees may not correlate with health status despite studies that suggest a change in health over time for the larger immigrant population. More than 71 million people worldwide have been forcibly displaced due to ethnic violence, natural disasters, failed governments, civil wars, and political oppression and are unable to live safely in their own countries . Most ofLittle is known about barriers relating to health care for refugees who live in multiethnic communities; most studies have examined single-ethnic community needs, perceptions of health providers, or comparisons of immigrant versus host populations . ClarkstLimited English proficiency (LEP) is a social determinant that creates a barrier to health care ; patientThere are 3 million refugees living in the US today whose health and wellbeing may be diminished by not being able to understand and use health information. Our first research question examines relationships between health status, English proficiency, health literacy, and length of time in the US controlling for age and gender. Our second research question examines differences in poor health status by LEP and low health literacy individually and in combination. The present study examined these barriers that may be affected through resettlement efforts and policy change . FurtherAdult refugees who live in and around Clarkston, GA, were recruited using several health clinics and refugee re-settlement agencies; of the 150 people who were approached, 136 agreed to be in the study. All participants were born overseas and were age 18 years or older. The consent form was read to participants and interpreters were available to assist with informed consent and survey administration. Interpreters were required for 110 participants with the primary languages being Swahili, Arabic, and Burmese. One researcher was fluent in Swahili and French and one was fluent in Arabic so they assisted with consents and surveys in addition to student volunteers and clinic and agency volunteers and staff. The Georgia State University Institutional Review Board approved this study.The 58-question survey included questions about demographics, health, health literacy, English language proficiency, social determinants of health, and barriers to getting health care in the US. Demographic questions were age, gender, nativity, length of time in US, years of schooling, marital and family status, employment, and income and sources of income. Questions measuring English language proficiency were derived from the Programme for the International Assessment of Adult Competencies, an Organization for Economic Cooperation and Development  householall, most, or some of the time) and adequate health literacy (do not need help). An English Proficiency Scale was derived from the English proficiency questions \u201cHow well would you say you read/write/ speak English?\u201d These were dichotomized into not at all/ not well and well/very well. Length of time in the US was measured as less than or more than 2.5 years, which is our sample median; we chose the median because our responses had extreme outliers that, even when transformed was still significantly skewed, and because, within a few years of arrival, many refugees have improved English skills and are in the workforce  Do you have someone to help you read hospital or clinic materials?; (2) Do you have problems learning about your medical condition because you have trouble understanding written information?; and (3) Do you have trouble filling out medical forms by yourself? These questions contained points from the Brief Health Literacy Screener , a generorkforce .We used SAS 9.4 for data analysis. Descriptive statistics included means, standard deviations, frequencies, and chi-square calculations. Logistic modeling used bivariate dependent and independent variables to assess the individual and combined importance of English proficiency, health literacy, and length of time in the US with gender and age as covariates.N = 136) came from 22 different countries; 88% were from nine countries: . Participants' average age was 39 years and one-third of the participants were male. Slightly more than one-half have been in the US for 2.5 years or less, and 40% were employed. Educational attainment was less than an average of 9 years. Most participants were below the poverty level, and less than 30% had health insurance. Approximately two-thirds had LEP and low health literacy (Table 1).Participants (r(134) = .77, p < .05, and a moderate positive correlation between health literacy and health status r(134) = .40, p < .05 and English proficiency and health status r(134) = .37, p < .05 (Table 2).There was a statistically significant, strong positive correlation between health literacy and English proficiency, 2 (6) = 37.238, p < .05. Of the six predictor variables that were included in the model, only two were significant: the interaction between health literacy and English proficiency and age (as shown in Table 3). Those with inadequate health literacy and LEP were 5.08 times more likely to have poor/fair health, and as age increases health status decreases by 4%.A logistic regression was performed to ascertain the effects of health literacy, English proficiency, length of time in the US, the interaction between health literacy and English proficiency, gender , and age on health status. In individual regressions, both health literacy and English proficiency had substantial point estimates odds ration 4  and odds ratio 3.6  but neither was significant. A binomial logistic regression was performed to ascertain the effects of health literacy, English proficiency, the interaction of health literacy and English proficiency, length of time in the US, age, and gender on health status. The model was statistically significant XThis study has two key findings: (1) both English proficiency and health literacy are individual barriers to poor health but some combination of the two may be more indicative of health-related challenges for refugees; and (2) length of time in the US for refugees may not correlate with health status despite studies that suggest a change in health over time for the broader immigrant population.English proficiency continues to be one of the most challenging aspects of resettlement for refugees. LEP is a widely documented barrier to health care and can have a compounding effect on other social determinants like income, employment, and transportation . A gap eWe found that refugees who are more proficient in English also report stronger health literacy skills. Health literacy is a specialized form of literacy that includes reading, writing, speaking, and listening skills along with an ability to engage in dialogue with a health care provider . People LEP and low health literacy often co-occur but are less often studied together . There aWe know that adults with low health literacy may not understand how the body works or understand health terms; for refugees, the problem may be further compounded by discordant cultural beliefs\u2014health and wellbeing viewed through refugee cultural beliefs may be dissimilar to those of Western medicine . The abiWe found no significant relationship between length of time in the US and health. The \u201chealthy immigrant effect\u201d theory posits that the longer immigrants stay in the US, the worse their health becomes as they assimilate; health status equalizes between immigrant and nonimmigrant levels within 10 to 20 years . Most reRegardless of length of time in the US, refugees with both LEP and low health literacy are a particularly vulnerable group and have a high prevalence of poor health . Our stuN = 136).This study has many strengths, including being the first collection of literacy and health literacy data at the individual-level for a diverse refugee community that has rarely been studied. One limitation is that English proficiency and health status are self-reported; people may not be fully aware of their language limitations or health issues. Second, the validity and meaning of self-reported health literacy skills may also vary across refugee groups . Third,"}
+{"text": "Strength and toughness are both of great importance for the application of polylactic acid (PLA). Unfortunately, these two properties are often contradictory. In this work, an effective and practical strategy is proposed by using carboxylated graphene oxide (GC) grafted with polyethylene glycol (PEG), i.e. GC-g-PEG. The synthesis procedure of GC-g-PEG is firstly optimized. Then, a series of PLA nanocomposites were prepared by the melt blending method via masterbatch. In comparison to that achieved over pure PLA, these nanocomposites are of higher crystallinity, thermal stability and mechanical strength. This is mainly attributed to well-tailored interface and good dispersion. Especially, while retaining the tensile strength of the original PLA, the elongation at break increases by seven times by adding 0.3 wt% GC-g-PEG. In viewet al. reinforced PLA with GO grafted with PLA (GO-g-PLA) via solution blending and then compression moulding. He found that, in comparison to the neat PLA, the elongation at break and tensile strength of PLA/GO-g-PLA nanocomposites was increased by 114.3% and 105.7%, respectively [Graphene, as one of the most attractive nanofillers, is composed of a single-atom-thick graphite sheet, which has gained more and more attention owing to its superior mechanical strength, giant specific surface area, and distinguished electronic and gas barrier properties ,15. It iectively . To datePolyethylene glycol (PEG) is non-toxic, biocompatible and easily dissolved in water and various organic solvents . More imTherefore, Williamson synthesis was used to convert some of the hydroxyl groups of GO into carboxyl groups, resulting in more a reactive position of GO for hydroxyl groups of PEG ,29. Here2.2.1.N,N-dimethylformamide , NaOH , ethanol (\u226599.5%) and PEG with a weight-average molecular weight of 2 \u00d7 102 g mol\u22121 were delivered by Aladdin Industrial Corporation, China. Chloroacetic acid (\u226599.5%) was purchased from the Tianjin Damao Chemical Reagent Factory. Deionized water (DI) was used for all experimental water. All chemicals were used as received without any further purification.PLA (trade name 4032D) was purchased from Nature Works LLC (USA). Natural graphite powder (325 meshes), 4-dimethylaminopyridine , dimethylcyclohexylcarbodiimide , 2.2.\u22121, maintaining the mass ratio as 50 : 1. After that, chloroacetic acid was added to convert the hydroxyl group into a carboxyl group. The carboxylation procedure of GO is illustrated in GO was synthesized from natural graphite powders via a modified Hummer's method ,31. Subsx\u2019, of which the x represents the aim loading ratio of chloroacetic acid to NaOH.In order to investigate the effect of chloroacetic acid content on the graft ratio of GO, a series of GCs with the same NaOH/GO feed ratio were prepared with different amounts of chloroacetic acid . For abbreviation, the GCs were denoted as \u2018GC2.3.With the aid of ultrasonication, 100 mg of dried GC was added into 500 ml of DMF, forming a stable GC/DMF solution. Then, 0.5 g of PEG was added and stirred for 15 min before adding DCC and DMAP. The mixture was stirred at room temperature for 3 days, followed by an exhaustive dialysis process against DI water to remove all small molecules and unreacted PEG. The obtained composites were denoted as GC-g-PEG. In comparison, GO-g-PEG was synthesized via the same procedure.2.4.The masterbatch was prepared by the solution method to ensure the dispersion of GC-g-PEG and GO-g-PEG. Taking PLA/GC-g-PEG  as an example, 40 mg of GC-g-PEG was added in 40 ml of ethanol, followed by a bath sonication procedure for 60 min. Meanwhile, 2 g of PLA was dissolved in 20 ml of chloroform with a stirring speed of 45 rpm until dissolved completely. Then, the ethanol/GC-g-PEG suspension was poured into the chloroform solution of PLA, and excessive ethanol was added until no precipitation could be observed. The solution was then transferred to a PTFE evaporator to volatilize the liquid phase. After completely evaporating the ethanol solvent in a vacuum oven at 60\u00b0C, the masterbatch can be obtained and cut into small pieces.x, where x represents the mass fraction of GC-g-PEG. Similarly, PLA/GO-g-PEG 0.4, PLA/GO and PLA/GC were prepared by the same method for comparison.Prior to blending, PLA was dried in an oven at 80\u00b0C, while GC-g-PEG was dried in a vacuum oven at 50\u00b0C for at least 12 h. Melt blending was then operated using a HAKKE RHEOMIX  at 175\u00b0C with a screw speed of 55 rpm for 5 min. Then, the mixture was made into a dumbbell shape via a hot-pressing procedure with the vacuum laminating machine under the condition of 5 MPa and 175\u00b0C for 5 min. For clarification, the nanocomposites are labelled as PLA/GC-g-PEG 2.5.\u22121 and 32 scans in the wave number range from 400 to 4000 cm\u22121 at room temperature. X-ray diffraction (XRD) measurement was carried out by a  X-ray diffractometer at room temperature. The diffracted intensity of Cu K\u03b1 radiation (\u03bb = 1.542 \u00c5) was recorded at a scan rate of 5\u00b0 min\u22121 from 5\u00b0 to 30\u00b0. The graft ratio of modified GO was calculated from thermogravimetric analysis (TGA) using a NETZSCH-TA4/TG209 thermoanalyser (Germany), which was conducted under N2 atmosphere from ambient temperature to 750\u00b0C (10\u00b0C min\u22121). Raman spectra were recorded on a RM 2000 Microscopic confocal Raman spectrometer  using a 523 nm laser beam. X-ray photoelectron (XPS) spectra were obtained with a XSAM800 X-ray spectrometer  with double anode, operating in an FAT mode, with a pass energy of 20 eV and a power of 120 W. AI K\u03b1 X-ray (1486.6 eV) was chosen as the source of radiation.The chemical structure of the GO and GCs was analysed by a Spectrum GX Fourier transform infrared (FTIR) system  with a resolution of 4 cm\u22121 and held at 200\u00b0C for 3 min to remove the thermal history. Then, the samples were cooled down to 20\u00b0C at a cooling rate of 10\u00b0C min\u22121. Finally, the samples were again heated up to 200\u00b0C at a rate of 10\u00b0C min\u22121. Glass transition temperature (Tg), cold crystallization temperature (Tcc) and melting point (Tm) are determined by the secondary temperature curve. The thermal stability of nanocomposites can also be evaluated by TGA, which is conducted with the scan range from 30\u00b0 to 750\u00b0 at a constant heating rate of 10\u00b0C min\u22121 and continuous nitrogen flow.The crystallization behaviour of PLA, PLA/GO-g-PEG and PLA/GC-g-PEG nanocomposites was studied by using differential scanning calorimetry  measurements on a DSC instrument. Under the protection of nitrogen, the samples were firstly heated up to 200\u00b0C at a rate of 10\u00b0C min\u22121, the load cell size is 10 kN and at least five splines were tested in each group. Scanning electron microscopy (SEM) was conducted using a Hitachi S-5500 scanning electron microscope (Japan) at an acceleration voltage of 15 kV, and all the samples were sputter-coated with gold. The transmission electron microscopy (TEM) characterization was performed using a FEI Talos f200X electron (USA) microscope at an operating voltage of 220 kV.The tensile properties of the samples were measured using the universal electronic tensile machine  with reference to ASTM D638. The tensile rate was 5 mm min3.3.1.x and GO were characterized via FTIR, XRD, TGA, XPS, Raman and TEM. Figure\u00a01a shows the FTIR spectra of GO and GCx dispersed in high-quality and non-scattering potassium bromide (KBr) discs. Typical characteristic peaks of GO at 1720 cm\u22121 (C=O in carboxylic groups), 1617 cm\u22121 (C=C in aromatic ring), 1380 cm\u22121 (C\u2013O in carboxylic groups), 1249 cm\u22121 (hydroxyl C\u2013OH) and 1046 cm\u22121 (C\u2013O\u2013C in epoxide) were observed, as displayed in figure\u00a01a [\u22121 over GC50 and GC25, corresponding to C=O in the carboxyl group, is sharper than that observed over GO. Conversely, reflection at 1720 cm\u22121 over GC75 and GC0 is weaker than that observed over GO. Meanwhile, the peak at 3411 cm\u22121 obtained over GCx is weakened in comparison to that detected over the original GO, indicating that some of the hydroxyl groups are converted into carboxyl groups [In order to optimize the condition for carboxylation, different amounts of chloroacetic acid  were examined by applying them through the identical procedure with the same alkali concentration. The synthesized GCigure\u00a01a . Interesl groups ,34.Figub, after graphite is intercalated with sulfate ions, the distance between the sheets increased to 8.17 \u00c5. When the chloroacetic acid is grafted onto the GO surface, spacing expansion between the layers is also noted. For instance, when the content of chloroacetic acid is 25 times that of GO, the diffraction peak shifts from 10.74\u00b0 to 10.44\u00b0, and the d-spacing increases from 8.17 to 8.41 \u00c5. The d-spacing further increases to 8.9 \u00c5 for GC50. However, when chloroacetic acid content keeps increasing, i.e. GC75, the enhancement of layer spacing cannot be observed. In addition, it is interesting to note that the diffraction peak over GC0 shifts towards the right and the d-spacing decreases to 7.81 \u00c5 in comparison to that observed over GO. On the other hand, when sodium hydroxide is added without chloroacetic acid, the interlayer spacing is further reduced, which may be owing to the reduction reaction of GO [In addition, as shown in figure\u00a01on of GO .c displays the thermal decomposition behaviours of the GO and GCs. As expected, two steps of weight loss over GO can be observed, which happens at 90\u2013150\u00b0C and 150\u2013230\u00b0C, respectively. The first stage can be attributed to the loss of the surface hydroxyl group and adsorbed water, and the second stage is mainly owing to the thermal decomposition of the relatively stable oxygen-containing functional groups in the GO structure. In comparison, one step of weight loss over GCs is displayed. The platform at 90\u2013150\u00b0C disappeared, demonstrating a decrease in mass loss at 90\u2013150\u00b0C, while an increased mass loss at 150\u2013230\u00b0C is detected. This further suggests that the hydroxyl groups of GO are partially converted to carboxyl groups. In addition, the grafting ratio of carboxyl groups is calculated to be approximately 5\u201310%. The corresponding Raman spectra of GO and GC50 are displayed in figure\u00a01d. It can be seen that both Raman spectra contain the D- and G-bands at around 1341 cm\u22121 and 1596 cm\u22121, respectively. The intense D-band corresponded to the vibration of sp3-bonded carbon atoms and related to the structural defects of GO, while the G-band was reflective of the in-plane vibration of sp2-bonded carbon atoms. The intensity ratio of the D- and G-bands (ID/IG) of GO is 1.07 and that of GC50 is 1.16, which indicates that the ordered structure was further disrupted during the modification process. This is probably owing to the fact that the graft reaction of the chloroacetic acid segments can increase the disorder of the system.It has been established that the hydroxyl groups on GO sheets begin to decompose at 110\u00b0C and all hydroxyl groups disappear at 150\u00b0C, the remaining oxygen-containing functional groups gradually decompose at 150\u2013230\u00b0C . Figure\u00a0Figure\u00a01a). The wrinkles of GC increased and transparency became lower (b). After reaction with chloroacetic acid, the morphology was apparently maintained.The morphologies of GO and GC50 were investigated by TEM. GO showed a typical wrinkle layer structure and high transparency a. The wrme lower b. After In order to quantify the conversion of carboxyl functional group, XPS was performed to analyse the carboxyl content of GO and GC50, as shown in Herein, GC50 was selected to be esterified with PEG. In order to check whether grafting reactions were conducted, FTIR, TGA and XRD were conducted. FTIR spectra, TGA thermogram and XRD pattern of the GO, GC-g-PEG and GO-g-PEG are presented in a. Two new peaks at 2928 cm\u22121 and 2838 cm\u22121 were observed over GC-g-PEG and GO-g-PEG, respectively, corresponding to \u2013CH2 of the PEG, indicating that PEG was successfully grafted onto GO and GC by the esterification reaction [\u22121. XRD patterns of the original GO as well as GO-g-PEG and GC-g-PEG are displayed in figure\u00a04b. After modified by PEG through the esterification reaction, two reflections at 7.75\u00b0 and 7.18\u00b0 over GO-g-PEG and GC-g-PEG were detected, which corresponds to d-spacing of 11.30 and 12.20 \u00c5, respectively. This further implies that PEG is grafted on GO and GC successfully, which is consistent with the FTIR results. Figure\u00a04c,d demonstrates the thermal decomposition behaviours of the GO-g-PEG and the original GO as well as GC-g-PEG and GC50, respectively. In comparison to the original GO and GC50, there is an extra weight loss step observed over GO-g-PEG and GC-g-PEG at 170\u2013250\u00b0C. This can be attributed to the thermal decomposition of the PEG molecular chain. Note, the processing temperature of PLA is around 170\u00b0C, for which GO-g-PEG and GC-g-PEG can be processed at this temperature.FTIR spectra of GC-g-PEG and GO-g-PEG dispersed in high-quality and non-scattering KBr discs are shown in figure\u00a043.2.a, pure PLA exhibits a relatively smooth fracture surface with no plastic deformation. However, when 0.4% GO-g-PEG was added, as illustrated in figure\u00a05d, the PLA surface became rough and dense, and no obvious interface debonding sign was observed. However, cracking of the surface was observed, showing limited interactions between PLA and GO-g-PEG, indicating only a partial compatibility between them. On the other hand, when 0.3% GC-g-PEG was applied, the boundary between the PLA matrix and the GC-g-PEG becomes indistinguishable (figure\u00a05b). This might be rationalized in terms that more PEG is grafted onto the GC in comparison to that achieved over GO, leading to stronger interfacial interaction between GC-g-PEG and PLA, which is completely infiltrated by PLA. Unfortunately, when 0.5% GO-g-PEG and 0.4% GC-g-PEG are added, more cracks and agglomeration can be observed, respectively. The aggregation of nanosheets can be attributed to the inevitable van der Waals force.Good dispersion of nanofillers in the polymer matrix is of great essence for high performance of nanocomposites ,39. SEM figure\u00a05The dispersion of GC-g-PEG was further evaluated by the TEM, which is shown in 3.3.Tg, Tcc, Tm, the melting enthalpy (\u0394Hm) and the crystallinity (Xc). Furthermore, the DSC heating curves of PLA nanocomposites with the different content of GO-g-PEG are shown in the electronic supplementary material, figure S2.As a semi-crystalline polymer, the crystallization behaviour of PLA plays a guiding role in the mechanical properties to some extent . The DSCTg and Tcc of PLA dropped from 62.3\u00b0C and 122.3\u00b0C to 56.2\u00b0C and 101.4\u00b0C, respectively. Similarly, when the GO-g-PEG content was 0.4%, Tg and Tcc also decreased to 56.6\u00b0C and 102.5\u00b0C, respectively. This is because the grafted PEG can be used as a low-molecular-weight plasticizer, which increases the lubricity of molecular chains and the free volume of molecular chains. Therefore, less energy is needed to move the molecular chain in comparison to pure PLA, which could result in a decrease in the Tg of the entire nanocomposite. However, when the loading of GC-g-PEG is further raised to 0.4%, the values of Tg and Tcc stop increasing, which may be owing to the inevitable agglomeration of the nanofillers, partially hindering the mobility of the molecular chain. The process of cold crystallization and crystal melting of pure PLA is shown in x and PLA/GO-g-PEG 0.4 exhibits a distinct bimodal phenomenon. The reason for this phenomenon is that an unstable crystal is formed at a low temperature (about 160\u00b0C), with increasing the temperature, thermodynamically, the unstable crystal regions will be partially rearranged to stabilize the crystal structure [Increasing the loading of GC-g-PEG from 0% to 0.3%, tructure .Xc of PLA can be determined by comparing the obtained \u0394Hm of PLA with its theoretical melting enthalpy (93 J g\u22121) [Xc of pure PLA is only 16.2%, as shown in Xc of 44.1% over PLA is achieved. With the content increasing, the Xc decreases to 40.5%, which is ascribed to the agglomeration of GC-g-PEG. The increase of Xc can be attributed to the fact that the mobility of PLA molecular chains can be improved by the plasticizing effect of PEG. Moreover, GO and its derivatives, as crystallization heterogeneous nucleating agents, could also promote the crystallization ability of PLA [Xc of PLA is 37.8%. More DSC curves with different GO-g-PEG contents are presented in the electronic supplementary material, figure S2 and table S1.In addition, the 3 J g\u22121) . The Xc y of PLA . The sam3.4.x were estimated by the tensile test. The typical stress\u2013strain curves are presented in It has been established that the mechanical performance of PLA could be improved when graphene-based derivatives are introduced, which is attributed to the large surface area and comparatively high modulus ,43. RegaWb) can be calculated from the integrated area under the stress\u2013strain curve, which is Wb rises from 20.1 to 155.8 MJ m\u22123, which is 11 times higher than that of pure PLA. When the content of GO-g-PEG is 0.4%, the Wb rises to 120.8 MJ m\u22123. However, when the content of added GC-g-PEG is further increased to 0.4%, the Wb decreases to 89.3 MJ m\u22123, which can be attributed to the inevitable agglomeration of nanofillers.Generally speaking, tensile toughness is a measurement of the energy absorbed by a material during its tensile fracture. The absorbed energy . When 0.3% of GC-g-PEG was added, the surface became rougher, and more pull-out and even microvoids were observed, indicating considerable tensile energy dissipation and a stronger shear yielding phenomenon (figure\u00a010c).figure\u00a010c, which can be rationalized in terms that the nanofillers will rupture under tensile loading. This leads to a decline in the capacity of load-carrying for nanofillers, in which the original load will be transferred to the surrounding matrix or unfragmented nanoparticles, thus increasing the local deformation of the surrounding matrix. It has been reported that energy dissipation and shear yielding of matrix occur once voids are created [From the above discussion, it is realized that the addition of GC-g-PEG or GO-g-PEG can improve the interface compatibility between GC/GO and PLA, and then the mechanical properties of PLA will be improved. This can be attributed to the fact that PEG acts as a transmitter during the stress transmission at the interface between PLA and GO. When external stress is applied to the material, the PEG at the interface can induce plastic deformation, resulting in the slippage of the PLA molecular chain. The obvious microvoids are demonstrated in figure\u00a010 created ,56. Ther created . The SEM3.5.T5), 50% (T50) and maximum (Tmax) decomposition temperatures can be speculated, which are shown in figure\u00a011a,b, neat PLA, PLA/GC 0.3 and PLA/GC-g-PEG 0.3 nanocomposites decompose in a one-step process. Furthermore, as shown in T5 value increases from 330 to 343\u00b0C when 0.3% GC-g-PEG content is applied in pure PLA, but no obvious change in T50 and Tmax can be observed. Interestingly, the addition of GC produced no effect on thermal properties of PLA. These indicate that the initial decomposition temperature of PLA can be increased about 13\u00b0C by adding a small amount of GC-g-PEG. The increase in thermal stability may be owing to the shielding effect of the nanosheets and the diffusion of volatile decomposition products [Some studies have reported that thermal properties of the polymer matrix can be improved by the addition of lower loadings (\u22640.5 wt%) of GO or its derivatives ,32. Simiproducts . Howeverproducts .Figure 4.In summary, the optimum process of carboxylation and the effect of GC-g-PEG content on PLA as well as the comparison of GO-g-PEG were explored. FTIR, XRD and TGA experiments showed that the maximum grafting rate of the carboxyl group could be achieved when the concentration of chloroacetic acid was 50 times of NaOH. XPS results showed that the carboxyl group content of GC50 on the surface increased to 15% compared with that obtained over original GO (6%). Then, novel PLA nanocomposites were prepared by using GC-g-PEG as a nanofiller through the masterbatch method. Because of the interaction between PEG and PLA, the compatibility between PLA and GC was improved and uniform dispersion was achieved. The study of crystallization behaviour shows that the addition of nanofillers can accelerate the cold crystallization process and improve the crystallinity of PLA from 16.2 to 44.1%, but the Tg of PLA was decreased from 62.3 to 56.2\u00b0C. The TGA test showed that the initial decomposition temperature of PLA/GC-g-PEG 0.3 was 343\u00b0C, which is obviously higher than that of PLA (330\u00b0C). More importantly, when the addition of GC-g-PEG was 0.3%, the elongation at break of PLA increased to 30.55%, which was 7.15 times higher than that of pure PLA, accompanied by a slight decrease in tensile strength, i.e. 7%. The good compatibility between GC-g-PEG and PLA and the uniform dispersion in the PLA matrix are mainly attributed to the improvement of the ductility of PLA while maintaining the tensile strength. When the external force loads on PLA, the PEG at the interface can bear the medium of stress transmission, and then stress yield occurs. These results show that GO or its derivatives grafted with PEG can maintain PLA strength while enhancing its own ductility as well as crystallinity and thermal stability. This work provides a promising application on medical materials, three-dimensional printing technology and so on."}
+{"text": "To examine the associations between hip muscle cross-sectional area and hip pain and function in community-based individuals with mild-to-moderate hip osteoarthritis.This study included 27 participants with mild-to-moderate hip osteoarthritis. Cross-sectional area of hip muscles, including psoas major, rectus femoris, gluteus maximus, gluteus medius and minimus, adductor longus and magnus, obturator internus, and obturator externus, were measured from magnetic resonance images. Hip pain and function were evaluated using the Hip Disability and Osteoarthritis Outcome Score (HOOS) categorised into 5 subscales: pain, symptoms, activity of daily living, sport and recreation function, and hip-related quality of life .n\u00a0=\u200918) were female. After adjusting for age and gender, greater cross-sectional area of adductor longus and magnus was associated with a higher HOOS score in quality of life  0.2\u20132.7, p\u2009=\u20090.02), activity of daily living  and sport and recreation function . There was a trend towards an association between greater cross-sectional area of psoas major and a higher quality of life score . The cross-sectional area of hip muscles was not significantly associated with HOOS pain or symptom score.Mean age of the 27 participants was 63.2 (SD 7.6) years and 66.7% (Greater cross-sectional area of hip adductors was associated with better function and quality of life in individuals with mild-to-moderate hip osteoarthritis. Greater cross-sectional area of hip flexors might be associated with better quality of life. These findings, while need to be confirmed in longitudinal studies, suggest that targeting the hip adductor and flexor muscles may improve function and quality of life in those with mild-to-moderate hip osteoarthritis. Osteoarthritis (OA) is one of the leading causes of disability worldwide, with disability-adjusted life years predicted to rise with the increasing age and prevalence of obesity in the population . Hip OA Hip muscles are critical for movement of the trunk and legs and redistribution of segmental power when walking, and may also be important for stabilizing the hip joint , 6. The There is a need for studies to comprehensively examine hip muscles of different functional groups as possible modifiable factors for improving the management of hip OA at an early stage of the disease, where the opportunity to alter patient outcomes remains. The aim of this study was therefore to examine the associations between CSA of hip muscles of different functional groups and patient-reported hip pain and function in individuals with mild-to-moderate hip OA.Individuals over the age of 45\u2009years were recruited between June 2011 and October 2014 through advertisements, word of mouth, and hospital orthopaedic waiting lists. Volunteers were screened using the Harris Hip Score (HHS)  and radiInclusion and exclusion criterian\u2009=\u200927). Individuals with Kellgren-Lawrence grade 4 and joint space width\u2009<\u20091\u2009mm or any major lower limb musculoskeletal or neurological conditions besides hip OA were excluded.Participants with hip pain in the last 3\u2009months, HHS \u2264\u200995 points, Kellgren-Lawrence grade 2 or 3 and/or joint space width\u00a0\u2264\u00a03\u2009mm in one or both hips were defined as having hip OA  was calculated.Hip pain and function were evaluated using the validated Hip Disability and Osteoarthritis Outcome Score (HOOS) . The HOOParticipants underwent magnetic resonance imaging (MRI) of the pelvis and leg  using a 3.0\u2009T MRI unit . Participants were positioned in supine position with body coil arrays superiorly placed on lower limbs and legs in 15\u00b0 of hip internal rotation, secured together with a strap. Hip muscle CSA was measured on axial images obtained using a T1 weighted 2-dimensional fast spin echo sequence . The CSA of hip muscles was measured from five regions  was considered statistically significant. All statistical analyses were performed using the IBM Statistical Package for the Social Sciences  software, version 24.Participant characteristics were tabulated. Multiple linear regression was used to examine the associations of hip muscle CSA with hip pain and function (HOOS scores) of the target hip, adjusted for age and gender. A 2 and 66.7% being female. The mean joint space width of the study hip was 2.39\u2009mm, and the majority (92.6%) of the participants had mild-to-moderate hip OA (i.e. Kellgren-Lawrence grade\u00a0\u2264\u00a03).The characteristics of participants are shown in Table\u00a0p\u2009=\u20090.02). There was a trend towards an association between greater CSA of psoas major  and flexors total  and better hip-related quality of life, achieving borderline significance. The CSA of other hip muscles was not significantly associated with hip-related quality of life.The associations between hip muscle CSA and HOOS scores are presented in Table\u00a0p\u2009=\u20090.04) and in sport and recreation . The CSA of hip muscles was not significantly associated with HOOS symptom score in multivariable analyses . Including fat infiltration in the regression models did not change the results (data not shown).There was low variation in the levels of fat infiltration of hip muscles, with fat infiltration of 1\u201310% or 11\u201350%  and quality of life. There was also a non-significant trend towards an association between greater CSA of hip flexors and better quality of life. These findings suggest that targeting the hip adductor musculature in treatment may lead to improved hip function and quality of life among individuals with mild-to-moderate hip OA.We found that greater CSA of hip adductors was associated with better functional outcomes in those with mild-to-moderate hip OA. The most significant and consistent associations were found for the hip adductors, including adductor longus and magnus. The hip adductor muscles play an important role in balancing the pelvis during standing and walking and for overall hip stability and injury prevention. Previous studies reported lower volume and CSA of hip adductor muscles and decreased hip adductor strength in people with hip OA compared with healthy controls , 22. OthThere was a trend that larger CSA of hip flexors would be associated with better quality of life in our study. Hip flexors are used for a variety of everyday functional activities such as advancing the lower extremity during gait, running, or lifting the leg when going up steps. Although decreased hip flexor strength has been reported in people with hip OA compared with healthy controls , 22, no There is evidence that larger CSA of hip muscles may result in greater muscle strength , 8, 22 wThis study had limitations. It is a cross-sectional study with a moderate sample size. Whether there is a temporal relationship between hip muscle CSA and functional outcomes could not be investigated. The moderate sample size limited the power of the study to detect an association between CSA of some hip muscles and pain and functional outcomes. The CSA of hip muscles was not significantly different between the right and left hips in the 11 participants with bilateral hip OA (data not shown). The most affected hip in participants with bilateral hip OA was the study hip for statistical analyses. The results did not change when the average CSA of right and left hip muscles were examined in those with bilateral hip OA (data not shown). Due to the lack of demarcation between muscles, some specific muscles could not be segmented individually and instead were grouped for CSA measurement, such as the adductors, and gluteus medius and gluteus minimus muscles. Identifying specific muscles could shed further light on which muscles contribute to better hip outcomes. Consistency with regards to anatomical positions for muscle CSA measurement was another issue. The slice thickness may have surpassed some regions due to the difference in terms of where the initial slice began or the difference in body size among participants. As shown in a systematic review, segmentation of CSA on a single slice increased volume errors . These tThis study found that greater CSA of hip adductors was associated with better function and quality of life in individuals with mild-to-moderate hip OA. Furthermore, a similar trending association was found between hip flexor CSA and quality of life. These findings, while need to be tested in clinical trials, suggest that targeting hip adductor and flexor muscles may have a beneficial effect on improving function and quality of life in those with mild-to-moderate hip OA.Additional file 1: Table S1. Relationship between hip muscle cross-sectional area and HOOS outcomes. Table S2. Fat infiltration of hip muscles."}
+{"text": "Nutrient reference values are important parameters that guide nutrition and public health work globally. Micronutrient requirements during the peri-conception period are generally increased, which is essential in ensuring maternal, fetal, and neonatal health. Nevertheless, the current dietary reference intakes (DRIs) may be limited in terms of the methods used and the populations included, particularly the DRIs for pregnancy and lactation. In this proposed review, we will examine the methods  and the population  in the studies used to inform the current DRIs. We will apply meta-science methods to this review, which involves formally reviewing the current evidence, and identifying opportunities to improve how we fund, perform, evaluate, and incorporate nutrition science into public health programs for better outcomes. In the 1990s, a group of experts proposed a then novel approach for setting new nutrient intake recommendations. These dietary reference intake (DRI) values were initially adopted in the US, in collaboration with Canada and the UK1.Nutrient reference values (NRVs) play an essential role in promoting the health and well-being of a population. Conceptually, NRVs are meant to assess the risk of deficiency and inadequacy while avoiding excessive intakes for healthy populations, and to be utilized in planning adequate diets for individuals and populations. The key components of NRVs are the population average requirement (AR) and the safe upper intake level (UL)2. However, the DRIs are more widely used. This review will focus on the DRIs developed by the US, UK, and Canada, and not the WHO NRVs, for two main reasons. First, the NRVs set forth by WHO/FAO, despite considering several contextual factors, have not been recently updated for micronutrients. While a few important updates have been made to the DRIs over the years, including calcium and vitamin D, and more recently sodium and potassium3. In contrast, the WHO NRVs have had updates only in sodium and potassium, as well as in calcium supplementation6. Second, the DRIs and NRVs are similar for most micronutrients in terms of AR (but not UL). A few micronutrients have DRIs but not NRVs , such as choline, vitamin E, phosphorus9.The World Health Organization (WHO) also provides NRVs  in a 2004 document, which is based on a 1998 convening joining the WHO and the Food and Agriculture Organization (FAO) of the United Nations10. Further, NRVs in pregnancy inform the composition of \u201cprenatal vitamins\u201d, or multiple micronutrient supplements (MMS), used in pregnancy to promote positive pregnancy outcomes11. The commonly used UNIMMAP supplement for pregnancy includes 15 vitamins and minerals at the level recommended by the DRIs.Nutrient reference values are especially important during pregnancy. There is a biological imperative for increased nutrient intake, and there are well-documented maternal and fetal consequences of inadequate nutrition during the periconceptional, pregnancy, and postpartum periods1. Women have historically been underrepresented in medical research. For example, drug trials to date have been conducted almost exclusively in young men12, and a recent review of clinical trials  in India found that less than 2% included pregnant women13. This is problematic because there is likely to be sexual dimorphism in the physiology, metabolism, and related toxicity and efficacy of supplements and drugs. Further, there are clear ethical imperatives to include pregnant women in research because women deserve access to well-studied interventions14. We hypothesize that nutrition science and the related evidence-based guidelines have similarly suffered from excluding women and pregnant women from research.Yet, there is growing recognition of the limited extent to which the DRI study populations represent all sub-populations, including pregnant and lactating women15. There are five broad areas of focus for meta-science including: the way research is performing, or methods; the way people communicate about research, or reporting; the way we verify research, or reproducibility; the way we conduct peer review, or evaluation; and the way we reward research, or incentives15. Meta-research is well suited to measure whether specific subpopulations are excluded from biomedical research and whether novel research methods are equally applied to problems or conditions that affect women, underrepresented minority populations, and people living in the global south.Meta-research, or meta-science, is an evolving field focused on systematic evaluation of the way science is produced and usedpopulation andnutrition-science methods used in studies informing the nutrient reference values for women, and for pregnant and lactating women.We aim to formally review the methods of research studies used to inform the DRIs and identify opportunities to improve how we fund, perform, evaluate, and incorporate nutrition science for improving public health. The objective of this meta-science study is to summarize theThis review is focused primarily on assessing the population and methods used in the studies informing the development of DRIs:1.Population (Women). To what extent do the studies informing the population average requirement (AR) and safe upper levels of intake (ULs) include female subjects? Is there variation in the subjects\u2019 race or ethnicity, nutritional or anthropometric status, and health or disease status?2.Population (Pregnancy and Lactation). To what extent do the studies informing the population average requirement (AR) and safe upper levels of intake (ULs) include women across the life course, specifically pregnant and lactating people?3.Methods (Study Design). Do studies utilize best-in-class methods including: controlled feeding studies and/or well-designed intervention and observational cohorts?4.Methods (Molecular): Do studies assess functional and metabolic outcomes? Do studies utilize integrated approaches, determining alterations in absorption, kinetics and whole-body nutrient losses using mixed methods approaches ?5.Methods (Modern). Do studies utilize modern methods including: metabolomics, proteomics, data analytics, or adaptive and/or target trial methods? Modern omics technologies include liquid chromatography mass spectrometry for metabolite profiling and protein profiling. Modern data analytic approaches include computational, statistical, and deep learning techniques to analyze omics data with high-dimensionality, correlation between features and zero-inflated data.Secondary research questions include:1.Representativeness  of the study population: To what extent can we generalize the findings from this study population to other populations?not assess the quality of the research or risk of bias in each study. Nor will it assess whether the interpretation and conclusions of the studies are accurate or appropriate.This review study willThe research will take part in four phases: 1) search, 2) screening, 3) data abstraction, and 4) data analysis.The search process will begin with identifying the indicators considered by the committee to establish the DRI. All references in the \u201cSelection of Indicators for Estimating the Requirement for [nutrient]\u201d section will be abstracted. We will also abstract references in the \u201cFindings by Life Stage and Gender Group\u201d Section, specifically for the \u201cAdults\u201d, \u201cPregnancy\u201d, and \u201cLactation\u201d sub-sections. Finally, we will abstract references in the \u201cTolerable Upper Intake Levels\u201d section. The full reference (as listed in the DRI document) and the section in which it appeared will be recorded.There are two exceptions in the search process requiring slight adjustments to the search strategy. First, for vitamin B2 (riboflavin), an additional section will be included, \u201cApproaches for Deriving the Estimated Average Requirement\u201d because this section provided an overview of B2-related references used to determine the EARs. Second, the latest (2011) calcium and vitamin D report has a different layout of the chapters and sections from other reports. We will abstract references from three chapters in this report: Chapter 4, \u201cReview of Potential Indicators of Adequacy and Selection of Indicators: Calcium and Vitamin D\u201d ; Chapter 5, \u201cDietary Reference Intakes for Adequacy: Calcium and Vitamin D\u201d; and Chapter 6, \u201cTolerable Upper Intake Levels: Calcium and Vitamin D\u201d. To ensure the completeness of the data, we will also abstract references in the \u201clife stages\u201d section of the 1997 calcium and vitamin D report, which determined adequate intakes (AIs) but not estimated average requirements (EARs) for these two nutrients (EARs were set in the 2011 report). The following five documents will be reviewed during the search:1.Institute of Medicine (IOM) 1998. Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid, Biotin, and Choline. Washington, DC: The National Academies Press.2.IOM 2000. Dietary Reference Intakes for Vitamin C, Vitamin E, Selenium, and Carotenoids. Washington, DC: The National Academies Press.3.We will focus on Vitamin A, Vitamin K, Iodine, Iron, and Zinc in this report)IOM 2001. Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Washington, DC: The National Academies Press. IOM 1997. Dietary Reference Intakes for Calcium, Phosphorus, Magnesium, Vitamin D, and Fluoride. Washington, DC: The National Academies Press.  Exclusion criteria1.e.g., in the Thiamin chapter, a few indicators were noted as \u201cNone of these was judged to be a dependable criterion of thiamin status).Studies for indicator selection: exclude the reference if a particular indicator was mentioned in the DRI report, but was not used to determine EAR/AIs. Other studies: exclude the reference if this study was mentioned in the report for a particular life stage / UL determination, but with additional comment about it not being used in setting the final DRIs. Life stages UL determination Study ID Full reference Funding source(s) Is the article an open-access publication?Inclusion/exclusion at full-text stage, reasons for exclusion include:Article cannot be found (through direct online search or through interlibrary loan request)This is a review article and do not provide primary data to help determine DRIsStudy methodsin vitro study, end of data collection, e.g., cell, organ, tissue) Type of study population: is this a human and/or non-human study? Cross-over trialCohort studyCase-control studyCross-sectional studyCase reportModeling, kinetics, and other secondary data analysis studyDescribe the study design brieflyWhat was the intervention / exposure / status?Is there a control group? Any outcome pertaining to women\u2019s health? Rigorous design:Is this a controlled feeding study?Is this a randomized controlled trial?Does this study have repeated measurements on the same participants / subjects over time? Molecular design:Does this study use stable isotope?Is this a balance study (measured input and excretion)?Does this study measure biomarkers for micronutrient status?Modern design:Does the study use any \u201c-omics\u201d method?Human populationWhere was the study conducted? (Country where study population resides)Is this a healthy population?Does this population share a common condition, comorbidity, or other characteristics? Total sample sizeAre women included in the study population? Are pregnant or lactating people included in the study population? What was the average/median age of the population (year)?Were women of reproductive age included (14-45y)?Is race/ethnicity reported?  Non-human subjectsSpecies/breed if animal studyTotal sample sizes (n)Are female subjects included? Are pregnant or lactating animals included? Is this a special group of subjects? Other information relevant to our review16. We will conduct univariate descriptive analysis ; between-group comparisons , including the Student\u2019st-test (or non-parametric test such as the Mann-WhitneyU test), chi-squared test; as well as trend test .After the completion of full-text data extraction, qualitative synthesis will be conducted for all included studies, separately for each micronutrient . The synthesis will prioritize the research questions in terms of population characteristics and methods used in the included studies. We will also import the cleaned dataset into a statistical software  for analysisPopulation (Women).% women overall and refs in indicator, AR, UL sections% women of reproductive age?% URM overall and refs in indicator, AR, UL sections% low / high BMI overall?% healthy subjects overallPopulation (Pregnancy and Lactation).% pregnant women/animals overall and refs in indicator, AR, UL sections% lactating women/animals overall and refs in indicator, AR, UL sectionsMethods (Rigorous Design).% studies used controlled feeding in indicator, AR, UL sections% studies used randomized controlled trials in indicator, AR, UL sections% studies had well-designed interventions in indicator, AR, UL sections% studies were observational cohortsMethods (Molecular Design):% studies used stable isotope methods in indicator, AR, UL sections% studies measured biomarkers for micronutrient status in indicator, AR, UL sectionsMethods (Modern Design).% studies used any -omics methods in indicator, AR, UL sectionsWe will publish the paper in an open access scientific journal. Depending on the amount of evidence we eventually review and generate, there may be more than one manuscript as a result of this project. We will upload our primary (raw) data upon completion of the review to an approved online repository . We will then share the repository DOI with the manuscript.No underlying data are associated with this article.1We excluded several nutrients from the proposed meta-review for the following reasons: i) not a micronutrient, including water, energy, carbohydrate, fiber, fat, fatty acids, cholesterol, protein, and amino acids; ii) an estimated average requirement (EAR) or adequate intake (AI) was not set for a given nutrient, including arsenic, boron, nickel, silicon, vanadium; iii) the UL is not determinable in current DRI, owing to lack of data of adverse effects, and concern regarding lack of ability to handle excess amount, including arsenic, chromium, silicon, sulfate, vanadium; iv) source of intake should be from food only to prevent high levels of intake, including arsenic, chromium, silicon, sulfate, vanadium; and v) not determinable owing to a lack of data of a specific toxicological adverse effect, including sodium and potassium. This study reminds us to re-evaluate existing recommendations along with developments in science and technology. Moreover, DRI is the foundation for determining intervention plans, evaluations, and policies in various sectors. The steps the authors took were excellent, clear, and replicable in other populations and age groups. In Phase #2: Screening, the authors may explain what the second researcher will do as a quality controller. It would be better if the two researchers screened simultaneously and matched the results; if there were differences, it could be directly discussed or invited a third researcher. If possible, include the name  of the investigator conducting the screening. In Phase #3: Data Abstraction, the authors may assess the reference used to observe the absorption/bioavailability. Whether it is enough with a balance study (measured input and excretion) only, or is there a reference to observe the micronutrient levels biomarker in circulation. This study focuses on women, pregnancy, and lactation. However, for this population, especially in low- and middle-income countries, it is encouraged to note the possibility of pregnancy and lactation in the adolescents' population. Therefore, it is recommended to be considered by the team, primarily if it is found in Phase #3 Data Abstraction.1) and Arimondet al. (20112). The authors are also suggested to elaborate on dietary reference values on specific nutrients from other sources such as Hotz , and is an important contribution to the literature. The authors are to be applauded for pre-publishing efforts a priori to review. The work plan is well organized, clearly written, and scientifically sound. Please clarify, is the AR or EAR being used in the global context to harmonize with WHO/FAO, and if the UL is specifically the Tolerable Upper Intake Level or used in the global context? Meta-research may be new to some readers, if possible consider expanding the last paragraph of the introduction. Consider inviting FNB staff to collaborate (suggest Anne Yaktine as a first point of contact), they are a tremendous resource and will have knowledge not found in the reference section of the reports. Assuming inclusion criteria refers to all forms of the micronutrients listed, if not clarify.et al. (20161). Efforts are being made to scan the literature to trigger if a DRI should be updated. You may wish to examine the evidence scanning paper by BrannonIs the study design appropriate for the research question?YesIs the rationale for, and objectives of, the study clearly described?YesAre sufficient details of the methods provided to allow replication by others?YesAre the datasets clearly presented in a useable and accessible format?Not applicableReviewer Expertise:Dietary assessment, nutrition epidemiology, NHANES, dietary supplements, precision nutritionI confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard."}
+{"text": "The aim was to explore the associations of religion and religious affiliations with CVD risk among Ghanaian non-migrants and migrants in Europe. (2) Methods: The 10-year CVD risk was estimated using pooled cohort equations for 3004 participants from the cross-sectional Research on Obesity and Diabetes among African Migrants (RODAM) study. Logistic regression analyses were conducted to assess associations between religion and elevated CVD risk (score \u2265 7.5) with adjustment for covariates. (3) Results: Religious men in Europe had a lower 10-year CVD risk compared with non-religious men , specifically men affiliated with Seventh-Day Adventism  followed by other affiliations  and Roman Catholicism . The opposite was found in Ghana, with religious women having higher odds for elevated 10-year CVD risk  compared with their non-religious counterparts, specifically women affiliated with Reformed Christianity  and other denominations . Associations were not significant for men in Ghana and women in Europe. Adjustments for social support, stress, and health behaviors did not meaningfully alter the associations. (4) Conclusions: Christian religious Ghanaian men living in Europe seem to have lower CVD risk compared with their non-religious counterparts, while Christian religious women in Ghana appear to have increased CVD risk. Further unravelling the contributing factors and the differences between sex and environmental settings is needed. Cardiovascular diseases (CVDs) currently account for one in three deaths worldwide ,2. ArounMigrant populations residing in high-income countries seem to be more affected by CVDs and associated risk factors than host populations . The ResReligious and spiritual (R/S) affiliations may be an underlying factor for CVD risk variations. R/S affiliations play an important role in lives of SSA (migrant) populations with the majority being affiliated with Christianity (63%) . These bAdherence to religion may change as a consequence of migration ,18. In tThe theoretical framework in We used data from the multi-centre cross-sectional RODAM study of which the rationale and design have been described in detail elsewhere . Data weRecruitment of participants in Ghana was carried out in two purposely-chosen cities and fifteen randomly selected villages in the Ashanti region. Response rates were, respectively, 76% and 74% in rural and urban Ghana. In Amsterdam 67% of the randomly drawn individuals from Municipal Health registers responded to invitations, 53% of whom agreed to participate. In Berlin and London, participants were recruited through Ghanaian organisations and response rates were, respectively, 68% and 75%. Altogether, 6385 Ghanaians participated by completing a structured health questionnaire 5898 of whom were physically examined. Ethical approval was obtained from local ethics committees at all study sites and all participants gave informed written consent.Structured questionnaires provided information on demographics, length of stay in a respective European country, and whether participants practised religion . When answering yes, follow-up questions included current religious denomination , Islamic, or other) and frequency of attending religious services in past 6 months . This was categorised into once a week or more, once every 2 weeks or once a month, or less than once a month or never.Based on the previously described theoretical framework, the following variables were chosen to be included in the analysis. Socioeconomic indicators (education and employment) were taken into account as potential confounders given their association with religious involvement and CVD risk . EducatiFasting venous blood samples were collected, manually processed and immediately aliquoted, and then temporarily stored at the local research location at \u221220 \u00b0C. The samples were then transported to the respective local laboratories for registration and storage at \u221280 \u00b0C and were subsequently transported to Berlin, Germany, for biochemical analysis to avoid intra-laboratory variability. Serum total cholesterol, serum HDL-cholesterol, serum LDL-cholesterol, and fasting glucose levels were determined using ABX Pentra 400 chemistry analyzer . Concentrations of total cholesterol and HDL-cholesterol were assessed using colorimetric test kits. Fasting plasma glucose concentration was measured using hexokinase. The definition of type 2 diabetes was based on World Health Organization (WHO) diagnostic criteria of fasting glucose \u22657.0 mmol/L, prescribed type-2-diabetes medication use, or self-reported diabetes . Blood pCVD risk score was calculated using the Pooled-Cohort-Equations (PCE) risk calculator developed for different ethnic groups . The PCEn = 3124) [n = 39), religious denominations (n = 24), or frequency of attending religious services (n = 57) were excluded leaving a total number of 3004 participants (n = 8). A subset analysis was conducted to examine whether associations between practising religion and CVD risk varied in rural or urban Ghana (for non-migrants) and in Amsterdam, Berlin, or London (for migrants). Statistical significance level was set at p < 0.05 for all tests.Data were analysed using STATA version 14.2. CVD risk was estimated for RODAM participants aged 40\u201370 with total-cholesterol ranging from 130\u2013320 mg/dL, HDL-cholesterol 20\u2013100 mg/dL, systolic BP 90\u2013200 mmHg, and without prior history of stroke and heart attack ( = 3124) . Particiicipants . Particin = 273) in Ghana practised religion compared to 83.4% (n = 649) in Europe. Smoking and alcohol use were higher among non-religious men compared with those practising religion in both Ghana and Europe. Men not practising religion reported higher physical activity levels than men practising religion. Slightly fewer type-2 diabetes cases were detected among men practising religion in Ghana and Europe. In Ghana, more religious men experienced negative life-events than non-religious men while in Europe fewer religious men reported negative life-experiences. CVD risk differed significantly between men in Europe as about 66% of non-religious men had elevated risk compared to 54% of religious men. The most frequently reported religious denomination in Ghana was Reformed Christianity (23%) and Pentecostalism in Europe (24%). n = 926) than in Ghana . No women reported smoking in Ghana while about 5% of the non-religious and 1% of the religious women in Europe smoked. None of the women in both Ghana and Europe reported using alcohol. More diabetes cases were detected in religious women in Ghana and Europe. Religious women in Ghana appeared less physically active and experienced more negative life-events than non-religious women. In Ghana, 18% of non-religious women had elevated CVD risk compared to 27% of the religious women. Similarly to men, the majority of women in Ghana were Reformed Christians (26%) and in Europe, Pentecostals (32%). Women tended to visit religious services more often than men .In men, those practising religion had lower odds of elevated CVD risk than men not practising religion . In EuroIn contrast, odds of elevated CVD risk were 53%  higher for religious women in Ghana compared with non-religious women after adjustment for education and employment. Additional adjustment for social support, stress, and health behaviors did not affect the odds for elevated CVD risk in women . Among Ghanaian women in Europe, there was no significant difference in the odds of elevated CVD risk for those who did and did not practise a religion . When the analysis was stratified by the various European sites, practising religion was significantly associated with CVD risk in Berlin only, although the direction of the associations was similar except for London after adjustments for health behaviors., SDA , and other denominations . Adjusting for health behaviors changed the significance of associations for Roman Catholics and other affiliations reaching statistical significance, which was not the case in earlier models. In Ghana, no association was found between religious denominations and elevated CVD risk for men.Women in Ghana who practised Reformed Christianity , SDA  or other denominations  had higher odds of elevated estimated CVD risk relative to their non-religious counterparts after controlling for education and employment status. Adjusting for social support, stress and health behaviors did not meaningfully alter associations with an OR of 1.73 (1.03\u20132.90) for Reformed Christians, 2.15 (1.00\u20134.62) for SDAs, and 2.81 (1.20\u20136.54) for other denominations. In Europe, no association was found between religious denominations and elevated CVD risk for women.This study revealed that practising Christian religion is associated with lower odds for elevated CVD risk among Ghanaian men living in Europe but not among men living in Ghana. In contrast, practising Christian religion was associated with higher odds of elevated CVD risk for women living in Ghana but not for women living in Europe. The association between religion and lower CVD risk among Ghanaian male migrants, particularly in Berlin, was reported previously in other populations. These studies demonstrated a favourable cardiovascular profile for religiously affiliated persons ,20,21. CA striking finding was the higher odds of elevated CVD risk among religious women living in Ghana, particularly in urban centres for which reasons are unclear. The data seem to demonstrate that regulation of health behaviors such as tobacco and alcohol consumption are unlikely to contribute to observed differences in associations between religion and CVD risk as very few Ghanaian women in both Ghana and Europe smoke and consume alcohol. Yet, it is possible that this elevated risk is related to religious fatalism  . FatalisThe main strengths of the RODAM study are the relatively homogenous study population and the use of well-standardised approaches across sites . The firOur findings suggest that Christian religious Ghanaian men living in Europe seem to have lower CVD risk compared with their non-religious counterparts, while Christian religious women in Ghana appear to have increased CVD risk. Further research is needed to gain more insight into the underlying processes and differences between sex and environmental setting in order to guide future CVD prevention programmes for this high-risk population. The study found that Christian religious Ghanaian men living in Europe seem to have lower CVD risk compared with their non-religious counterparts, while Christian religious women in Ghana appear to have increased CVD risk.The contributing factors and differences between sex and environmental settings need to be further unravelled.Insight into these underlying processes can guide future CVD prevention programmes for this high-risk population."}
+{"text": "There are many commercially available kits to create PRFM, but they are often expensive and uneconomical. This research will test a modified method of making ideal PRFM from PRP without any commercial kits. The modified method will include determining the minimum level of CaCl2 used, the type of centrifuge, and the speed and duration of centrifugation. By performing a modified preparation method on five samples of whole blood, it was found that the ideal PRFM could be made by mixing PRP with 25 mM CaCl2 and centrifuging it at a speed of 2,264 \u00d7 g for 25 min at room temperature. The PRP and PRFM platelet counts of this method tend to be lower than the platelet counts found in other studies. Although visually comparable, further study is needed to compare the performance of PRFMs made with this method and PRFMs made with commercial kits.One bioproduct that is widely used in the wound healing process is platelet-rich plasma (PRP). PRP is a liquid solution with high autologous platelet concentration, making it a good source of growth factors to accelerate wound healing. Recent development in PRP had created a new product called platelet-rich fibrin matrix (PRFM), which has a denser and more flexible structure. PRFM is the newest generation of platelet concentrate with a fibrin matrix that holds platelet in it. The key concept in creating PRFM from PRP is the addition of CaCl Otorhinolaryngology specialists in plastic reconstruction have reported successful use of exogenous growth factors and PRP in clinical settings. Sclafani reported that the release of growth factors in wound healing was primarily carried out by platelets, echoing that PRP plays a vital role in wound healing. The influence of growth factors on endothelial cells and fibroblasts will increase within 7 days after injury and disappear after 14 days , 3. In cPlatelet-rich fibrin matrix (PRFM) is the latest generation of platelet concentrates with simple preparation without biochemical ingredients (bovine thrombin). PRFM is a slow polymerization of fibrin in PRP, resulting in a PRFM structure that resembles natural fibrin . This spLaboratory experiments looking at usage of PRFM in specific media show that there was an increase in levels of platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and transforming growth factor beta (TGF\u03b2) on the first day, followed by a gradual decrease on the next day , 5. This2 and centrifugation at 1,100 \u00d7 g for 6 min , a by-product in PRFM production, has a platelet content of 0/\u03bcl, representing the fact that all platelets are attached to the PRFM fibrin matrix at the bottom of the tube. Measurement of TGF\u03b21 will be conducted to reassure that growth factors are still available and viable inside the end product. The modified method will also try to make PRFM production more economical by creating PRFM from PRP through improvised methods without using a commercial PRFM production kit. At the same time, this study will determine the minimum amount of CaCl2) before centrifuging the solution to form the PRFM. The trials will evaluate whether the modified method can prepare ideal PRFM to accelerate the wound healing process. Improvement in wound healing is essential in reconstructive surgery and will affect the successes of operations both aesthetically and functionally.This study will carry out trials processing human whole blood, making PRP and adding in 1 M of calcium chloride . The study design and protocol are made and implemented following the Helsinki Declaration. Volunteers recruited from the study were given an explanation, given an information sheet, and signed the informed consent form before participating in the study.Whole blood samples (8 ml per tube) were collected from five healthy volunteers . The volg for 5 min at room temperature. The centrifuge process resulted in three separate layers: a clear yellowish plasma thrombocyte on top (consisting of a PPP layer on top and a PRP layer in the bottom), a cell selector gel layer with leucocyte in the middle, and a red blood cell layer on the bottom. A small amount of PPP and PRP was taken from each specimen to analyze thrombocyte count using Celtac-a automatic cell counter. PRP process is determined to be successful when the thrombocyte count in PPP is 0/\u03bcl. The tube was then inverted gently back and forth three times to mix the plasma, thrombocyte, and leucocyte, forming the final PRP. PPP was mixed with PRP to ensure a larger volume of end product used for PRFM preparation. A total of five PRP specimens will be produced at the end of this step and will be used to produce PRFM.This study utilizes a commercial PRP kit, the RegenKit\u00ae A-PRP\u00ae . This instruction directly follows the protocol presented in the kit. PRP can also be produced using other PRP kits or methods. Blood that had been taken from volunteers were immediately centrifuged using RegenLab 642VFD PRP Fixed Angle Centrifuge . The centrifuge from RegenLab was designed to prepare PRP and was programmed to run at 1,500 \u00d7 2. The maximal speed of this centrifuge is 4,500 RPM . Six milliliters of PRP were taken from four different PRP specimens using a micropipette (extra care was given to avoid taking the red blood cell layer) and moved to four separate 10 ml cylindrical centrifuge tubes . Each tube was then added with CaCl2 1 M until a final concentration of 70, 45, 25, or 15 mM of CaCl2 was achieved in each tube. All four tubes were then centrifuged at 2,264 \u00d7 g for 25 min at room temperature. The resulting product will consist of two layers, the PRFM and PPP.This experiment concentrates on creating PRFM using methods proposed by O'Connel, which is a modified Fibrinet  PRFM kit method . Orthope2 was then analyzed using the Celtac-a automatic cell counter. As previously mentioned, an ideal PRFM is the one with a 0/\u03bcl thrombocyte count in their PPP. The lowest concentration of CaCl2 that produces an ideal PRFM was then determined as the minimum recommended concentration of CaCl2.PPP from all four PRFM specimens with different concentrations of CaCl2 needed had been obtained from the previous steps, the fifth PRP specimen was assigned to an alternative PRFM preparation method to produce a coin-shaped PRFM. This particular shape was sought after because in several reconstructive surgery, a round coin-shaped PRFM with larger surface area is practical in certain clinical conditions. Six milliliters from the fifth PRP specimen was taken using a micropipette and put into a wide-mouth Wheaton bottle (diameter 30 mm), followed by addition of the CaCl2. The Wheaton bottle was then centrifuged with a Beckman CS-6R centrifuge at 3,800 RPM  for 25 min , and fixated in 2.5% glutaraldehyde in PBS for 1 h at 4\u00b0C. The samples were then washed for the second time with 0.1 M cacodylate buffer (pH 7.3) followed by fixation with 1% osmium tetroxide (OsO4) and 0.1 M cacodylate buffer for 1 h at room temperature (22\u00b0C \u00b1 2\u00b0C). The samples were then dehydrated in serial ethanol and dried before being placed on an aluminum sheet with silver adhesive paint coated with a layer of 4 nM of gold inside an Edward S150B argon atmosphere apparatus . The samples were then observed at 0\u00b0 with SEM Stereoscan 200  at 20 kV. Three different fields of view were examined for each sample, and the diameter of thrombocyte and the size of fibrin fibers were examined.TGF\u03b21 in PRFM were analyzed using a TGF\u03b21 immunoassay kit . The immunoassay kit works by identifying reactions of growth factors in the sample with a TGF\u03b21 monoclonal antibody in the microtiter plate wells. The bond between TGF\u03b21 and anti-TGF\u03b21F was identified via a TGF\u03b21 polyclonal antibody that is labeled with an enzyme. With additional substrates, these reactions will produce a varying intensity of colors identified with spectrophotometry at 450 nm. The intensity of the color is directly correlated to the concentration of protein analyzed. Compared to a standard solution with a known concentration, the concentration of TGF\u03b21 in the PRFM sample can be analyzed.*TGF\u03b21 complex that formed in the first incubation forming a sandwich of anti-TGF\u03b21*TGF\u03b21*anti-TGF\u03b21.HRPO. A substrate is added to form a blue color that will change into yellow after a stop solution is added. The intensity of color will correlate with the amount of TGF\u03b21 inside the sample.PRFM specimens analyzed were put into the microtiter plate wells coated with TGF\u03b21 monoclonal antibody and incubated. TGF\u03b21 in the sample will bind to the antibody inside the well. After washing the well to remove excess substances, the TGF\u03b21 polyclonal antibody with a horseradish peroxidase (HRP) label was added. Second incubation was done, during which the polyclonal antibody will bind to the anti-TGF\u03b21Blood was drawn from all volunteers and was processed right away using the methods above. All five volunteers had a normal hematologic profile, no chronic comorbidities, and normal CBC  examinations. Comparison of the amount of thrombocyte in whole blood, PRP, and PPP in the PRP specimen can be seen in 2 to determine the minimal amount in which ideal PRFM (the ones with 0 \u03bcl of thrombocyte in its PPP). The schematic shown in 2 is the minimum amount needed to produce ideal PRFM. The fifth specimen was used to produce a coin-shaped PRFM to represent diversity in the practical application of PRFM in different clinical conditions  and centrifugation to produce PRFM without the need for additional exogenous thrombin. The addition of CaCl2 and centrifugation to PRP will convert fibrinogen to fibrin, and the fibrin cross-links to form a matrix that contains viable platelets . This scaffold-like fibrin matrix is essential as a place for platelet adhesion. This scaffolding helps localize platelets and ultimately increases the concentration of growth factors to the desired point or location for tissue regeneration .2 and the centrifuge settings needed to obtain the ideal PRFM. To achieve ideal PRFM, PRP obtained was mixed with 25 mM CaCl2, then centrifuged again at a speed of 2,264 \u00d7 g for 25 min at room temperature. The centrifugation will result in two layers, the PPP and PRFM. The platelet level in PPP was 0/\u03bcl; thus, it can be assumed that this method produces ideal PRFM with all the platelets from PRP adhering to the matrix fibrin. Commercial kits usually include CaCl2 solutions in their package, though most choose not to disclose the amount or concentration of CaCl2 used in their set.In this experiment, we obtained a formula for making PRFM without using a costly reagent kit. The ideal PRFM requires that maximal platelets are trapped in the PRFM; the PPP platelet count of 0/ml can prove this. This experiment also found the minimum amount of CaCl2 in a Wheaton bottle before starting the centrifugation. In his study, 18 ml of whole blood can create 7\u20138 ml of PRP, which in turn yields a 35 mM round PRFM membrane (The PRFM protocol for this study is based on a previous experiment by O'Connell, which breaks down the creation of PRFM from whole blood. O'Connell created PRFM by inserting PRP and CaClmembrane .The platelet count results for whole blood, PRP and PPP after the first centrifugation showed that the platelet count in PRP was lower than that of the whole blood . In geneg for 5 min for separating centrifuge and 1,500 \u00d7 g for 15 min is best to produce optimal PRP (Studies that looked upon platelet, leucocyte, and erythrocyte yield on PRP production shed more light on the conundrum regarding the number of platelets being yielded. A review by Marxref concluded that a platelet count of 1 million in a 6-ml aliquot could be considered a benchmark for therapeutic PRP, again showing that the amount of platelet inside the PRP in our experiment falls short in terms of platelet counts to be considered as benchmark PRP . It seemBased on SEM examination, it can be seen that the PRFM obtained has a microscopic fibrin fiber matrix resembling a mesh . All plag for 60 min will precipitate platelets and the formed fibrin polymer. The dissolved protein will remain in the plasma, resulting in TGF\u03b21 and the activated platelets not precipitating in PRFM. It is known that both cytokines and growth factors secreted from cells have a short half-life, which means that a higher level of TGF\u03b21 in PRP does not mean that PRP is better than PRFM (Other properties analyzed in the PRFM produced are TGF\u03b21, a growth factor responsible for controlling and promoting cell growth, proliferation, and differentiation . Contrarhan PRFM . CompariThe weakness of this study is a lack of actual observation and comparison toward actual clinical use of the PRFM produced through the modified method proposed. A follow-up study looking at how the PRFM produced through this method fares compared to PRFM produced by commercial kit would further raise the credibility of using the proposed method as an alternative way of preparing PRFM, especially in limited-resource settings. A continuation of the study with more extensive and more varied samples may also yield valuable knowledge and further understanding of the procedure. Further modification and adjustment in the PRP creation method also need to be done to improve platelet yield during PRP production; as previously stated, omitting dilution, changing the centrifugation speed, or using alternative PRP creation kits or methods might counter this problem. Nevertheless, the study had proven that this modified method could produce ideal PRFM with somewhat comparable quality to the ones produced using a commercial kit.2 and centrifuging at a speed of 2,264 \u00d7 g for 25 min at room temperature can reliably produce ideal PRFM comparable in quality to the commercial kit. Further follow-up study is needed to compare the performance of PRFM produced by the modified method to those produced with commercial kits.The proposed modified method by mixing PRP with 25 mM CaClThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MR: study concept, design, ethical submission, data collection, data analysis, interpretation, and writing the paper. AH: study concept, design, lab examination, data analysis, and interpretation. LS: study concept, design, lab examination, and data analysis. MY: data analysis and interpretation and writing the paper and submission. 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": "In this paper, we propose a hierarchical statistical model for a single repairable system subject to several failure modes (competing risks). The paper describes how complex engineered systems may be modelled hierarchically by use of Bayesian methods. It is also assumed that repairs are minimal and each failure mode has a power-law intensity. Our proposed model generalizes another one already presented in the literature and continues the study initiated by us in another published paper. Some properties of the new model are discussed. We conduct statistical inference under an objective Bayesian framework. A simulation study is carried out to investigate the efficiency of the proposed methods. Finally, our methodology is illustrated by two practical situations currently addressed in a project under development arising from a partnership between Petrobras and six research institutes. The challenges in the production of offshore oil wells have been increasing over time, either due to the increase in technical difficulties because of the greater complexity of the areas to be explored, or due to improvements in the rules of the regulatory bodies in order to increase safety. There are two key pillars that should guide an oil well project: safety and productivity.Petr\u00f3leo Brasileiro S.A.), which is Brazil\u2019s largest oil and gas producer, has invested in technological innovation projects in order to minimize these losses and increase oil and gas production. Annelida is one of these Petrobras\u2019 innovation projects, which has been developed in partnership with the main Brazil\u2019s research centers. It regards an in-pipe robot that will be used at a near future to remove hydrates and paraffins that form in pipelines and can cause problems in oil and gas flow  the return and forward locomotives subsystem  with the intensity described by a power law. The resulting method is generally referred to as the Power Law Process (PLP). The PLP is convenient in many ways, especially for its flexibility, easy implementation, and the interpretability of its parameters , 10.Considering the fault-causing mechanisms known, it is also important to observe how to repair such failures, including preventive maintenance. In this context, the books of Crowder , PintiliUnder a Bayesian perspective, the inference of a problem is on the basis of the posterior distribution of the quantity of interest, which combines the information provided by the data with the available prior information. The elicitation of an appropriate prior distribution becomes the main task for Bayesian statisticians in practice. Subjective priors, which always depend on the experts\u2019 belief, are not easy to derive in a limited time period. Therefore, given little prior information, we prefer to use objective (non-informative) priors to make inference.a priori information, reflecting the vague nature of a priori knowledge. To obtain such prior, the expected Kullback-Leibler divergence between a prior distribution and a posterior distribution was maximized. The posterior distribution obtained using this prior has interesting properties, such as invariance and consistency in marginalization and sample properties , where T = 60. The first is a system subject to 3 failure causes each with 2 subcauses; and the second is a system subject to 2 main causes each one subject to 2 subcauses which, in turn, are subject to 2 other causes which, in the end, are also subject to 2 causes, in a 4-level structure. These structures can be seen in Figs In what follows, we present the results for two distinct structures of a single system, both under the assumption that the components system is observed in the fixed time interval  when compared to the asymptotic ones (B).As can be seen in Tables et al. , where T = 209 days, which relates to an operating time close to 5,000 hours.In both subsystems, the parameters of the model proposed here were associated with the Severity (S), Occurrence (O) and Detectability (D) indices. Thus, it was possible to take random realizations (based on Algorithm 1) of the failure times that represent the perspective provided by the technical team at FMEA, from the perspective of reliability. This approach ensures the addition of information at this early stage of the project. It was also assumed that both subsystems are observed in the fixed time interval  with high probability, the system must remain in operation (without any failure) for at least a minimum number of days; (ii) on the other hand, the median lifetime of the first failure is expected to be around another number of days. These requirements were determined by considering the expected number of annual missions, the size of the step taken by the robotic unit inside the oil pipelines, its respective speed and the estimated time for the hydrate block to melt. Such estimates also allowed to assess the time needed per mission and, therefore, the minimum desired lifetime.Based on the reliability requirements for the pressure vessel system, we consider that with high probability (\u224895%) the first failure should not occur before 68 days. Similarly, the median of first-failure time should be around 136 days. In this way, we can plot the reliability curve associated with the proposed model . This cuThe project designed 11 pressure vessels connected by connecting cables, as shown in An illustration of the data set generated can be obtained in full via an individual request for the authors. Due to the approximately linear behavior in the Duane plots, for each failure mode see , we haveThe summary for the parameter estimates see  express Some subsystems did not have an increasing intensity function (which would indicate a degradation process), however, the failure intensity in the initial times was higher, which results in the high occurrence of failures in the first moments of activity and, therefore, significantly reduces the component\u2019s survival time. This occurred more frequently in the Heatpipe and Carbon Fiber Protection subsystems. This information cannot be perceived directly on the parameter estimates see , howeverFrom versus the number of failures estimated over time can be used to assess the quality of the model\u2019s fit. In The graph of the observed number of failures In this section, we return to the situation described in Section 1. We obtained a suitable data set for this problem using a similar approach as proposed in the previous section, i.e., based on the limited but available information provided by the revised FMEA and FTA tools see .The required reliability for the traction system is graphically exposed in The Duane plots built for each failure mode see  have an In a practical context, if the failure times came from some missions, the BMAP estimates of the adjusted model for the traction system see  would shFrom these results see , we idenThe estimated reliability and intensity functions for the first-failure times are shown in The goodness of fit can be seen by the comparison between the observed and estimated number of failures along the time. These graphs, for each component, can be found in In this paper, we have continued the study started in , presentThe model structured in the proposed way allowed to highlight analytically and graphically the reliability associated with the first-failure times of each one of the subsystems  of two arbitrary systems that illustrate the use of modeling. Namely, the pressure vessels set and the traction system of the developing system that has served as a practical motivation for this theoretical development.As future works, we intend to evaluate the quality of these estimators in a context with outliers, their behavior  when exposed to data from an imperfect repair regime. In addition, we wish to evaluate the change in reliability based on the increase in redundancy of some subsystems. We also intend to assume that repairs are either perfect or imperfect, and model the dependence among the failure modes via shared frailty models.S1 FigFor the failure modes.(TIF)Click here for additional data file.S2 FigNumber of observed and estimated failures per component.(TIF)Click here for additional data file.S3 FigS = Severity, O = Occurrence, D = Detection.(TIF)Click here for additional data file.S4 FigFor the failure modes.(TIF)Click here for additional data file.S5 FigNumber of observed and estimated failures per component.(TIF)Click here for additional data file.S1 Data(ZIP)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file."}
+{"text": "Effective teaching methods are vital for cultivating advanced professional skills in nurses and equipping them with the necessary training. Problem-based learning (PBL) and self-directed learning (SDL) have been consistently used in nurse education. Therefore, their effects on nursing students\u2019 academic performance warrant comparison. This study compared the effects of PBL and SDL on an adult nursing university curriculum. Participants in this quasi-experimental study with a pre-post non-equivalent control group design were 106 third-year nursing students divided into the PBL and SDL groups. Data collection, conducted from April to June 2019, included a pre-test before an eight-week intervention, followed by a post-test. Changes in the scores of each group were analyzed for learning motivation, self-directed learning ability, self-efficacy, learning confidence, learning satisfaction, and academic performance using paired and independent t-tests. The PBL group scored higher on learning motivation, self-directed learning ability, and academic performance than the SDL group. Based on these results, the PBL method was more effective than the SDL method in an adult nursing curriculum. To maximize the learning effect in adult nursing education, it is necessary to apply SDL education, including the PBL method, with a clearer learning process. Today\u2019s medical environment requires nurses to have clinical reasoning abilities, collaboration skills, and the ability to identify and solve patient problems. The foundation of these skills includes the capacity to accurately and comprehensively understand basic medicine and professional nursing, and appropriately apply this knowledge in clinical settings . To ensuProblem-based learning (PBL) is a teaching and learning method that uses real-life situations to allow students to acquire the knowledge, skills, and attitudes needed to identify patient problems and develop the necessary solutions. Previous studies have identified PBL as an appropriate learning method in nurse education ,3,4. A PNursing students are required to engage in theoretical education and learning, as well as practice in clinical settings, to acquire extensive curricular knowledge. After entering the workforce, they also need to commit to lifelong learning, because they must adapt to dynamic changes in clinical settings. Within this context, nursing students adopt the self-directed learning (SDL) method and study proactively. SDL is a process through which learners identify their desire to learn, set their own learning goals, secure the resources necessary for learning, implement appropriate learning strategies, and assess their learning outcomes . One impDespite the above-mentioned findings, existing studies have had a rather fragmented focus on PBL and SDL methods ,6,10,13.PBL and SDL methods may differ according to topics covered, learning context, and educational objectives, but they usually involve certain established steps. To achieve educational goals through PBL, the tutor sets learning goals and forms small groups. The groups then receive goal-based case scenarios, which they must find their own ways to resolve. Subsequently, each group presents the results of their case-based problem-solving and receives feedback from their tutor and peers, followed by a discussion ,19,20.To achieve educational goals through SDL, the tutor sets the learning goals. Learning can be undertaken in groups, but is mainly carried out individually. The tutor provides learning topics and materials , and the students acquire knowledge of the concerned topic either according to the strategy suggested by the tutor or their individual plans. Ultimately, students present their learning outcomes and receive feedback from the tutor ,21,22.This quasi-experimental study employed a pre-post non-equivalent control group design.This study was conducted at Semyung University, located in Chungcheongbuk-do, South Korea, between April and June 2019. Convenience sampling was used, and students were recruited through posters placed on the nursing department\u2019s bulletin board in March. Of the 110 third-year nursing students who participated, 54 were in Class A , and 56 were in Class B .Based on an independent t-test, each group required at least 51 participants, assuming a two-sided significance level (\u03b1) of 0.05, power (1\u2212\u03b2) of 0.80, and an effect size of 0.5 . Thus, tClass A was assigned to the PBL group and Class B to the SDL group. Both groups then completed a written pre-test in early April. For eight weeks, Class A was exposed to PBL-related course content while Class B was exposed to SDL-related course content. Subsequently, a post-test was completed in mid-June.The learning objectives and content of the adult health nursing course were designed according to Bloom\u2019s taxonomy. Teaching tools and strategies were selected for each objective. The PBL- and SDL-specific objectives and content are presented in The PBL process, organized by a tutor, centers on each team solving problems based on patient scenarios on a specific topic. The objective of the PBL process is for students to solve patient case problems, apply nursing processes, and learn relevant theories. Students were divided into groups of no more than 10 each.SDL methods are the result of self-designed or group study, and focus on self-learning of a given topic. The objective of the SDL process is for students to learn theories on a given topic.Effective training on the application of PBL and SDL requires trained tutors or experts. In this case, the tutors selected for both PBL and SDL were staff nursing experts from different departments. The three tutors received training from the nursing education department through workshops on successful PBL and SDL that also described the role of the tutor. The PBL and SDL methods are shown in To assess learning motivation, Hwang\u2019s 27-question instrument , which iThe SDLA scale, developed for university students by Lee et al.  and compKim and Park\u2019s  academicTo measure learning confidence, an eight-item instrument developed by Hur et al.  was usedTo assess learning satisfaction, an instrument developed by Hur et al. , which iAcademic performance is an evaluation of the learner\u2019s acquisition of information or skills from specific classes . In thisA final questionnaire was developed to evaluate the extent of students\u2019 knowledge of the respiratory system, which is a part of the adult nursing care curriculum. Thirty questions were developed by an experienced panel of nurses and nursing professors. A correct answer was awarded one point, and a wrong answer was awarded zero points. The total possible score ranged from 0 to 30.Data were collected after the Institutional Review Board of \u201cS\u201d University approved the study protocol (SMU-2018-03-001-01). The employed instruments were approved for use by their respective authors. Research assistants explained the study objectives, methods, and the fact that there was no penalty for withdrawing from the study to the volunteers, who participated after providing written informed consent. Participants received small gifts for their participation. The PBL and SDL methods were cross-applied to the experimental and control groups after the post-test.p \u02c2 0.05 was considered significant.Data were analyzed using SPSS 21.0 . To analyze participants\u2019 general characteristics, we computed frequencies, percentages, means, and standard deviations. Chi-square, Fisher\u2019s exact, and t-tests were used to test for homogeneity between the PBL and SDL groups with regard to general characteristics and pre-intervention dependent variables. After each learning method was applied to the PBL and SDL groups, the Kolmogorov\u2013Smirnov normality test was performed and its conditions were satisfied. Subsequently, a paired t-test and an independent t-test were performed to examine differences in learning motivation, SDLA, self-efficacy, learning confidence, learning satisfaction, and academic performance, before and after the intervention. Thirty questions representing SDL and PBL were included in the final examination, and comparisons were made through an independent t-test and a chi-square test. For each tool, student scores (maximum of 30) were categorized into high (\u226524), moderate (18\u201323), and low (<17). For all analyses, After excluding four students\u2014two members of the SDL group and one member of the PBL group who did not take part in the post-test, and one member of the PBL group who took a leave of absence during the semester\u2014the data from 106 participants  were analyzed. Participant characteristics are shown in The t-test results showed no significant difference between the PBL and SDL groups in any of the pre-intervention dependent variables, thus confirming between-group homogeneity .t = \u22122.262, p = 0.031), SDLA , self-efficacy , and satisfaction in learning . There were differences in the SDL group\u2019s pre-test and post-test scores for SDLA  and self-efficacy . A comparison of the PBL and SDL groups\u2019 pre-test and post-test scores showed statistically significant differences in learning motivation , SDLA  and satisfaction in learning . Based on categorization of the scores according to high, moderate, and low, the PBL group had a higher distribution of participants scoring \u201chigh\u201d than did the SDL group, and a lower distribution of participants scoring \u201clow\u201d; the difference was statistically significant (\u03c72 = 2.146, p = 0.046; The PBL group\u2019s average score was 22.85 \u00b1 3.74, while that of the SDL group was 20.94 \u00b1 3.39. The difference between the two groups was statistically significant (This study aimed to compare the effectiveness of the PBL method which is used to solve problems in patient cases, and the SDL method which is used for self-learning on a topic, in adult nursing student education. The PBL group demonstrated a significant increase in learning motivation, SDLA, self-efficacy, and satisfaction in learning. The PBL and SDL groups did not differ statistically in self-efficacy, learning confidence, and academic performance. The PBL group\u2019s respiratory system knowledge, as demonstrated by scores on the final test, however, was significantly higher than that of the SDL group. This result warrants further review. The implication of this study is that, to maximize the effect of SDL education, it is necessary to devise an educational method that includes the PBL method as well, as it was more effective than the SDL method in nursing education.Regarding learning motivation, the SDL group\u2019s post-test score was much lower than that of the PBL group. Studies have reported that nurses who used the SDL method experienced learning difficulties at many stages, resulting in a lack of learning motivation . FurtherBoth the PBL and the SDL groups demonstrated an increase in post-test scores on the SDLA scale, compared to pre-test scores. SDLA is a quality that is expected of healthcare graduates . Both SDThe term \u201cself-directed\u201d in SDLA may sometimes be misleading; it could imply that no help is needed in learning . When thRegarding self-efficacy, we found a significant increase in the post-test scores of both groups. This finding is supported by previous studies with nursing students , medicalAdult nursing care is a vast and challenging curriculum that demands the coverage of a wide range of topics in a brief period. The application of SDL  and PBL In the PBL group, the pre-post difference in learning satisfaction scores was significant; this was not the case in the SDL group. A previous meta-analysis revealed that PBL required professors to be professionally skilled in clinical reasoning and in preparing students to navigate this method . Thus, iThe pre-post score difference in academic performance was also non-significant. However, in the final test, the PBL group obtained a higher average score and a higher distribution of \u201chigh\u201d scores compared to the SDL group, demonstrating a more positive impact of PBL on academic achievement. Findings from other studies also show that PBL  and SDL In a systematic review of effective SDL strategies in medical students, the included studies focused on setting goals and monitoring the situation and social relations, not on the SDL process and self-assessment support, thus limiting the effectiveness of SDL . The sysDespite its strengths, this study has certain limitations. First, owing to the use of five variables to confirm the difference in the effects of PBL and SDL, the questionnaire contained a large number of items. This could have led to fatigue during the pre- and post-test, reducing the accuracy of participants\u2019 responses. Second, this study was conducted in only one school, which limits the generalizability of the results. Finally, the eight-week intervention period may not have been sufficient to demonstrate changes in learning motivation, SDLA, self-efficacy, learning confidence, and learning satisfaction, or to improve students\u2019 academic performance.This study attempted to explore the effects of applying the SDL and PBL methods to adult nursing education. SDL is an important aspect of lifelong learning in an integration-based curriculum. However, the SDL method was less effective than the PBL method in nurse education in an adult nursing curriculum. In future studies, we will continue using well-planned PBL methods for nursing student education and make recommendations for the development and application of evidence-based SDL methods. Based on this study, a further exploration of PBL curriculum development in nursing education is suggested. We also suggest that future research should explore the development of the SDL learning process by including the PBL method."}
+{"text": "Streptococcus\u00a0gallolyticus subspecies gallolyticus is a known pathogen that causes infective endocarditis, and most cases involve the left heart valves. We present the first reported case of prosthetic tricuspid valve endocarditis caused by this microorganism. Relevant literature is reviewed.Streptococcus\u00a0gallolyticus subspecies gallolyticus was grown from its culture. Prolonged antibiotic treatment was initiated.A 67-year-old Jewish female with a history of a prosthetic tricuspid valve replacement was admitted to the emergency department because of nonspecific complaints including effort dyspnea, fatigue, and a single episode of transient visual loss and fever. No significant physical findings were observed. Laboratory examinations revealed microangiopathic hemolytic anemia and a few nonspecific abnormalities. Transesophageal echocardiogram demonstrated a vegetation attached to the prosthetic tricuspid valve. The involved tricuspid valve was replaced by a new tissue valve, and Streptococcus\u00a0gallolyticus should be considered as a rare but potential causative microorganism in prosthetic right-sided valves endocarditis. The patient\u2019s atypical presentation emphasizes the need for a high index of suspicion for the diagnosis of infective endocarditis.Based on this report and the reviewed literature,  Staphylococcus\u00a0aureus (S.\u00a0aureus) is the most common pathogen , in addition to the presence of schistocytes in the peripheral blood smear. Additional abnormal laboratory results included increased levels of C-reactive protein (CRP) and an elevation of cholestatic liver enzymes. Urinalysis demonstrated hematuria and mild proteinuria, without clinical or laboratory findings of acute kidney injury. A mildly elevated level of rheumatoid factor was noted. Chest X-ray and fundoscopy revealed no pathological findings. Transthoracic echocardiogram demonstrated a 1.1\u00a0cm vegetation on the tricuspid valve. Transesophageal echocardiogram demonstrated severe tricuspid stenosis as a complication of the attached vegetation, as well as an additional 1.1\u00a0cm vegetation on the pacemaker electrode . A native aortic valve was the most prevalent site of infection (28%), followed by a coinfection of native aortic and mitral valves (18%). An isolated infection of a native mitral valve was reported in 16% of the cases and a native tricuspid valve infection in only 2%. Prosthetic tricuspid valve involvement has not been reported in any of the cases, or in any other case report. Furthermore, infection of pacemaker electrodes was also extremely rare, with only two reports in the reviewed studies (0.4%) and a few case reports.To the best of our knowledge, this is the first reported case of Streptococcus\u00a0gallolyticus infections are also associated with colonic neoplasms. A recent study from a multicenter registry found colorectal tumors in 69% of S.\u00a0bovis IE patients, with a clear predominance of benign lesions (78%) [S.\u00a0bovis complex, S.\u00a0gallolyticus has the strongest association with colonic neoplasm, and this association is higher for IE compared with other sites of S.\u00a0gallolyticus infection [S.\u00a0bovis bacteremia.ns (78%) . Among tnfection . TherefoA history of prosthetic heart valve implantation constitutes a significant risk factor for IE, with the greatest risk during the first 6\u201312\u00a0months after the valve replacement. Prosthetic valve endocarditis (PVE) accounts for 7\u201325% of IE cases in developed countries, with a similar risk for bioprosthetic and mechanical valves at 5\u00a0years after\u00a0the valve replacement . AntecedS.\u00a0aureus is the most common pathogen causing tricuspid IE, a disease that primarily affects IVDA [S.\u00a0bovis pacemaker IE were observed [S.\u00a0bovis was found in only 3% of patients with IE involving intracardiac electronic devices (including pacemakers and implantable cardioverter defibrillators) [As mentioned before, cts IVDA . Besidesobserved . In anotllators) .S.\u00a0bovis should be considered as a rare but potential causative organism in cases of right-sided IE.IE is a complex infectious disease characterized by a highly variable clinical presentation, heterogeneous patient populations, and various causative microorganisms. The patient presented here was admitted to the emergency room because of nonspecific complaints, and her physical examination demonstrated no significant findings. Initial laboratory tests revealed mild hemolytic anemia and presence of schistocytes in peripheral blood smear. In the absence of fever, heart murmurs, or peripheral IE manifestations, the existence of new mechanical hemolysis in a patient with a biological prosthetic valve was the major clinical hint for IE and prompted the need to obtain blood cultures in the emergency room. This case demonstrates that IE should be suspected and ruled out in cases of new or worsening hemolysis in patients with intracardiac risk factors for IE. Based on this report and the reviewed literature,"}
+{"text": "Compared to their predecessors, the next generations of aircrafts will be more electrified, require more electrical power and operate at higher voltage levels to meet strict weight and volume constraints. The combined effect of low-pressure environments, increased voltage levels and compact designs intensifies the risks of premature insulation degradation due to electrical discharge activity. This paper studies the resistance to surface discharges of PTFE (polytetrafluoroethylene) and ETFE (ethylene tetrafluoroethylene), two insulation materials widely used in today\u2019s aircraft wiring systems due to their outstanding properties, such as a wide temperature operation range and a high dielectric strength. The study is carried out in a low-pressure chamber, which was pressurized within the pressure range of 10\u2013100 kPa that includes most aircraft applications. There is a compelling need for experimental data to assess the resistance of insulation materials to surface discharges at a very early stage as a function of the environmental pressure. Data on resistance to surface discharges in low-pressure environments for aeronautical applications are lacking, while most standards for insulation systems are based on tests under standard pressure conditions. The results provided in this work can be useful to design wiring systems for future more electric aircrafts, as well as to design fault detection systems for an early detection and identification of faults related to surface discharges. Therefore, the data and analysis included in this paper could be of great interest to design and develop insulation systems for wiring systems and standard assessment methods, as well as to design fault detection strategies for the early detection and identification of surface discharges for future generations of more electric aircrafts. Distribution voltage levels will increase on new aircraft models due to their progressive electrification as a result of recent developments in more electric aircrafts (MEA) and all electric aircrafts (AEA). The combined effect of higher voltages, compact designs and extreme environmental conditions found in next-generation aircrafts pose insulation systems in a challenging position. Operation at higher voltages in combination with the extreme environmental conditions typical of aircraft systems greatly increases the risk of electrical discharge occurrence . As deriCommercial airliners typically fly at altitudes in the range of 9.5 to 11.5 km, although they can climb up to 15 km , and difAccording to the IEC 60112:2020 standard . AlthougAlthough there are different international standards to determine the resistance of solid electric insulation materials to electric discharges ,17,18,19\u00ae) [By using field-grading materials in the insulation layer or by adding insulating filler compounds, it is possible to reduce the electric stress at the expense of adding complexity, weight, increasing the diameter of the cable ,21, and \u00ae) .This work studies the surface discharge behavior of PTFE and ETFE insulation materials commonly used in current wire insulation aircrafts under 400 Hz in the pressure range of 10\u2013100 kPa, which is representative of most aircraft applications. It also presents a low-pressure test bed designed to qualify wire insulation materials under this pressure range to simulate the operating environment of unpressurized areas of aircrafts. The experimental results presented in this work allow a fair comparison of the performance of the two insulation materials studied and show the vital role that pressure plays in the inception of surface discharges. Due to the development of MEA aircrafts, there is an urgent need to generate experimental data to design compact wire insulation systems as well as to develop test systems to assess the resistance of insulation materials to surface discharges, taking into account the variable pressure range found in aircraft systems. Currently, there is a scarcity of experimental data related to the resistance of wire insulation materials to surface discharges under low-pressure conditions found in aircrafts, in part because most standard tests for qualifying insulation systems are performed under standard pressure conditions. Therefore, it is necessary to understand and address the role of wire insulation materials for preventing surface discharge occurrence, in order to optimize the reliability of insulation systems operating at low pressure. The results derived from this work may be valuable not only for designing wiring insulation systems for next-generation MEA aircrafts, but also for developing failure detection methods for the early detection of surface discharge activity.The organization of this document is as follows. This section details the components of the experimental setup, including the sensor used to detect the UV radiation emitted by surface discharges, the wire electrodes and insulation materials used, as well as the components of the low-pressure chamber used for the tests performed under different pressures and the variable high-voltage source.Before complete breakdown, electrical discharges produce partial discharges, which in air gaps are perceived as corona discharges  in the mThe UV spectrum includes wavelengths in the range of 100\u2013400 nm, which is divided into UVC (100\u2013280 nm), UVB (280\u2013315 nm), and UVA (315\u2013400 nm) spectral ranges. It is an accepted fact that ozone in the stratosphere absorbs most of the extraterrestrial radiation that falls within the UVB and UVC ranges. Solar blind sensors allow one to avoid the interference of sunlight, so they fall in the category of optical sensors that can only detect UV radiation with wavelengths less than 280 nm . Thus, sDue to the abovementioned characteristics, the solar blind R9533 UVTRON sensor  was used to detect discharge activity at a very early stage due to different relevant features, including small size, very reduced power consumption, high sensitivity, pressure range compatible with the requirements of this work and reasonable cost. This sensor incorporates a gas-filled tube inside, in which an electron avalanche multiplication phenomenon occurs due to a high-voltage difference applied between the photocathode (negative electrode) and the anode (positive electrode).The main features of the gas-filled tube UV-sensitive sensor used in this work are summarized in To optimize the performance of the R9533 UVTRON sensor, a suitable driver circuit is required. In this way, by applying a low-voltage to the driver circuit, the sensor can operate safely. In addition, the signal processing circuitry built into the driver circuit cancels out sporadic background noise, thereby minimizing the probability of false detection events. To this end, the commercial C10807  driver circuit was used to supply the R9533 sensor. Previous internal tests were carried out in the AMBER laboratory of the Universitat Polit\u00e8cnica de Catalunya. In such tests, the performance and sensitivity of the solar blind R9533 UVTRON sensor was compared with that of a single-loop antenna sensor and a back-illuminated CMOS imaging sensor. The results verified the sensitivity and accuracy of the UV-sensitive solar blind sensor in detecting UV radiation emitted by the discharges at a very early stage .This section describes the specific electrodes used to determine the performance to surface discharges of PTFE- and ETFE-insulated wires. To this end, wire electrodes are used to reproduce in the laboratory the surface discharges occurring in real aircraft environments. It should be noted that this paper focuses on detecting surface discharges at a very early stage, long before significant consequences can arise. The wire electrodes were damaged following the procedure detailed in the European standard EN 3475-603:2018 , which dA cable-stripping tool  was used to cut the insulation layer, which is shown in In order to compare the arc tracking performance of various specimens, it is very important to ensure that the cuts made on the different specimens are as similar as possible. To this end, the width of the cuts and the distance between them were measured with the help of digital photographs. Using a 54 Mpixels digital camera (approximately 9000 \u00d7 6000 effective pixels), resolutions of around 10 micrometers can be achieved. Even so, as it is not possible to perform identical notches in the insulation layer, three replicas of the pairs of insulated wire samples were made for each type of insulation; these replicas allowed for the evaluation of the dispersion of the results.It is also noted that both analyzed wires have the same size (AWG 24) and insulation thickness, so the results can be compared.As the experiments presented in this work are realized in a wide pressure range that covers the 10\u2013100 kPa interval, a low-pressure chamber is required. For this purpose, a 375 mm \u00d7 260 mm (height \u00d7 diameter) cylindrical stainless-steel container was used, with a methacrylate lid sealed with a silicon rubber gasket to prevent air from entering the chamber from outside. This chamber also includes two vacuum-tight access ports, the first one for the high-voltage cable and the second one for the wires that supply the solar blind UV-sensitive sensor.3/min manufactured by Bacoeng, Suzhou, Jiangsu, China) was used to reduce the pressure in the low-pressure chamber. The pressure inside the low-pressure chamber was measured with an analog manometer integrated with the chamber. The experiments were carried out at a temperature of 25 \u00b0C and the humidity was limited below 25%. More details of the low-pressure chamber can be observed in A vacuum pump is required to vary the pressure inside the low-pressure chamber within the pressure range of 10\u2013100 kPa. To this end, a single-stage BA-1 vacuum pump  was linked to a VKPE-36 single-phase high-voltage transformer manufactured by Laboratorio Electrot\u00e9cnico . It should be noted that during the tests, the output frequency of the SP300VAC600W power supply was adjusted to 400 Hz, the characteristic distribution frequency in current aircrafts. As the SP300VAC600W power supply has a sensitivity of \u00b10.1 V and the high-voltage transformer steps up the voltage by a factor of 100, the minimum voltage step on the high-voltage side is 10 V.The results presented in this section have been obtained using the experimental setup described in The experimental data presented in this section are based on the corona extinction voltage (CEV), i.e., the minimum value of the voltage at which corona activity (UV radiation emission) is detected with the solar blind sensor. To determine the CEV value at each pressure level, the following procedure is applied. First, the voltage is gradually increased starting from 0 kV until the solar blind sensor detects corona activity, this being the corona inception voltage. The voltage is then further increased by approximately 10%. Next, the voltage is gradually reduced until the solar blind sensor detects no UV radiation coming from the analyzed mating wire electrodes. The minimum voltage at which UV radiation is detected corresponds to the CEV value. It is worth noting that because under CEV conditions, i.e., at the very early stage of discharge activity, due to the very low energy level of discharges and noisy aircraft environments, the analysis of UV emissions is very convenient. Otherwise it is very complex to detect the phenomenon by measuring electrical signals.This section presents the CEV values obtained with the PTFE-insulated wires described in The results presented in Once again, the results presented in The comparative results presented in For a better interpretation of the results presented in The results presented in The results presented in Future aircraft designs will be more electrified, requiring more electrical power and higher distribution voltage levels to meet stringent weight and volume constraints. The combination of low-pressure conditions, higher voltage levels, and dense form factors in next-generation aircrafts increases the risks of premature insulation degradation due to surface discharges.This paper has investigated the resistance to surface discharges of two insulation materials intended for aircraft wiring systems, namely PTFE and ETFE. For this, artificially damaged insulated wire electrodes have been used, so that surface discharges can be effectively replicated on the different electrodes and analyzed and detected at a very early stage. The discharges have been detected using a small-size low-cost solar blind UV sensor, which detects the UV radiation emitted by the discharge activity without any interference from sunlight. The analyzed PTFE- and ETFE-insulated wires have been tested in a low-pressure chamber in the pressure range between 10 kPa and 100 kPa, which accounts for most aircraft applications. The experimental data presented in this work show that, under the conditions of this study, PTFE insulation outperforms ETFE in terms of resistance to surface discharges, because the average value of CEV (corona extinction voltage) of PTFE-insulated wires is 16.5% higher than that of ETFE-insulated wires. The results provided in this work can be useful to design wiring systems for future more electric aircrafts, as well as to design fault detection systems for early detection and identification of faults related to surface discharges."}
+{"text": "Recent research on the effects of COVID-19 on school closures has mainly focused on primary and secondary education, with extremely limited attention to early childhood education (ECE). To address this gap, we identify the extent to which parents and caregivers with pre-primary school-aged children were engaged in their children\u2019s learning during school closures in Ethiopia. Our focus on Ethiopia is of particular relevance given that ECE provision has expanded dramatically in recent years, aimed at ensuring children are prepared for primary school. Using data collected through a phone survey with 480 parents and caregivers, the results revealed that learning disruption due to COVID-19 school closures is likely to be substantial and will probably widen existing inequalities further. Many poorer households and those where parents or caregivers are not literate, are less likely to have child-oriented learning resources, and home learning activities between parents and children in these households are limited. The study highlights that greater attention needs to be paid to mitigate the threats of COVID-19 on Ethiopia\u2019s recent gains in ECE, to prevent the pandemic from further reinforcing inequalities between children from advantaged and disadvantaged households. The outbreak of COVID-19, with its associated economic and social challenges, has led to serious consequences for the schooling of children worldwide. A recent study indicates that the interruption of early childhood education (ECE)ECE in Ethiopia has expanded dramatically in recent years with the government\u2019s commitment to providing access to all 6-year-old children, with the intention of expanding ECE gradually to include 4- and 5-year olds. In line with other countries worldwide, schools in Ethiopia were closed due to the pandemic on 16 March 2020. More than 26 million students from over 47,000 schools nationwide were affected by the closures, including 3.2 million young children who had been participating in ECE. However, while primary and secondary have received attention in the government\u2019s COVID-19 response planning  demonstrate that those exposed can suffer life-long negative impacts, such as poor cognitive development Kelly,  and loweIn the light of the scant attention paid to early childhood development during the ongoing COVID-19 crisis, Yoshikawa et al.  highlighLopez Boo et al.  have simA conducive learning environment at home has been widely documented as critical for children\u2019s acquisition of foundational skills , Ethiopia ranks near the bottom, at 173 out of 189 countries UNDP, .Ethiopian children officially enter Grade 1 of primary school at the age of seven. Since the 1990s, Ethiopia has made remarkable progress toward achieving the Education for All goal of universal primary education. In 1992, almost four out of five children were out of school, but by 2016, the net enrolment rate reached 100%  research in Ethiopia. During the 2019 ELP survey, household respondents were asked to provide mobile phone numbers so that they could be contacted in the follow-up surveys if they moved from their sample location. At least one valid phone number was obtained for 1,985 out of 3,219 households (62% of households). This was the basis for the sampling frame for the 2020 phone survey. It is therefore important to note that the views captured in the phone survey are inevitably confined to parents and caregivers who possess a mobile phone. This could create some bias in responses, given that our previous analysis has shown that poorer households are less likely to possess a phone . In addition, we purposively sampled according to children\u2019s ECE enrolment status (to ensure a mix both of those who had attended O-Class and those who had not prior to school closures), gender, and caregivers\u2019 literacy (measured according to whether they were able to read a sentence during the face-to-face interview conducted in November 2019).Second, we used a three-stage selection process to obtain a sample disaggregated by children\u2019s O-class enrolment status, gender, and caregivers\u2019 literacy. Table The phone survey drew on core modules developed by the ELP Systems Research Program at the World Bank, from which the Ethiopia ELP team selected items that were most relevant to the context. The core module included a set of items drawn from the caregiver section of the Measuring Early Learning Quality and Outcomes (MELQO) assessment. This is a globally validated, standardized assessment which is used to\u00a0measure child development and learning , degree questions , and questions where participants had to rank responses, to allow for a wide range of information to be collected. The instruments underwent an iterative revision processes within the team, notably based in Ethiopia, to make them most relevant to the country-specific context. Given that many of our participants were likely to be in precarious circumstances, we decided not to include any questions asking sensitive information through the survey. To check the clarity of the questions, we conducted a pilot with teachers and parents living in Addis Ababa. The pilot survey confirmed that participants could understand the questions, and so no major revisions were needed. The instruments were translated into three local languages, including Amharic, Afan Oromo and Tigrigna.Prior to conducting the phone surveys, ethical approval was obtained from the Ethical Review Board of the College of Education and Behavioural Studies at Addis Ababa University, and from the Faculty of Education at the University of Cambridge. We were particularly mindful of the disruption that COVID-19 was having on the lives of those involved in the study. All participants provided informed verbal consent. They were presented with the option to participate in the phone interview immediately, or to arrange a suitable time for the researcher to call back. Participants received compensation (100 ETB phone credit) once the interview was completed, drawing on best practice from within Ethiopia, and in line with ethically sound procedures Morrow, . All datOf the 480 households selected at the initial stage, 58 (or 12%) households were substituted with other households on the replacement list, which was prepared before data collection in case some respondents could not be reached. Where the initial sample was replaced, this was most commonly due to failure to obtain a response from the respondents, failure to acquire the correct contact details of the participants or, in a very small number of cases, because those contacted declined to participate.In terms of response rates, about 98.5% of respondents with whom the research assistants made initial contact, agreed to participate in the research. The fact that we had previously conducted face-to-face interviews with them, is likely to have contributed to the high response rate in the current study. The majority of the calls were uninterrupted (65.6%), a few were interrupted but subsequently continued and completed (15.4%), and a very small number were interrupted and continued at a different time (0.9%). A poor network connection was often the reason for some of the interrupted calls.For the data analysis, we merged data collected via the phone survey with the respondents\u2019 background information collected during the ELP survey conducted in November 2019. This enabled us to analyse the data to show differences in caregivers\u2019 COVID-19 responses across various groups of respondents . Descriptive analysis presented in the next section was undertaken using Stata 16.Parents and caregivers have faced serious financial constraints as a result of the pandemic. The survey results show that nearly 80% percent of parents and caregivers living with pre-primary school-aged children reported that their family experienced a loss of household income since the pandemic, with poorer families being disproportionately affected (81% in poorest households compared with 71% in richest households). About four in 10 households reported that they had experienced a shortage of food since the outbreak of COVID-19, which raised concerns about insufficient food and nutrition for children. Despite the prevailing economic burden of COVID-19, less than half of households reported that they had taken some measures to try and mitigate their financial difficulties. Among those who used coping measures, two-thirds reported cutting household spending on food and non-food purchases, 16.1% received assistance from friends or families, while some received remittances (5.2%), or assistance from the government (4.2%).Our first research question addresses the extent to which parents and caregivers had access to learning resources and information during school closures due to COVID-19. Figure\u00a0Figure\u00a0This indicates that learning disruption is likely to be substantial, given that\u00a0many households have limited learning resources of relevance to young children. Not surprisingly, parents reported that the biggest challenges they have faced in home-based learning included a lack of home learning materials (51.5%), followed by a lack of radio, TV, or tablet for remote education (14.2%), a lack of information and knowledge on child development (13.4%), and a lack of radio/TV educational programmes for young children (9.2%).In addition, most parents and caregivers have received little information from schools or local governments on how to support their children\u2019s learning during school closures. Only 10% of caregivers with children enrolled in O-class reported that they have been in contact with ECE teachers or school principals. More affluent households were more than twice as likely to be in contact with ECE teachers or school principals (15%) than poorer households (6%). Importantly, caregivers who had communicated with teachers or principals were more likely to be engaged in their child\u2019s learning activities at home.Our second research question assesses the extent to which parents and caregivers have been able to support their children\u2019s learning activities and play at home during school closures. Figure\u00a0As shown in Table Urban families were more likely to support their children to read books obtained from schools than rural families. Similar to the urban\u2013rural divide in terms of access to technology, rural families helped their children to listen to educational radio programmes, while urban families helped their children to watch educational TV programmes. It should be noted that the Ethiopian government did not provide explicit radio or TV educational programmes for pre-primary school-aged children, unlike the radio programmes they provided for primary school children.Almost half of the educational activities for pre-primary school-aged children were supported primarily by their mothers, with around one-quarter of older siblings and fewer than one in five fathers having this responsibility. Mothers in rural households were more likely to have primary responsibility for supporting child learning, even though they were less likely to be literate, and thus more likely to face challenges in supporting their children\u2019s learning at home.Positively, around three-quarters of caregivers reported that they play more often with their child since the COVID-19 crisis than they did previously, with about half of caregivers reading books, telling stories, or singing songs more often to their child while staying at home Fig.\u00a0. HoweverAlong with families experiencing unexpected disruptions to their daily lives and welfare, some parents and caregivers also observed increased levels of stress and anxiety in their child, during school closures. Table At the same time, parents and caregivers were more likely to pay attention to their children\u2019s psycho-social wellbeing. More than half of parents and caregivers reported that they care about children\u2019s emotions by comforting them when s/he was feeling sad or cried, asking how the child feels, and listening to what he/she said, more often than they used to before the COVID-19 pandemic.This study aimed to explore the effect of COVID-19 on children\u2019s early learning continuity, from the perspectives of parents and caregivers. By conducting a phone survey of 480 Ethiopian households with pre-primary school-aged children between August and September 2020, we examined the extent to which parents and caregivers had access to learning resources and information during school closures caused by the COVID-19 pandemic, and the extent to which they have been able to support children\u2019s learning, play, and wellbeing at home. In this section, we link the responses to our research questions, to existing literature and information collected in other countries that are facing similar challenges.Our analysis shows that, in addition to the health and economic burdens from COVID-19, households have faced difficulties in supporting children\u2019s learning at home. Parents and caregivers have had limited access to learning resources at home during school closures. Although remote learning strategies have emerged as the prominent means to ensure learning continuity, more than half of the families surveyed reported that they did not have access to either a radio or TV, with striking disparities between urban and rural, and the poorest and richest households. Moreover, despite the evidence that the availability of child-oriented books can play an important mitigating role for continued learning (Dowd et al., The challenges in equipping homes with learning resources are likely to be reinforced by a lack of information and support for parents from schools and governments, both of which are prevalent in Ethiopia and other low-income countries. We found that only one in ten caregivers and parents reported that they have been in contact with their child\u2019s school. Even at the primary level, only one in five low-income countries reported providing regular phone communication between parents and schools during school closures, which has the potential to reinforce parental involvement in a child\u2019s home learning (UNESCO et al., Another challenge has arisen due to a lack of prioritization of ECE in policy planning within the education sector. The Ethiopian government has no remote learning policies and programmes for ECE in their COVID-19 response plan, contrary to those in primary and secondary education (Ministry of Education, Our second research question focuses on the extent to which parents supported children\u2019s learning activities and play at home during school closures. Only about half of parents and caregivers reported that they had been involved in supporting their children\u2019s educational or learning activities. While it is apparent that younger children require additional support from parents to engage in learning and play at home at a time of social isolation, this is unlikely to be attainable, especially for parents and caregivers who have never been to school, lower-income households, and those living in rural areas. Notably, we found that many parents have struggled to balance their responsibilities for childcare, household chores, or paid employment, with a disproportionate burden placed on mothers to support their child\u2019s learning.Parents and caregivers reported their efforts to create a supportive home environment, with stimulating activities such as more frequent playtime with their children, telling stories, and singing songs more often during the closure period. More than half of them also expressed how much they care about the child\u2019s psycho-social development through these increased interactions with their children. It is important to note that the current study is confined to self-reported responses of caregivers, with a need for further research on how parents and caregivers have interacted with their children, as well as the psycho-social wellbeing of caregivers themselves. During the influenza A-H1N1 pandemic, parents who faced quarantine with limited social interactions showed higher symptoms of mental ill-health, compared to those who underwent less strict social isolation in the U.S and Canada (Sprang & Silman, Moreover, a complex array of factors elevated by COVID-19\u2014social isolation, parental stress and adversity, and uncertainty\u2014has an impact on the psycho-social wellbeing of children. In our survey, parents often reported that their child was less motivated to learn, and expressed their stress and anxiety through more frequent crying, being less talkative, or aggressive behaviours. Some emerging evidence shows alarming patterns of children\u2019s mental health during the spread of COVID-19. To illustrate this in other contexts, since schools were closed, children and youth were more likely to show mild to severe mental health problems such as depression, anxiety, and sleep disorders in China (Duan et al., Although this is beyond the scope of the current study, more attention should be paid to the indirect effects of school closures, such as reduced community cohesion, domestic violence, food insecurity, and widespread job losses, which are interconnected with the disruption in early learning continuity. Our results indicate that one in six families reported an increased incidence of child punishment since school closures, particularly for boys and those living in rural areas. This highlights the importance of putting measures in place to respond to child protection risks, that are likely to be heightened in the context of the pandemic. There is also a greater risk of child malnutrition, as many school feeding programmes have been halted during the closure period, and families tend to reduce food consumption to cope with economic hardship caused by the pandemic. As shown in previous evidence from Ethiopia, malnutrition influences children\u2019s ability to engage in education activities (Woldehanna et al., This study presents important insights based on the voices of caregivers in the Global South. The focus on caregivers of pre-primary school-aged children is particularly valuable, as very little attention has been paid to the continuity of education for this age group during the COVID-19 pandemic. The study benefited from the use of globally validated measures adapted to the Ethiopian context by a team with extensive experience of undertaking research on ECE in this setting. It succeeded in achieving high response rates thanks to the team\u2019s engagement with the respondents in a survey prior to COVID-19. Despite these strengths, there are also important limitations that should be acknowledged. First, given it was not feasible to conduct face-to-face surveys or direct observations, we were unable to collect systematic data on the types of home learning environments, and the interactions between parents and children during school closures. Such information would have allowed us to understand whether and how different home environments mitigate or exacerbate the learning loss induced by the COVID-19 school closures. Second, the study\u2019s sample is limited to six of the 11 regions in Ethiopia. While purposive sampling allowed us to ensure our sample mirrored the national average ratio of the urban and rural population (World Bank, Schools in Ethiopia began to re-open from October 2020 on a staggered basis, after a long period of learning disruption which began in March 2020. In the absence of the government\u2019s support for remote learning for pre-primary school-aged children, and limited parental involvement in children\u2019s learning at home, many children are likely to have had little to no education during school closures. This is likely to have serious longer-term consequences: for example, a recent study shows that the loss of opportunity to learn due to COVID-19, including the absence of instructional time by qualified teachers, could results in a loss of 0.6\u00a0years of schooling and a reduction of $872 in yearly earnings, on average, for each student from today\u2019s cohort in primary and secondary school (Azevedo et al., Our findings highlight that young children and their families received very limited support from the education system in Ethiopia during school closures, especially those in poorer households and those living in rural or remote areas. Introducing strategies to promote parental involvement in children\u2019s learning will continue to be important even after students return to school. To prevent a reinforcement of learning gaps across socio-demographic groups, these strategies need to be designed from the perspective of the challenges that parents face, including with respect to access to technology, limited availability of books and other materials in households, and a lack of guidance for parental engagement in children\u2019s learning. Attention to the most vulnerable groups, particularly poorer households and those in which parents and caregivers are not literate, requires more comprehensive support such as the provision of cash transfers and essential supplies for families, and a strengthening of community-based platforms for parents and young children, as they are more likely to face even more challenging contexts.Lastly, special attention needs to be paid to prioritising ECE in the government\u2019s and aid donors\u2019 COVID-19 response planning and financial commitment. Moreover, this public health crisis calls for coordinated response plans across health, nutrition, education, and social protection sectors to provide support for families that are particularly important in early childhood. These are critical issues that apply to Ethiopia, with important lessons for other low-income contexts facing similar challenges."}
+{"text": "Rangifer tarandus) populations have experienced significant recent declines throughout Qu\u00e9bec, Canada, and are considered of concern, threatened or endangered. Here, we calculated the ancestral and contemporary patterns of genomic diversity of five representative caribou populations and applied a comparative population genomics framework to assess the interplay between demographic events and genomic diversity. We first calculated a caribou specific mutation rate, \u03bc, by extracting orthologous genes from related ungulates and estimating the rate of synonymous mutations. Whole genome re\u2010sequencing was then completed on 67 caribou: from these data we calculated nucleotide diversity, \u03b8\u03c0 and estimated the coalescent or ancestral effective population size (Ne), which ranged from 12,030 to 15,513. When compared to the census size, NC, the endangered Gasp\u00e9sie Mountain caribou population had the highest ancestral Ne:NC ratio which is consistent with recent work suggesting high ancestral Ne:NC is of conservation concern. In contrast, values of contemporary Ne, estimated from linkage\u2010disequilibrium, ranged from 11 to 162, with Gasp\u00e9sie having among the highest contemporary Ne:NC ratio. Importantly, classic conservation genetics theory would predict this population to be of less concern based on this ratio. Interestingly, F varied only slightly between populations, and despite evidence of bottlenecks across the province, runs of homozygosity were not abundant in the genome. Tajima's D estimates mirrored the demographic models and current conservation status. Our study highlights how genomic patterns are nuanced and potentially misleading if viewed only through a contemporary lens; we argue a holistic conservation genomics view should integrate ancestral Ne and Tajima's D into management decisions.The loss of genetic diversity is a challenge many species are facing, with genomics being a potential tool to inform and prioritize decision\u2010making. Most caribou ( Therefore, it encapsulates a variety of factors that have shaped the ancestral genetic variation that is still observed in the current population census  might not accrue if the bottleneck is short or very recent: in fact changes in inbreeding depression and genetic load may often be negligible and return back to equilibrium, dependent on the size of the bottleneck and speed of population growth  populations that were once historically distributed throughout Qu\u00e9bec and neighbouring provinces are declining in NC , genetic diversity (F and ROH) and the Ne/NC ratio , and compared the results among ecotypes to characterize population history and their current trajectory.Past population bottlenecks can have negative genetic consequences due to drift and inbreeding Lande,\u00a0, but the22.1Bos taurus, Oryx gazella, Capra aegagrus hircus, Equus caballus, Elaphurus davidianus, R.\u00a0tarandus, Ovis aries, and Odocoileus virginianus from the Ensembl Genome Browser and GIGA Science database  and related ungulates. We obtained coding sequences (CDS) and peptide sequences of eight species: se Table\u00a0: these wse Table\u00a0. PorthoMse Table\u00a0 was useds et al.\u00a0 with gapN/dS, was estimated for each branch using the CODEML function in PAML/4.9  across the province of Qu\u00e9bec . NC values represented the estimated number of adults in the population and were collected from reported estimates via aerial surveys and population monitoring for Rivi\u00e8re\u2010aux\u2010Feuilles and Rivi\u00e8re\u2010George . FST was estimated for each population pair using \u2010\u2010weir\u2010fst\u2010pop vcftools/0.1.13. Euclidean distance in kilometers was calculated between the centroid of each population range and isolation by distance was tested by running a Mantel test using the R package ecodist/2.0.7.Genome wide \u03b8\u03c0 and \u03bc estimates were input into the equation: \u03b8\u03c0\u00a0=\u20094 Ne\u03bc to solve for ancestral Ne of each population  equation of Waples\u00a0(R2) with the sample size correction of Waples et al.\u00a0: Standard neutral modelTSingle population change at time 1; Model 2 : A single, instant change; Model 3 : Gradual change.TTwo independent population size changes at 1Tand 2; Model 4 : Instantaneous size change followed by gradual change; Model 5  Two independent, instant changes; and Model 6 : Gradual change followed by an exponential change.Inference of demographic history for each population was based on the folded site frequency spectrum (SFS) and modelled using a diffusion\u2010based approach executed through Diffusion Approximation for Demographic inference , which is consistent with other mammal lineages . The Gasp\u00e9sie population showed the least amount of nucleotide diversity among individuals (0.0019). The populations with the highest nucleotide diversity among individuals were the TRAF and TRG populations. Ancestral Ne ranged from 12,030 (Gasp\u00e9sie) to 15,513  to 162 , is concordant with previous genetic studies, showing high gene flow between these two populations , which is expected as it has the smallest range, and the St. Lawrence River has generated prolonged isolation. The relationship of genomic variance to latitude , and the highest observed inbreeding rate was observed in the Gasp\u00e9sie population (F\u00a0=\u00a00.31). An increased level of inbreeding can be a product of population declines and isolation, which is consistent with southern populations , flycatchers (Tyrannidae), and finches  than estimates of Yannic et al.\u00a0(r2), with the discrepancy here likely due to reduced r2 values in microsatellite data , as these markers are typically selected based on linkage disequilibrium and hypervariability.The ancestral and contemporary e Figure\u00a0, the simc et al.\u00a0 for the Ne estimates can change drastically in the course of a few generations  is consistent with recent bottlenecks, significantly reducing current\u2010day NC, while having little effect on the ancestral, long\u2010term Ne or nucleotide diversity. The ancestral Ne/NC ratio and demographic models  are strongly correlated to Tajima's D (this study; Peart et al.,\u00a0D an easily generated metric that is informative for conservation.The actual size of the population, as well as contemporary 4.3FROH were not necessarily informative for assessing a population's current or ancestral genetic diversity (see also von Seth et al.,\u00a0Ne/NC might be also misleading (Ferchaud et al.,\u00a0Ultimately, the population statistics measured in this study are all metrics of diversity reflecting past demographic events that are used to gauge the future adaptive potential. The analytical toolbox has expanded to incorporate genotype likelihoods (e.g. \u00c7elik & Tuncali,\u00a0NC values as their main source of population data (IUCN Standards and Petitions Committee,\u00a0Ne and Tajima's D given they are derivated from genome\u2010wide metrics of diversity and there is clear connection to demographic history and conservation status. This approach has fundamental implications for understanding populations which have not been intensely studied and recently discovered species that do not have accurate census data or population histories. Diversity estimates alone correlate to life history tactics (Ellegren & Galtier,\u00a0D (this study; Peart et al.,\u00a0NC. The Gasp\u00e9sie and Saguenay populations should be a management priority. The genetic signatures are still relatively positive and suggest that if the time spent in the current bottlenecks is minimized, so too will be the impact on genomic diversity and adaptive potential.The majority of governing bodies use The authors declare no conflicts of interest.Benefits GeneratedBenefits from this research accrue from the sharing of our data and results on public databases as described above.Appendix S1Click here for additional data file."}
+{"text": "The appraisal of the formulae was conducted on the basis of entrapment efficiency percent (EE%), particle size (PS) and zeta potential (ZP). In addition, the spherical shaped optimal formula (F5) exhibited EE% of 86.1\u2009\u00b1\u20092.9%, PS of 228.9\u2009\u00b1\u20098.5\u2009nm, and ZP of \u221239.8\u2009\u00b1\u20091.3\u2009mV. The sorted optimum formula (F5) exhibited superior dissolution behaviors, and boosted Caco-2 cells cellular uptake by a round 4.7 folds relative to RSV dispersion. In addition, F5 demonstrated a complete in vitro suppression of SARS-CoV-2 at a concentration 0.48\u2009\u03bcg/ml with 6.6 times enhancement in antiviral activity relative to RSV dispersion. The accomplished molecular modeling heavily provided proof for the possible interactions of resveratrol with the key residues of the SARS-CoV2 Mpro enzyme. Finally, F5 could be proposed as a promising oral panel of RSV for curation from SARS-CoV-2 infection.Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) predisposed to the emergence of worldwide catastrophe that impels the evolution of safe and effective therapeutic system. Polyphenols as resveratrol (RSV) exhibit a well evidenced antiviral activity. Unfortunately, like most phenolic nutraceuticals, RSV suffers from restrained solubility and massive degradation in GIT and liver which in turn prohibit its clinical use. Herein, PEGylated bilosomes (PBs) contain PEGylated edge activator along with the traditional components as  were proposed to boost both permeability and bioavailability of RSV. The investigation of the prominent effect of the diverse variables on the characteristics of the vesicles and picking of the optimum formula were conducted via construction of 2 This virus can be also categorized under the enveloped positive-sense single-stranded RNA viruses, unfortunately, it is a highly contagious virus with high transmission rate especially from human to human  is in charge of the replication and transcription of the SARS-CoV-2 via the transformation of the polypeptides into functional proteins is a well-known phytoceutical compound related to the polyphenols stilbenoids group. Stunningly, it offers interesting and multiple therapeutic and biological applications  involvement in the vesicular structure allow the attainment of vesicles exhibiting greater stability relative to liposomes and niosomes; releasing bile salt modified vesicular system identified as Bilosomes (Bs)  was the source of Resveratrol (RSV) and Span 60, while ITX Biomedicals  was the source of Cholesterol. Sodium glycholate (SGC) and Sodium deoxycholate (SDC) were purchased from BASF Co. . Absolute ethyl alcohol sodium hydroxide and potassium dihydrogen orthophosphate were acquired from El-Nasr Chemical Co., Cairo, Egypt. SERVA Electrophoresis GmbH., Heidelberg, Germany was the source of  VISKING\u00ae dialysis membrane. All chemicals and solvents were of analytical grade and were used as received.2.2.2.2.1.3 factorial analyses utilizing Design Expert\u00ae software version 13 . The design resulted in eight runs considering the investigated factors: bile salt type at 2 levels (A- SGC or SDC), edge activator type and amount (EA type and amount) (B- Brij 20 or Brij 72) and (C-15\u2009mg or 30\u2009mg) respectively, meanwhile EE% (Y1), PS (Y2) and ZP (Y3) were picked as the dependent variables.The investigation of vast fabrication aspects of RSV-loaded PEGylated bilosome was conducted via 22.2.2.Ethyl alcohol injection technique was employed in fabrication of RSV loaded PBs  RSV . Each sample was measured three times and the results were represented as the mean value.2.2.4.According the highest EE% and ZP values together with minimum PS the election of the optimum formula was employed using the software Design Expert\u00ae version 13. Additionally, the ANOVA assessments were involved in figuring out and investigation of the prime consequences of the studied variables on the pre-determined responses, where the significance of each variable was explored and based on the highest desirability values the optimal formula was elected for further assessments  and for the prohibition of the vesicular lysis and aggregation, mannitol (5% w/v) was incorporated. At 80\u2009\u00b0C the dispersion of optimum formula was preserved for overnight succeeded by under vacuum drying for 24\u2009h  with a Cu Ka radiation detector. The scan was performed in 2 \u03b8 range of 3.0\u00b0\u201340\u00b0 at a scan rate of 1\u2009min.2.2.5.3.The surface properties along with the morphology of the selected RSV-PBs optimum formula were explored by using transmission electron microscopy . A droplet of formula dispersion was negatively stained using 1% phosphotungstic acid succeeded by copper coating carbon grid, after that this was self-dried to attain a thin film. TEM was then used to scan the copper sheet.2.2.5.4.For a comprehensive view on the release pattern of RSV from the fabricated RSV loaded PBs compared to RSV suspension (20\u2009mg of RSV in 10\u2009ml PBS) dialysis technique was employed  was used to compare the significance of the results.For a period of 3\u2009months and at 4\u2009\u00b0C, the optimum formula was preserved for studying the impact of short term storage of the physical characteristics of the formula. At zero time and at the end of 3\u2009months, the samples were withdrawn and assessed for PS, ZP, EE (%). The effect on the parameters was studied in comparison with the freshly prepared formula utilizing one-way ANOVA analysis and level (2.2.6.2.2.6.1.1% (w/v) penicillin, 1% (w/v) nonessential amino acid 10% (v/v) FBS, 1% (w/v) sodium pyruvate and streptomycin 1% (w/v) were added to MEM/EBSS and then used as growth medium for Caco-2 cells. The cells were later cultivated in 1% (w/v) streptomycin, 10% (v/v) FBS and 1% (w/v) penicillin contained in DMEM. In final, the incubation of cells was implemented with 5% CO2 supply at 37\u2009\u00b0C and 90% relative humidity  then for 72\u2009h they were cultured. The cells were subjected to  concentrations from each of the optimum formula and the drug dispersion for 12\u2009h. Afterwards, the replacement of the medium with 200\u2009\u03bcl of the MTT solution (0.5\u2009mg/ml in PBS) and for 4\u2009h the cells were incubated. The supernatant was then aspired and the formazan crystals were dissolved using dimethyl sulfoxide (200\u2009\u03bcl/well). , Varioskan Flash multiplate reader was employed to assess the absorption at 570\u2009nm and the relative cell survival was computed , the Caco-2 cells (5\u2009\u00d7\u200910 3 cells per well) were seeded. Every 2\u2009days the medium was replaced with fresh one in week 1, after that the daily change of the medium was performed until Day 14. Before the cells being incubated with 100\u2009\u00b5g/ml of both RSV dispersion and RSV loaded optimum formula for different durations , for 0.5\u2009hr, they were pretreated with freshly prepared PBS at 37\u2009\u00b0C; for complete detailed procedure, see the 2.2.7.In biological safety cabinet level 3, the titration of hCoV-19/Egypt/NRC-3/2020 isolate (SARS-CoV-2 virus) was performed using Vero-E6 cells . The Vero-E6 cells were infected in 96-well tissue culture plates utilizing serially diluted virus post confluency. In a 96-well plates, the cells were harvested and then preserved in humidified incubator under 5% CO2 at 37\u2009\u00b0C. The cells monolayer was washed twice; consequently, the cells were then infected at 37\u2009\u00b0C for 72\u2009h using the serially diluted virus. The fixation of the cell monolayers was conducted using 3% paraformaldehyde followed by staining using crystal violet (0.1%). Finally, Reed and Munch equation was involved in the calculation of virus titer  was explored . Docking simulations were achieved using AutoDock Vina with exhaustiveness of 8. The Mpro active site was determined with a grid box  with the size 25\u2009\u00d7\u200935\u2009\u00d7\u200925\u2009\u00c5 (spacing 1\u2009\u00c5).PubChem database was used to download reversatrol as sdf format, and subsequently was subjected to energy minimization protocol Discovery Studio (DS) 5.0 client (Accelrys). SARS\u2010CoV-2 Mpro structure  was obtained from the protein data bank  was attained in almost all responses which assured the validity of the designed model to lead the design space  could be noticed for formulae containing SDC compared to those containing SGC and this can be explained by the higher lipophilicity of SDC (HLB = 17.6) relative to that of SGC (HLB = 23.1) which predisposed to proper intercalation of the lipophilic RSV within the hydrophobic core of the vesicles and consequently resulted in higher drug entrapment , a significant higher EE% (p\u2009=\u20090.0004) in formulae composed of Brij 72 relative to those composed of Brij 20. This could be justified on the basis of degree of lipophilicity, whereas Brij 72 (HLB = 4.9) is more lipophilic than Brij 20 (HLB = 15.3) led to higher entrapment of the lipophilic RSV , ANOVA results declared that the values of EE% were significantly higher (p\u2009=\u20090.001). Higher amount of EA predispose to the introduction of more pores within the vesicular structure rendering it more leaky, additionally, the fluidity of the bilayer may be also increased as a consequence of the incorporation of higher amount of EA, subsequently a decline in EE% values could be noticed  relative to those prepared using SGC and this could be justified by the level of hydrophilicity, as BS of lower hydrophilicity SDC (HLB = 17.6) than SGC (HLB = 23.1) leading to a drop in surface free energy of the system, in addition, the increase in hydrophilicity of the BS subsequently increase the water uptake within the vesicles leading to the formation of larger PS , the formulae prepared using SDC exhibited lower PS (p\u2009=\u20090.0008). As previously mentioned, the EA of higher PEG units possesses higher steric hindrance effect and better capability to delay the precipitation of the vesicles (Brijo20 and Brij72 contain 20 and 2 PEG units), respectively . This can be explained by the ability of Brij to stabilize the vesicles, as on using EA with high amount was sufficient enough to wrap the vesicles and hinder their aggregation due to the steric stabilization induced by PEG units. Moreover, the surface active nature of Brij augmented in lowering the interfacial tension of the system, thus increasing its stability and resulted in formation of vesicles of lower PS. In contrary, all these effects will be diminished on using lower amounts of EA and consequently led to enlargement in PS  higher ZP values  than those prepared using SDC. This can be justified by the fact that the higher acidity of the glycine conjugate in SGC than the unconjugated homologues as SDC which in turn resulted in densify the negative charges on SGC containing vesicles , ANOVA results revealed that formulae prepared using SGC acquired significant (p\u2009=\u20090.0013) negative impact on ZP values. This may be due to the PEG moieties present in the structure of each surfactant, where Brij 20 comprises 20 PEG repeated units while Brij 72 comprises 2 units and it was previously recorded that increasing the number of anionic PEG unit leads to the localization of densified negatively charged coat of PEG surrounding the vesicles that consequently increase the repulsion force between the vesicle and rendered them segregated and stable  decline in ZP values. This can be explored by increasing the amount of EA led them to be localized at the surface of the vesicles obscuring the negative charges of the bilosomes , herein it was F5 with a desirability = 0.715. F5 exhibited an EE% of 86.1\u2009\u00b1\u20092.9%, PS of 228.9\u2009\u00b1\u20098.5\u2009nm, and ZP of \u221239.8\u2009\u00b1\u20091.3\u2009mV. Furthermore, the observed and the predicted outcomes of the responses of F5 were compared for assuring the validity and the rationality of the design. A high and adequate agreement between the observed and predicted outcomes can be depicted from the attained results 3.4.3.4.1.The degree of crystallinity of a substance can be explored adopting X-ray diffraction technique. The diffraction spectra of pure RSV, blank and RSV loaded optimum formula (F5) were displayed in 3.4.2.The TEM image of F5 declared that the vesicles exhibited round shape devoided from any abnormality 3.4.3.p\u2009<\u20090.05). This pattern could be justified by the fact that the carrier system acts as a reservoir for restricted and prolonged release of the drug followed a fast primary release phase, where the total drug released in the first 2\u2009hours were 32.1\u2009\u00b1\u20092.2%. Meanwhile, the poor wettability and solubility of the drug may be behind the diminished release of the drug from RSV suspension. Furthermore, the presence of bile salts and the assembled PEG units of Brij as EA surrounding the vesicles predisposed to enhanced drug solubilization and higher RSV releases, as they will augment the drug solubility and increase the hydrophilicity of the system, thus the drug could be easily dispersed in the release media without any agglomeration , owing to the steric stabilization accomplished due to the involvement PEGylated EA which act as a coat aided in prohibition of drug leakage and vesicular agglomeration, thus promoting the vesicular stability  was noticed which could be justified by the presence of high amounts of SAA and bile salts concerning (F5). Finally, the 0.1\u2013100\u2009\u03bcg/ml was the safe concentration range used for cellular uptake assessment  was investigated using MTT assays. From 3.5.2.P\u2009<\u20090.05) higher by around 4.7 folds than that of RSV dispersion, 2366.24\u2009\u00b1\u2009135.3\u2009ng/ml 495.65\u2009\u00b1\u200921.9\u2009ng/ml, respectively. This significant improvement in cellular internalization after formulation was attributed to the tiny PS of the vesicles loaded with the drug, in addition to the components involved in the formulation as bile salts and surfactant exhibited a vital impact on the alteration of cellular membranes permeability   cytotoxic effect was explored adopting MTT technique and from the results, it can be depicted that the CC50 values were 4.7 and 33.7\u2009\u03bcg/ml, respectively (p\u2009<\u20090.05) augmentation of bilosomal formulation in the activity of F5. The aforementioned outcomes revealed that RSV formulation using PEGylated bilosomal system attained the capability of boosting the antiviral activity and promoting the clinical of RSV, stunningly, at a minute concentration reached to 0.48\u2009\u03bcg/ml, the complete suppression of the virus was achieved.Using Vero- E6 cells, ectively . Based o3.7.It was obvious that resveratrol could pertain its antiviral activity by significantly diminishing the proteolytic activity of SARS-CoV-2 main protease (SARS-CoV-2 Mpro) (Jo et\u00a0al., In depth, resveratrol is perfectly stuck to the S1 binding site , where a4.3 full factorial experiment for oral delivery of RSV. F5 was sorted out as optimum formula with desirability value 0.707. Moreover, F5 exhibited spherical shape with high drug EE%, ZP and small PS. Additionally, Caco-2 cells permeability study revealed the superiority of F5 over RSV dispersion by around 4.7 folds\u2019 increase in cellular uptake. Stunningly, F5 exhibited enhanced antiviral activity against SARS-CoV-2 by 6.6 folds relative to RSV dispersion. The conducted modeling study revealed that resveratrol is perfectly oriented toward the catalytic Cys145-His41 dyad within the active site of main protease. Consequently, resveratrol pertains high affinity to prohibit the SARS-CoV-2 Mpro enzyme. Finally, the aforementioned outcomes affirmed that F5 was capable of overcoming its severe first-pass metabolism and other oral problems associated with its oral administration. Thus, F5 could be proposed as a potential carrier for oral delivery RSV with accentuated anti-SARS-CoV-2 activity.In the conducted study, PBs were fabricated adopting 2Click here for additional data file."}
+{"text": "To a great extent, F4 was able to significantly suppress the inflammatory response and oxidative stress resulted from MERS-CoV infection on comparison with RSV dispersion. Finally, the potentiality of PEMLs as nano-panel with boosted both antiviral and oral bioavailability for RSV could be deduced based on the outcomes mentioned herein.Resveratrol (RSV) is a phytoceutical polyphenolic compound exhibiting a well evidenced wide range of therapeutic activities. Unfortunately, its diminished aqueous solubility and extensive metabolism in gastro intestinal tract (GIT) and liver prohibit its biological activity and systemic availability. Herein the conducted study PEG stabilized emulsomes (PEMLs) were customized to enclose RSV aiming to boost its biological availability and antiviral activity. PEGylating the vesicles not only grant the promoted steric stability of the system but also being beneficial in exaggerating the intestinal permeability and extending the period of circulation of the drug, hence its targeted clinical use. The Investigation of the influence of predetermined variables on the physical characterization of formulae  was implemented utilizing Design Expert\u00ae software. (F4) with desirability value (0.772), picked to be the optimal formula, which is fabricated utilizing 35\u2009mg compritol as the lipidic core and 60\u2009mg 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-Mpeg-2000). The dominance of the F4 relative to RSV dispersion was affirmed by the data acquired from ex-vivo and pharmacokinetic studies. In addition, F4 exhibited significant lower EC Those remedies were capable of efficiently hitting the target without imparting any negative consequences on the host cells. One of the most virulent infection is middle east respiratory syndrome (MERS), or \u201cCamel flu\u201d which initially was recognized in 2012 in Saudi Arabia , stilbene derivative, originating from the skin of red grapes and in polygonum cuspidatum. On the basis of well evidenced data, RSV is capable of exerting multiple biological and pharmacological functions as anticancer, anti-inflammatory, antidiabetic, antioxidant and neuroprotectant  considering vast predetermined variables utilizing 22.2.1.supplementary material: S1.See 2.2.With minor modifications, RSV-loaded PEMLs were tailored adopting the thin film hydration approach  in the casted PEMLs dispersion was determined: primarily, the dispersion of 1.00\u2009mL aliquot of the RSV-charged PEMLs (enclosing 2.00\u2009mg of the drug) was replenished using distilled water (5.0\u2009mL) followed by agitation for 2\u2009min and finally centrifuged for 1\u2009h  at 15339\u2009g with cooling at 4\u2009\u00b0C  for the assessment of the mean droplet size, zeta potential (ZP) and PDI and they were investigated in triplicate , lipid core amount (X2), DSPE-Mpeg-2000 amount (X3). Meanwhile, EE% (Y1), PS (Y2), ZP (Y3) were chosen as dependent variables. According to the implemented ANOVA statistical analysis, the main impacts of the variables and their significance were outlined. The election of the optimum RSV-charged PEMLs was implemented on the basis of the criteria of the highest ZP, EE% and the minimum droplet size along with the highest desirability value the optimum formula and then it was involved in further assessments  adopting 22.5.2.5.1.The optimized RSV-loaded PEML formula solidification was implemented using Lyophilization  in presence of cryoprotectant namely Mannitol (5% wt/vol) to prohibit the vesicular damage and aggregation. The optimum formula dispersion was preserved at \u221280\u2009\u00b0C for overnight followed by drying under vacuum for 24\u2009h . The calibration of the equipment was conducted using Purified indium (99.9%). The thermal behavior of the samples was scanned in a temperature range 20\u2013400\u2009\u00b0C by an incremental rate of 10\u2009\u00b0C/min  was utilized for the visualization and determination of the vesicular configuration. The phosphotungstic acid solution (2% wt/vol) stained vesicle\u2019s droplet was then adhered to a carbon grid with copper coating and left to dry to attain a thin film. The acquired copper sheet was visualized by using the TEM  attained after the dilution of 1\u2009mL of the crude formula using 1\u2009mL Sorensen phosphate buffer (pH 7.4) and 1\u2009mL of RSV dispersion (1\u2009mg/mL) were placed in a 2.5-cm diameter 10-cm glass cylinder with a presoaked cellulose membrane fitted at the bottom of the cylinder. Then the glass cylinder was placed in 900\u2009mL dissolution media  at 37\u2009\u00b0C after being carefully hanged onto the shaft of the dissolution tester  revolved at speed of 50\u2009rpm  followed by replacement of the withdrawn volume using an equal volumes of fresh Krebs-Ringer solution. The amounts of RSV in the receptor solution were computed utilizing Hitachi LaChrome Elite HPLC fitted with L-2130 pump with built in degasser, L-2455 photo diode array detector (DAD), L-2300 column oven, a Model Series L-2000 organizer box and Rheodyne 7725i injector with a 20\u2009mL loop, Column: reversed phase C18, 4.6\u2009\u00d7\u2009100\u2009mm, 5\u2009\u03bcm, Xterra, USA, utilizing mixture of methanol and potassium dihydrogen phosphate buffer (pH 3.8) in a ratio 40%:60% vol/vol as a mobile phase which flow at a constant rate of 1.0\u2009mL min\u22121 and the drug was detected at the wavelength 320\u2009nm  of the optimized RSV-charged PEML versus RSV dispersion was computed adopting the following formula:0 is the initial concentration, and A is the total surface area of the ileum.where F is permeation flux that indicate the amount of RSV permeated across the membrane per unit time, C2.7.2.7.1.5 cells/mL) the cells were assembled cells and then incubated in 5%CO2 for 24\u2009h at 37\u2009\u00b0C. Subsequently, the investigated compounds in different concentrations were incubated with the cells for an extra 24\u2009h and each experiment was repeated three times. The supernatants were then detached from the cell monolayers and rinsed 3 times with sterile phosphate buffer saline (PBS). The harvested cells were allowed to be stained with MTT solution (20\u2009\u00b5L of 5.0\u2009mg/mL stock solution) and incubated for 4\u2009h at 37\u2009\u00b0C in each well followed by aspiration of the medium. The incorporation of 200\u2009\u00b5L acidified isopropanol (0.040\u2009M HCl in absolute isopropanol = 0.073\u2009mL HCL in 50\u2009mL isopropanol) in each plate in order to dissolve the attained formazan crystals. Multi-well plate reader was involved for the determination of the formazan solutions absorbance\u2019s at \u03bbmax 540 vs the 620\u2009nm reference wavelength. The cytotoxicity percentile relative to the untreated cells was estimated utilizing the following equation:Tiny modification was adopted during the assessment of the cytotoxic activity of the samples under investigation on Vero E6 cells utilizing dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)  was deduced  were cultivated for 24\u2009h at 37\u2009\u00b0C  were then incubated for 1\u2009h at 37\u2009\u00b0C with the safe concentration of the samples under investigation. The samples of the investigated formulae admixed with 2% agarose and 3\u2009mL DMEM were placed with cells challenged with virus (100\u2009\u00b5L/well) for1\u2009h as a proper contact time for viral adsorption. The plates were allowed to be solidified and incubated at 370\u2009C for 3 to 4d till the evolution of viral plaques. The cells were stained with 0.1% crystal violet in distilled water after being subjected to 10% Formalin for 2\u2009h. On another hand, the wells that were determined as control (untreated virus) were incubated with Vero E6 cells then, the relative percentage of decline in plaques count to that in the control wells was estimated as follows:MERS-CoV viral titers were determined adopting plaque loaded on Vero E6 cells assay. As previously conducted, in six-well plates Vero E6 cells  enclosed in 50\u2009\u00b5L of viral solution enclosing was intranasally inoculated in the animals under investigation  of the Theodore Bilharz Research Institute  was the source of the forty 10-week-old female C57BL/6 mice of weight 150\u2009\u00b1\u200910\u2009g. The accommodation of the experimental mice for seven days was in standard polypropylene cages and kept under optimum laboratory circumstances concerning light with 12-h of dark/light cycles, temperature and humidity. The mice were allowed to freely access the water and diet Uninfected (Control) Group 1: received (2\u2009mL/kg) saline intraperitoneally (i.p.) once followed by daily oral administration of saline at dose of 5\u2009mL/kg.5 pfu/50\u2009\u03bcl of virusMERS infected (untreated) Group 2: the infection achieved via intranasal inoculation of 10RSV suspension treated Group 3: post infection, an oral daily dose of 10\u2009mg/kg utilizing infant feeding syringe  at day 3 and day 6 for anesthesia, succeeding their viral infection, they were terminated by intraperitoneal overdose of sodium pentobarbital (30-50\u2009mg/kg) (on day 6). The excised lungs were immediately gathered and rinsed with normal saline followed by drying using tissue paper. Furthermore, the weights of the harvested lungs were recorded, then they were supplemented via 0.1% SDS, Roche complete ULTRA Tablet, physiologic saline (1.0\u2009mL) and protease inhibitor . Afterwards, the harvested lungs were homogenized utilizing a glass tissue homogenizer , and finally centrifuged for 10\u2009min at 7826\u2009g. The harvested aspirates were preserved at \u221220\u2009\u00b0C or at \u221280\u2009\u00b0C for subsequent analysis : Twelve male Wistar albino rats (220-250\u2009g) were accommodated standard polypropylene cages and were preserved under optimum laboratory circumstances of free accessibility to the standard laboratory water and diet ad libitum, temperature, humidity, and light. The rats were subcategorized into two groups each of six. Group (I) animals received an oral dose of RSV dispersion enclosing 20\u2009mg/kg of RSV dispersion in PBS whose PS was 697.4\u2009nm \u00b1 42.1 and PDI 0.813\u2009\u00b1\u20090.12, while, Group (II) animals received an oral dose of what corresponding to 20\u2009mg/kg of RSV contained in the dispersion of optimum RSV-charged PEML formulation  with the statistical software of SPSS version 14 and p\u2009<\u20090.05 was the level of significance  surpassed the EE% values of those composed of tristearin; this may be attributed to the disparity in the hydrocarbon chain length between compritol a blend of different glyceryl esters, including the diester glyceryl dibehenate, of the C-22 saturated fatty acid, behenic acid and the saturated tri acyl ester with stearic acid C-18 , the outcomes of ANOVA analysis revealed that the EE% of compritol based formulae significantly (p\u2009=\u20090.0092) improvement in the endorsement of RSV, this came in accordance to that previously mentioned in  from 35\u2009mg to 70\u2009mg results in significant(p\u2009=\u20090.0085) in EE% values could be noticed, this may be attributed to the increased possibility of interference with the vesicular structure and development of pores within the membranes  from 30\u2009mg to 60\u2009mg on EE%, a significant  can be denoted. The RSV-loaded PEMLs PDI values revealed in 3.3.2.In general, the particle size of the drug exhibits a great impact on the overall fate of the drug in the systemic circulation besides its the impact on the drug\u2019s activity, thus the therapeutic activity will be promoted on suppressing the size owing to the boosted intestinal permeation and extended drug residence duration. From p\u2009=\u20090.01) larger PS relative to those composed of tristearin. This may be attributed to the greater fatty acid chain length involved in compritol relative to those of the stearic acid involved in tristearin; besides, the lower EE% of tristearin formulae leading to diminishing their PS.For the lipid core type (Factor X1), the formulae composed of compritol exhibited significant (p\u2009=\u20090.0058) in PS, which may be owing to the increased tendency of the particle to agglomerate due to the subsequent elevation in the viscosity of attained formulated emulsion on utilizing greater amount of lipids : the lipid core amount, the increment in the lipid core amount results in a significant elevation (p\u2009=\u20090.0008) suppression in PS, owing to the increased steric bulkiness, hence steric hindrance achieved on increasing the content of PEG attained on increasing the amount of DSPE-mPEG 2000 predisposed to suppress the particle agglomeration tendency and restrain the vesicular aggregation probability, thus constrains the particle size growth and boosts the stability of the system , the higher amount of DSPE\u2013mPEG-2000 resulted in a significant . Values of \u00b130\u2009mV are adequate to impart proper electrostatic repulsion forces between the vesicles, which in turn will boost the vesicular electrical stability  on ZP. As the formulae comprising compritol exhibited higher ZP values than those of tristearin, this may be attributed to the greater engulfment for RSV in compritol \u2013based formulae resulted in the allocation of higher numbers of hydroxyl groups associated with RSV  in the vesicles. Moreover, the higher amount of lipid (Factor X2) will significantly (p\u2009=\u20090.0122) elevate the ZP values owing to the dispersion of more RSV (negatively charged compound) through the lipid core which will increase the net electronegativity of the vesicles.Regarding (Factor X1): type of lipid core alteration predisposed to a significant positive impact (p\u2009=\u20090.0113) higher ZP values. This may be attributed to the alignment of the anionic DSPE\u2013Mpeg  in the form of electronegative coat that wrap the lipid core will densify the electronegativity at vesicular surfaces  predisposes to significant . Additionally, the discrepancy percentage of the predicted from the observed values was calculated for each variable: EE%, PS and ZP and collectively added to 3.6.3.6.1.In an attempt to assess the alteration physical state and the extent of crystallinity of RSV, DSC study was employed on the pure drug and post being loaded on the nano-carriers. DSC attitude of the pure RSV, blank lyophilized formula relative to that of the lyophilized RSV-loaded optimum PEMLs (F4) are represented in 3.6.2.3.6.3.p\u2009<\u20090.05). This can be justified by the impact of the drug encapsulation in the vesicles which serve as a drug pool for restrained drug release pattern superseded an initial burst phase. While, in case of RSV dispersion the high possibility of drug agglomeration and precipitation owing to its poor wettability and solubility may be behind its restrained release. Moreover, the PEGylated sheath surrounding the vesicles which predispose to higher drug solubilization capability and elevated RSV releases as a consequence of inclusion of DSPE in formulation which will impart increased hydrophilicity and solubilization power for the drug due to its PEG moieties  was employed to figure out the extent of drug release, solubilization and stability configuration after being incorporated into the vesicles. 3.6.4.supplementary materialTable S6, it can be deduced that the short term preservation of F4 for 3\u2009months at 4\u2009\u00b0C and at 25\u2009\u00b0C did not impart significant (p\u2009>\u20090.05) alterations to the parameters under assessments: EE%, PS and ZP. The steric hindrance acquired as a consequence of the involvement of PEGylated lipid aided in prohibition of drug leakage and vesicular agglomeration, thus promoting the vesicular stability . The apparent permeability coefficient (APC) of RSV was 2.28\u2009\u00d7\u200910\u22124\u2009cm/min, on another hand that of the F4 formula was elevated significantly (p\u2009<\u20090.05) by around 3.8 times to 8.64\u2009\u00d7\u200910\u22124\u2009cm/min. RSV intestinal lavage was assessed adopting the non-everted gut sac method for RSV dispersion relative to RSV-loaded optimum formula (F4) in an attempt to prospect the in-vivo attitude, kinetic and availability of the drug to biological system. The superiority of F4 flux J3.8.3.8.1.p\u2009<\u20090.05) safety profile by around 3.7 folds relative to RSV dispersion. However, the Plain F4 exhibited cell viability exceeding 90% which assure the safety of the formulation ingredients. Moreover, the assessment of TC50 will outline the safe range of concentrations for being employed in further investigations besides, granting the exclusion of any host cell toxicity throughout the treatment course.A promising antiviral drug should compromise between the antiviral activity at lower concentration, meanwhile, imparting cytotoxicity on using higher concentrations. The host cell cytotoxicity of F4 was found to be (9.2\u2009\u00b5g/mL) and was (2.5\u2009\u00b5g/mL) for the RSV suspension ; thus F43.8.2.50 = 0.0127\u2009\u00b5g/mL) and (EC50 = 0.34\u2009\u00b5g/mL) respectively. These results denoted that RSV impart its antiviral activity even at diminished concentrations, moreover, this activity was significantly promoted (p\u2009<\u20090.01) on drug fabrication as Nano-vesicles. This boosted antiviral activity may be attributed to attachment and adherence of the drug loaded PEGylated nano-carriers to the viral cells permitting the drug accumulation surrounding the viral cells and creating a state of thermodynamically concentration gradient, hence RSV loaded lipid nano-carriers can diffuse easily through the viral membranes. To a great extent, it was previously reported that the composition of the lipidic nanovesicles as lipids and surfactants aid in the promotion of drug permeation to viral cellular membranes and serving the drug in solubilized form  regarding selectivity of F4 relative to the RSV dispersion.As a simple, reproducible and quantitative method, Plaque inhibition assays were implemented for determining the level of reduction in the (cytopathic activity) viral infectious capability via measuring the inhibition in cell culture formed plaques owing to the infection via serial dilutions of the virus. 3.9.3.9.1.The experiment timeline was started 6\u2009h post infection and terminated day 6 post infection as previously mentioned that the virus required at least 6\u2009h to be well recognized and noticed in the respiratory tract of the infected mice and on day 7 the virus could be barely detected  elevation of TNF-\u03b1 and IL-6 levels to 91.7\u2009\u00b1\u20099.8 and 195.5\u2009\u00b1\u200931.2\u2009pg/mg protein, respectively after infection instead of 24.1\u2009\u00b1\u20095.30 and 48\u2009\u00b1\u20098.7\u2009pg/mg protein respectively in the control group were observed denoting the huge inflammatory storm emergence associated with the infection. Meanwhile, their levels were suppressed to 54.1\u2009\u00b1\u20095.7 and 115.3\u2009\u00b1\u200937.2\u2009pg/mg protein respectively after administration of RSV dispersion. Stunningly, the levels of TNF-\u03b1 and IL-6 were significantly suppressed (p\u2009<\u20090.05) 31.3\u2009\u00b1\u20096.2 and 63.25\u2009\u00b1\u20098.1 respectively after administration of RSV loaded PEMLs (F4). In addition, 12.2\u2009\u00b1\u20091.3 was the level of SOD in BALF of control group and was significantly diminished to 5.05\u2009\u00b1\u20090.9\u2009U/mg protein in viral infected groups. On another hand, the elevation of SOD 4.5\u2009\u00b1\u20091.2 and 11.1\u2009\u00b1\u20092.6\u2009U/mg protein in the case of groups treated with RSV dispersion and F4, respectively could be depicted. Also the depletion in the serum levels of GSH to 6.5\u2009\u00b1\u20091.4\u2009\u00b5M post viral infection instead of being 9.85\u2009\u00b1\u20092.3\u2009\u00b5M in control group, while the treatment with RSV was able to restore GSH levels reaching to 9.6\u2009\u00b1\u20093.1\u2009\u00b5M and 11.1\u2009\u00b1\u20092.2\u2009\u00b5M in case of RSV dispersion and F4 respectively. These outcomes were in line with the findings that previously reported . The significant(3.10.p\u2009<\u20090.05) boosted than those of the RSV dispersion: The Cmax of F4 was 29.3\u2009\u00b1\u20092.8\u2009\u00b5g/mL significantly (p\u2009<\u20090.05) higher than that of the RSV dispersion (7.2\u2009\u00b1\u20091.1\u2009\u00b5g/mL). The Tmax value of 4\u2009h for the F4 was two-fold higher than that of the RSV dispersion, likely owing to the extended liberation of RSV throughout the vesicular membrane. Moreover, F4 computed AUC [0-24] was 397.2\u2009\u00b1\u200938.7\u2009\u00b5g*h/mL which was superior significantly (p\u2009<\u20090.05) relative to RSV dispersion AUC [0-24] 85.42\u2009\u00b1\u200921.3\u2009\u00b5g *h/mL). The enhanced relative bioavailability of F4 relative to RSV suspension resulted from the capability of the lipidic carrier to invade the intestinal membranes, moreover the PEG sheath aid in extending the residence time in the circulation and granting the sustainability of its biological activity. From According to the outcomes acquired the ex-vivo and pharmacokinetics, it can be concluded that PEMLs was capable of resolve the pitfalls (constrained permeability and diminished aqueous solubility) that reside the bioavailability of RSV. Moreover, the formulation was able to boost the antiviral activity of RSV and in prohibiting the inflammatory storm and oxidative stress as consequences following the infection. Thus PEMLs can be claimed as potential panel to be included in the remedy protocol either in protection or resolving of the virus and the succeeded consequences .4.Based on the study design which results in construction of eight formulations, the analysis of their related in-vitro characterizations: EE%, PS and ZP and picking F4 as an optimum formula according to the predetermined criteria of being of highest EE, ZP and minute PS, PEMLs can be successfully utilized as prosperous panels for oral delivery of RSV with boosted antiviral activity. Furthermore, the outcomes acquired the ex-vivo and pharmacokinetics, it can be concluded that PEMLs were capable of resolving the pitfalls (constrained permeability and diminished aqueous solubility) that reside the bioavailability of RSV. Moreover, the formulation was able to boost the antiviral activity of RSV and in prohibiting the inflammatory storm and oxidative stress as consequences following the infection. Thus PEMLs can be claimed as potential panel to be included in the remedy protocol either in protection or resolving of the virus and the succeeded consequences.Click here for additional data file."}
+{"text": "Decellularization of tissues and organs has recently become a promising approach in tissue engineering and regenerative medicine to circumvent the challenges of organ donation and complications of transplantations. However, one main obstacle to reaching this goal is acellular vasculature angiogenesis and endothelialization. Achieving an intact and functional vascular structure as a vital pathway for supplying oxygen and nutrients remains the decisive challenge in the decellularization/re-endothelialization procedure. In order to better understand and overcome this issue, complete and appropriate knowledge of endothelialization and its determining variables is required. Decellularization methods and their effectiveness, biological and mechanical characteristics of acellular scaffolds, artificial and biological bioreactors, and their possible applications, extracellular matrix surface modification, and different types of utilized cells are factors affecting endothelialization consequences. This review focuses on the characteristics of endothelialization and how to optimize them, as well as discussing recent developments in the process of re-endothelialization. Increased organ failure due to chronic infections, diseases, and genetic problems has elevated organ transplant requirements in recent years. According to the Global Observatory of Donation and Transplantation, approximately 150 thousand organs were transplanted worldwide in 2019 . Also, tTissue engineering and regenerative medicine, aiming to design, produce, and achieve an efficient and functional organ, play a crucial role in overcoming immunity limitations and transplantation obstacles . DecelluOne of the critical limitations of the D&R methods is undesired organ nutrition, blood supply, and lack of functional vessels, which may make the graft unsuitable for transplantation and further hamper achieving functional organs . FunctioMultiple strategies and processes have been adopted for this goal and to respond to the challenges described above, each of which separately plays a vital role in the endothelialization process . DecelluUltimately, on the track of acellular scaffold recellularization and endothelialization, the identification of the optimal decellularization method along with scaffold features, the bioreactor utilization approach, cytokine, and growth factors (GFs) quantities, appropriate cell type, and cell density are the most critical determinants of organ engineering . In thisThe endothelialization outcome depends on preserving ECM microstructure and component composition during decellularization. Thus, the more intact the ECM remains, the more desirable tissue engineering outcomes are achieved .In this regard, decellularization on a measurable scale is determined as: 1. less than 50\u00a0ng of dsDNA per mg of ECM (dry weight), 2. less than 200 bp retained DNA fragment length in acellular ECM, and 3. the absence of visible nuclear material under hematoxylin and eosin (H&E) and 4\u2032,6-diamidino-2-phenylindole (DAPI) staining  . Hence, One of the significant advantages of utilizing ramping perfusion is flow fluctuation that opens up collapsed capillaries and re-endothelializes these unreachable vessels to promote accessibility and recellularization . The invNonetheless, a broader numerical range has also been employed for endothelialization. For instance , In Kap in vitro and in vivo. Further, it can improve cell survival and strengthen cell-cell and cell-ECM connections , fucoidan, chitosan , antibodAccording to the bioinduction characteristics of decellularized materials and their regenerative effects, including the role of ECM on stem cell niches, angiogenesis, and tissue-specific differentiation, ECM components were used in the direction of surface modification. In this regard, fibronectin, have been utilized to coat the surfaces of engineered vascular grafts to promote autologous recellularization and graft function by facilitating the adhesion of ECs to ECM . Fibronevia portal vein perfusion . Heparin immobilization, as one of the most studied GAGs and an anticoagulant, is utilized to modify an acellular surface scaffold to increase endothelial hemocompatibility and biocompatibility . BesidesConsidering the positive impacts of other materials and the synergy of molecules on each other, producing chimeric molecules is a more practical method to modify the acellular surfaces. Particularly, heparin has been conjugated with other proteins to generate functional molecules to increase ECs attachment. Jiang B et al. linked a collagen-binding peptide to heparin which led to more and more extended attachment of ECs to the acellular vascular bed and reduced platelets activation . HeparinOne of the more feasible methods to modify an acellular scaffold is polyelectrolyte multilayer film utilization . IntermiHA is the other saccharide that has been used for surface modification purposes. It has been shown that different aspects of cell behavior uniquely depend on the size of surrounding HA fragments. It seems that ECs respond to HA oligomers (<10\u00a0kDa) more than long-chain HA components (1000\u00a0kDa) . AlthougAs the third category of surface modifiers, antibodies (Abs) are conjugated to the components of ECM present in the acellular structure, including collagen. On the other hand, they bind to ECs that promote cell-ECM adhesion as well as strengthen their attachment to the vascular bed due to having a particular identification site for ECs proteins . The binAnother promising surface modification approach to improve the integration of transplanted materials and promote cell-scaffold collaboration is biofunctionalization with GF-related molecules, especially proangiogenic GFs . AngiogeStudies have shown that several molecules involved in inflammatory and angiogenic cascades are preserved in the acellular ECM after decellularization and the ECM acts as a reservoir for GFs . In addiExogenous GFs have been utilized in various ways to promote endothelialization. In the most basic method, several studies used GFs in solution by adding them to the cell culture medium or perfusion culture . AlthougIn the first approach, studies have demonstrated that aside from the proangiogenic GFs preserved in the acellular scaffold, the stem cells used in recellularization secrete GFs and cytokines, which are helpful in re-endothelialization. VEGF and bFGF are secreted from MSCs over paracrine mechanisms, which can enhance ECs function . ECs canA prominent approach to preserve proangiogenic GFs concentration in a specified and practical range is to control GF release. One of the used methods is encapsulation in the microspheres . EncapsuTaken together, proangiogenic GFs have significant effects on re-endothelialization and supporting angiogenesis. Many methods have used GFs to provide a basis for the endothelialization of acellular vasculature; future experiments are still needed to determine their exact effectiveness.Active vascular remodeling, an action that imitates lumen formation during vascular development, is needed to generate a perfusable vascular lumen from decellularized vessels . ConsideIn vivo implanted arteries showed partial ECs coverage with platelets adhered to the bare cites at 1\u00a0month and a similar structure to native vessels with no detected thrombosis 3\u00a0months post-transplantation . The study achieved an endothelialized vascular network without any observed leakage in decellularized livers . FurtherSupporting cells, such as MSCs, pericytes, and perivascular cells, play a variety of roles, including improving ECs viability and proliferation and inducing angiogenesis by producing various GFs and ECM components as well as stabilizing newly formed vessels . TherefoMSCs, as one of the supporting cells, have been utilized in acellular scaffold endothelialization due to their complementary role in enhancing angiogenesis and modulating host immune responses . It has in vitro differentiation and in vivo vascular remodeling. Pericytes can also communicate with the ECs via direct contact and the paracrine signaling pathway. Moreover, they are associated with EC differentiation and proliferation, which play a crucial role in EC maturation, and lead to a better angiogenesis process. In this regard, umbilical cord pericytes have been utilized to functionalize the conduit made of ECM. Observations indicated the pericytes\u2019 ability to enhance HUVECs migration, ECM remodeling, and vessel formation . Their results showed the deposition of cells on the surface of vascular structures 1\u00a0day after graft transplantation. They represented vessels covered by HDMECs after 5\u00a0days in perfusion-based culture . FurtherDespite the progress made in the development of re-endothelialization approaches to create an efficient and functional endothelialized vasculature in tissue engineering, there remain certain bottlenecks that must be addressed. These barriers are mainly present in the D&R processes.At present, decellularization methods have been successful in eliminating cellular components and DNA contents from the cellular scaffold. However, these protocols have largely ignored the effect of scaffold microstructure and its components on subsequent re-endothelialization . ResearcIn the realm of recellularization, artificial and biological bioreactors have provided endothelialized acellular vasculature; however, complete endothelialization has yet to be achieved. The lack of specific settings in artificial bioreactors  for each organ, as well as immunogenic challenges arising from transplanting acellular structures, are obstacles that should be fully addressed in further research.Surface modification was also discussed as another influential issue in the endothelialization of acellular structures. Adhesive molecules and GFs have been used to improve ECM structure and endothelialization levels . Despitein vivo studies should be conducted to evaluate the compatibility and function of re-endothelialized constructs.Moreover, alternative approaches should be explored to improve re-endothelialization results. Exosomes, which are extracellular vesicles containing RNA, DNA, proteins, and lipids, may serve as potentially beneficial for complete endothelial coverage . Further"}
+{"text": "In plant cells, almost all kinds of abiotic stresses are able to raise cytosolic Ca2+ levels, and the spatiotemporal distribution of this molecule in distant cells suggests that Ca2+ may be a universal signal regulating different kinds of abiotic stress. Ca2+ is used to sense and transduce various stress signals through its downstream calcium-binding proteins, thereby inducing a series of biochemical reactions to adapt to or resist various stresses. This review summarizes the roles and molecular mechanisms of cytosolic Ca2+ in response to abiotic stresses such as drought, high salinity, ultraviolet light, heavy metals, waterlogging, extreme temperature and wounding. Furthermore, we focused on the crosstalk between Ca2+ and other signaling molecules in plants suffering from extreme environmental stress.Plants have evolved many strategies for adaptation to extreme environments. Ca CBLs interact with CBL-interacting protein kinases (CIPKs) to form a CBL/CIPK signaling network, which plays a key role in the plant response to abiotic stress. These networks may contain many interactions, with CBLs activating CIPKs and CIPKs phosphorylating CBLs. Phosphorylation is the major mechanism affecting downstream proteins .,20.2+-si2+, acting as a secondary messenger, plays an essential role in the plant response to abiotic stresses; it can not only transmit and recognize various regulatory signals but also participate in gene expression and normal protein functions cyt increase caused by NaCl in the wild-type, hyperaccumulation of cytosolic Ca2+ in PCA1 mutants remained high and did not return to prestimulus [Ca2+]cyt levels. Therefore, Ca2+ pumps contribute to the production of stress-induced Ca2+ signatures [2+]i increases 1 Arabidopsismonocation-induced cyt increases rapidly to induce calcium signaling cyt and cytoplasmic alkalization and activate anion channels in both wild-type and mutant plants, thus causing the stomata to be closed. However, compared with that in wild-type Arabidopsis, exogenous Ca2+ could not induce stomatal closure or activate anion channels on the plasma membrane in attpc1-2 mutants. Taken together, we can conclude that AtTPC1 protein is involved in both stomatal closure and plasma membrane anion channel activation and is regulated by exogenous calcium signals in guard cells; however, it is not regulated by ABA and MeJA cyt when high-temperature stress occurs, while the expression of PhCAM1 and PhCAM2 is not obviously changed after EGTA is added, implying that the Ca2+ signaling system and CAM play a major role in the regulation of resistance to high-temperature stress in Pyropia haitanensis cyt increase. The defense response occurs by catalyzing the elicitor peptide precursors into mature peptides located on the cytoplasmic side of the vacuole membrane; in turn, these peptides are recognized by the cytoplasmic vacuolar membrane-targeted receptor-elicitor peptide receptors cyt increase and ROS accumulation, which may be interdependent cyt. Furthermore, a slightly increased [Ca2+]cyt can not only act on the membrane skeleton and significantly reduce the deformability of cells but is also involved in the lipid redistribution of the membrane and the decline in membrane stability cyt in a precise and complex way so that they can respond to a series of complex upstream signals accurately and exclusively and ensure signal transduction sensitivity and specificity concurrently. Furthermore, because crosstalk between Ca2+ and other signaling molecules is vital for the stress response, the mechanism of stress perception and the system of signal transduction at the biological level should be investigated. Therefore, the next important task for Ca2+ signaling research is to determine which physiological reactions are involved in the various Ca2+-targeted proteins downstream of calcium signaling and which downstream molecules are regulated to affect gene expression. Moreover, with recent advances in techniques and the development of molecular biology, cell biology, genetics and other disciplines, the role of Ca2+ signaling will certainly be elucidated more thoroughly.Although the many molecular mechanisms behind Ca"}
+{"text": "NKX3.1 gene were found in the immunostain smear of the Virchow\u2019s node, which led to the identification of metastatic prostate cancer.In this case report, we describe the effectiveness of fine-needle aspiration of Virchow\u2019s node for the diagnosis of metastatic prostate cancer in a 62-year-old male without any medical history, negative urinary tract symptoms, and a normal digital rectal examination. The patient presented with respiratory distress, diffuse lymphadenopathy, and high levels of prostate-specific antigen. Multiple studies were done with inconclusive results until positive findings of the  Prostate cancer is the second most common cancer and the second leading cause of cancer-related death in American men . ProstatWhile different examinations alerted the primary care team of different etiologies of cancer, they had begun to rule out that the primary cancer was from the prostate because the clinical examinations, including rectal examination and transrectal ultrasound, were deemed normal despite alarmingly high PSA levels (402 ng/mL) throughout the patient\u2019s stay in the hospital.PSA is a protein produced by the epithelial cells of the prostate gland. It serves to liquefy the semen in the seminal coagulum allowing the sperm to move freely. PSA is present in the serum in small quantities in healthy people but is elevated in case of prostate cancer or other prostatic disorders . In our On the primary examination, the patient presented with a firm, non-tender lump in the left supraclavicular region between the two heads of the sternocleidomastoid muscle (Virchow\u2019s node). The patient did not have any other palpable nodes. Virchow\u2019s node correlates with metastatic cancer in the gastric regions, and, in rare cases, it can be present because of prostate cancer . Thus, tFNA is a diagnostic procedure used to investigate lumps or masses. This technique is performed by inserting a thin hollow needle into the lump and collecting its cells which are then stained to be examined under the microscope. The FNA biopsy is a very safe minor surgical procedure that can be done by the palpation of the mass, or with the assistance of images such as ultrasound or computed tomography (CT) scan. Puncturing tissues through a fine needle is a diagnostic technique that has developed mainly in the last century and is accepted globally . In conjA 62-year-old male presented to the emergency department complaining of progressive dyspnea and dry cough for two months. Two weeks before the emergency admittance, the patient was admitted to a peripheral facility for the same symptoms and had been clinically diagnosed with viral vs. bacterial pneumonia. He was treated with antibiotics and discharged home, but the symptoms persisted. After several visits to his primary provider who ordered a positron emission tomography-computed tomography (PET-CT) skull base to mid-thigh scan, there was mass-like substantial fluorodeoxyglucose (FDG) uptake in the proximal sigmoid colon, and numerous FDG-avid lymph nodes were seen in the abdomen, pelvic, and left the supraclavicular area, along with scattered ground-glass and interstitial opacities in the lungs.2, elevated D dimer, and PSA of 402 ng/mL. The viral panel including coronavirus disease 2019 was negative. He was also found with abnormal chest X-ray (CXR) and CT chest findings with diffuse ground-glass opacities on both upper lungs and multiple lymphadenopathies. No evidence of segmental pulmonary embolism was noted. There were no lower urinary symptoms and no previous colonoscopy. The patient reported no marked decrease in weight and no fever.The patient arrived at the emergency room (ER) with dyspnea and oxygen saturation of 87% at room air. No previous relevant medical history was noted. On initial examination, labs were unremarkable except for arterial blood gas (ABG) analysis with low PaOFigure The patient was a Cuban immigrant who had lived in Miami for over 10 years. He used to work in a metallic factory many years ago. The patient was married and lived with his family. He had never smoked and sporadically consumed alcohol at social gatherings. He denied the use of illicit drugs, and there was no family history of cancer. He was not taking any medications.On physical examination, he was awake, alert, and oriented to person, place, or time. He was not in acute distress and had good nutrition. Examination of the head, eyes, ears, nose, and throat was unremarkable. Small hard nodes were palpable on the left supraclavicular area. Bilateral basal crackles were heard on both lungs, but no clubbing, cyanosis, or edema was present. The cardiovascular examination was normal, and his abdomen was normal. On rectal examination, the size of the prostate was normal, and no induration or nodules were present. Hemogram was normal, and no rheumatologic common positive markers were noted. Erythrocyte sedimentation rate and C-reactive protein were not elevated. Blood cultures were also negative.Upon CT examination of the abdomen/pelvis, extensive retroperitoneal lymphadenopathy, with nodal conglomerates at the level of the kidneys was observed. At the same level, periceliac adenopathy, aortoiliac bifurcation adenopathy, left internal iliac, and left obturator adenopathy were also present.A conglomerate of enlarged hypoechoic lymphadenopathy measuring approximately 3.2 cm in the left supraclavicular region was observed with the help of a linear ultrasound.Consideration of metastatic cancer was acknowledged, and colonoscopy, bronchoscopy, and fine-needle biopsy from the left supraclavicular node were requested. Bronchoscopy findings were not relevant, thus the epiglottis, vallecula, and the right and left pyriform sinuses, as well as the arytenoids, were deemed normal-appearing. Additionally, no endobronchial lesions were noted on bronchoscopy. Results from bronchoalveolar lavage were also negative for infection and malignancy.The gastroenterology team completed a colonoscopy, and several polyps and a non-obstructive sigmoid mass were observed and biopsied. Carcinoembryonic antigen was negative. Biopsy was negative for malignancy.Figure NKX3.1, consistent with carcinoma of prostate origin in the FNA biopsy from the left supraclavicular node.Urology did not proceed with prostate biopsy as the patient was positive for malignancy in the FNA. Immunostaining was positive for NKX3.1, consistent with carcinoma of prostate origin.FNA and ThinPrep and smears were performed for the left supraclavicular lymph node (Virchow\u2019s node), which were markedly positive for malignancy Figure . ImmunocTreatment for the newly diagnosed prostate cancer and elevated PSA was initiated with oral bicalutamide 50 mg daily for two weeks before administering outpatient androgen deprivation therapy (ADT) to prevent an androgen storm. The patient was then started on ADT with leuprolide 22.5 mg injections every three months plus docetaxel  every three weeks for six courses.Differential diagnosisMetastatic colon cancer was considered in the differential due to positive PET-CT scan findings. In addition, sarcoidosis of the prostate was mentioned as a rare possibility considering diffuse lymphadenopathy and because it could have elevated PSA levels.\u00a0Laboratory findingsTable TreatmentAfter confirming the diagnosis on FNA biopsy, treatment was initiated for the patient with oral bicalutamide 50 mg daily for two weeks. After being released from the hospital he was on ADT to prevent androgen storm with leuprolide 22.5 mg injections every three months plus docetaxel  every three weeks for six cycles.Outcome and follow-upAfter three weeks of the initial treatment, the patient noted less dyspnea and his oxygen saturation improved. His performance status returned to normal. After five months of treatment, his PSA was 2.6 g/mL.\u00a0He was followed for 16 months with marked improvement, and a normal PSA was noted. After 19 months of treatment, the patient is grateful for the treatment; he considers himself a survivor and is following treatment diligently. He has expressed gratitude toward his primary care team and is hopeful for the future. It has to be noted that his mood has always been cheerful throughout the treatment, and he remains in good spirit.The diagnosis of metastatic prostate cancer is a challenge due to the similar presentation with cancers originating from other organs, including the gastrointestinal tract, pancreas, or biliary tract, as well as the liver or the lungs. To guide interventions, a logical clinical diagnosis is needed to establish the most appropriate workup. These logical statements could be guided by epidemiology including the answer to the question \u201cwhat are the most common primary cancers in patients with Virchow\u2019s node?\u201d In association with the possible answer, we must consider the results of the puncturing with FNA and cytology which can give us the most effective method to reach the diagnosis.Virchow\u2019s node, a left supraclavicular lymph node, was first described by German pathologist Rudolf Virchow in 1848, as a sign of metastatic malignancy, mainly from gastric cancer. The Troisier\u2019s sign is used interchangeably with Virchow\u2019s node. The mechanism of this lymphadenopathy is due to tumor embolization from the primary sites through the thoracic duct, which eventually involves Virchow\u2019s node and the cancer cells become trapped and become enlarged. Several studies have established its association with various malignancies, including gastrointestinal, pulmonary adenocarcinoma, prostate cancer, and lymphoma, among others. The initial presentation of a prostate adenocarcinoma as a left supraclavicular mass is unusual. The appearance of a left supraclavicular mass is not generally related to prostate cancer because its incidence is very low, being reported between 0.4% and 1% of all cases of metastatic prostate cancer\u00a0. It is aNKX3.1 gene, which is a superior indicator of metastatic prostate cancer compared to the PSA levels and the prostatic acid phosphatase (PAP) on cell smears. It is superior because the NKX3.1 is a nuclear maker while the PSA and the PAP are cytoplasmic markers, making the latter less reliable for the identification of metastatic prostate cancer.The case presented has shown highly significant levels of PSA, as clearly stated in the laboratory data, and this progress has been complemented by the utility of immunostaining in the detection of metastatic prostate cancer . It has NKX3.1 homeobox gene should be requested. A good physical examination is imperative to determine the presence of nodes. With the ever-evolving medical technology, the easy accessibility of ultrasound can be used to identify nodes.If Virchow\u2019s node is present, FNA is the most effective tool to determine the etiology of metastatic cancer. We must exercise logical reasoning to establish a clinical diagnosis that limits additional examination and extensive workup done on the patients. If metastatic prostate cancer is suspected, immunostaining to detect the"}
+{"text": "WVTR) value of (53.0 \u00b1 2.8 g/m2/h) and water vapor permeability (WVP) value (1.74 \u00b1 0.08 g mm/h/m2), delayed erosion (18.69 \u00b1 4.74%), high water uptake, smooth and homogenous surface morphology, higher tensile strength (56.84 \u00b1 1.19 MPa), and increased glass transition temperature and enthalpy . The in vivo data on day 16 of post-wounding indicated that the wound healing occurred faster with significantly increased percent re-epithelialization and enhanced collagen deposition with optimized MB-3 film application compared with the untreated group. The study concluded that the microwave-treated polymer blend films have sufficiently enhanced physical properties, making them an effective candidate for ameliorating the diabetic wound healing process and hastening skin tissue regeneration.This study aimed at developing the microwave-treated, physically cross-linked polymer blend film, optimizing the microwave treatment time, and testing for physicochemical attributes and wound healing potential in diabetic animals. Microwave-treated and untreated films were prepared by the solution casting method and characterized for various attributes required by a wound healing platform. The optimized formulation was tested for skin regeneration potential in the diabetes-induced open-incision animal model. The results indicated that the optimized polymer film formulation (MB-3) has significantly enhanced physicochemical properties such as high moisture adsorption (154.6 \u00b1 4.23%), decreased the water vapor transmission rate ( Skin tissue regeneration following wounds  has been a challenging task for biomedical scientists, especially the aesthetic outcome ,2, and aWound healing is a cascade of related molecular events that work jointly to reinstate cellular function and tissue integrity . These ePolymeric films have long been used as a cost-effective strategy to hasten skin tissue regeneration following damage. Hence, various polymers have been used for the purpose, such as cellulose , sodium Crosslinking is described as the generation of physical or chemical linkage between polymer chains, which is generally an easy way to amend the biological, degradation, and mechanical properties of the polymer . This isTo address the demerits associated with the use of chemical cross-linkers, physical methods of crosslinking were introduced, such as ultraviolet irradiation , electroMicrowaves are electromagnetic waves characterized by frequency in the range of 300 MHz to 300 GHz  and haveSodium alginate (SA) is a water-solvable hydrocolloid that is obtained from brown seaweed and is composed of (1\u20134)-linked -d-mannuronic acid and -l-guluronic acid units . The sigSodium Carboxymethyl cellulose (NaCMC), a biopolymer, is one of the derivatives of cellulose that is developed by substituting the hydroxyl group (-OH) with the carboxymethyl (-COOH2CH-) group, where both units are linked to each other by \u03b2 1 and 4 glycosidic linkages ,63. It pBoth sodium alginate and Na-CMC have been extensively studied from the perspective of wound healing ,74,75,76This project aimed to develop physically cross-linked sodium alginate and NaCMC films through microwave treatment, analyze them for physicochemical attributes, and perform in vivo testing in diabetes-induced animal models for rapid healing following open incision wound infliction. Blended sodium alginate/NaCMC films have been explored as prospective combinations that can be physically crosslinked using the microwave. Their combination was optimized by varying the microwave treatment time while keeping the concentration of both polymers constant. The optimized combination was tested for its ability to regenerate skin tissue in diabetic animals following open incision wound infliction.Polysorbate 80 , disodium hydrogen orthophosphate (purity ~99%), sodium chloride (purity ~99%), sodium carboxymethyl cellulose , and monobasic potassium phosphate (purity ~98%) were procured from Sigma-Aldrich , while hydrochloric acid (purity ~35%) was purchased from Merck, Darmstat, Germany. Sodium alginate  was bought from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China. Polyethylene glycol-400  and glycerol (purity ~99%) were kindly provided by Bio-Labs . All chemicals were used without any further processing or purification.w/w) solutions. Both solutions were then added with glycerol (2% w/w), tween 80 (0.1% w/w) and PEG-400 (0.05% w/w) and thoroughly mixed to ensure homogeneity. Both polymer solutions were mixed in a ratio of 60:40  and subjected to microwave treatment for different time intervals (1 and 3 min) at fixed power of 500 watts and a fixed frequency of 2450 MHz, utilizing a commercially available microwave oven . Following microwave treatment, a total of 50 g of bubble-free polymer mixture was transferred into petri dishes (\u00d8 34.30 mm) and dried in a convection oven  at 40 \u00b0C for 72 h or until complete dryness. Bi-polymeric blended films composed of sodium alginate and Na-CMC were developed by solution casting technique as described earlier . BrieflyThe dried polymeric films were detached from petri dishes and kept in a desiccator until they were subjected to several physicochemical characterization tests. The untreated blend films were developed similarly for comparison. The formulation ingredients and microwave treatment conditions are depicted in 4) at virtual relative humidity (RH) of 0% for 48 h. Following complete desiccation, the dried film pieces were weighed again and incubated again with a saturated solution of potassium sulfate in a desiccator at 25 \u00b1 2 \u00b0C with relative humidity maintained at 97 \u00b1 2% for an additional 48 h to allow the films to become fully hydrated and were weighed again. The percent (%) moisture adsorption was determined using the following equation.An already-reported method  was usedWt = Final weight after rehydration, and Wi = Initial weight after dehydration.The test was repeated three times and results were averaged with \u00b1 standard deviation.WVTR) through the polymeric film was assessed by the ASTM method with some modifications using a plastic bottle with its mouth area determined [2). The thickness of the films was determined using a micrometer screw gauge. The values were placed in equations to determine the WVT, which was estimated by dividing the slope of a linear regression of weight loss vs. time by film area, and the WVP (gm/m2 s Pa) was determined by using an equation.The water vapor transmission rate  and \u0394P is the partial water vapor pressure difference (mmHg) through two sides of the film sample .The experiment was repeated three times, and results averaged with \u00b1 SD.E%) and percent water uptake (WU%). The test was executed in triplicate, and the results were averaged with standard deviation.Briefly, film pieces were cut, each having a 3 \u00d7 2.5 cm dimension. Then dry film pieces were weighed and placed in 20 mL PBS of pH 7.4 in a petri dish. After that, it was incubated at 37 \u00b1 3 \u00b0C in a convection oven . The film pieces were taken from the petri dish at a specific time interval (every 5 min), blotted dry, and weighed again. The same was repeated for the entire incubation time of over 20 min. After 20 min, the buffer solution was discarded, and the same film pieces were dried in an oven at 40 \u00b1 2 \u00b0C for 5 days. After 5 days, the oven-dried weight was taken, and the following relations were used to calculate percent erosion  = dry weight of film taken at time t, and Wt = wet weight of film at time t.Scanning electron microscopy was used to analyze the film\u2019s surface morphology using an ultra-high-resolution field-emission scanning electron microscope . Each film was sliced into a 3 \u00d7 3 mm piece, which was then attached to a stub via double-sided adhesive carbon tape. The samples were then placed in the microscope chamber after being subjected to a 5-min gold sputter coating procedure , followed by SEM examination at a 10-KV accelerating voltage. Using the smartTiff tool, the photographs of corresponding parts were taken at magnification powers of 100, 500, 1000, 2000, and 3000\u00d7 [Using a universal testing device , the polymeric films\u2019 ultimate tensile strength was assessed at 25 \u00b1 1 \u00b0C. The polymeric film\u2019s tensile strength was measured employing a texture analyzer after being trimmed into rectangular-shaped strips. For each film sample, three rectangular-shaped strips of 7.5 cm in length and 3.5 cm in width were trimmed, and they were then fastened between the machine\u2019s grips. The initial grip distance was set to 50 mm, and the crosshead speed was set to 5 mm/min. The sample was pulled with a 50 N load . The mos2 gas with a 40 mL/min flow rate and a heating scan rate of 10 \u00b0C/min from 0\u2013400 \u00b0C. Each film peak\u2019s transition temperature and enthalpy (\u2206H) values were calculated in triplicate, and the results were averaged.The films\u2019 thermal characteristics were assessed using differential scanning calorimetry  . A 5\u20137 m\u22121 wavenumber range. Results were averaged after three analyses of each sample.An ATR-FTIR spectrophotometer   capturedn = 8 for each group), i.e., untreated and polymeric film groups. The rats were anesthetized by I/P injection of a mixture of xylazine (10 mg/kg) and ketamine (100 mg/kg), and the back hair of the rats was shaved. An open incision wound was inflicted on a mid-dorsal thoracic section of the rats with the help of sterilized forceps and surgical scissors. Following the infliction of the wound, the treatments were applied to the injured part, covered with sterile gauze, and adhered with 3M adhesive tape. The untreated group received only the gauze application, while the polymeric film group received only the  film piece. The treatments were applied daily until complete wound healing was observed. The institutional Ethical Review Board approved the animal study protocol, vide reference number: 502/QEC/GU, dated: 29 March 2019, Gomal University Pakistan.Healthy male Sprague\u2013Dawley rats with a weight range of 200\u2013250 g were procured and acclimatized/adjusted for 14 days with an easy approach to water and food at a temperature of 19 to 23 \u00b0C with a 12-h dark-light cycle. Before the diabetes induction, the rats abstained from eating for 24 h with free access to water. They were weighed, and their fasting blood sugar levels were determined using a glucometer . A single dose of freshly prepared streptozotocin solution was intraperitoneally injected in rats at a dosage of 50 mg/kg body weight of the animal. The blood sugar levels of the animals were monitored starting on day 3 of the streptozotocin injection , and ratThe photographs of wounds were taken by a Canon D5200 camera  on days 0, 3, 7, 14, and 16 post-administration to record the surface morphology of the wound. The wound size was investigated by Image J software . The % re-epithelization was then estimated using the following relation.To estimate changes produced in the lipid and protein regions of skin with film treatment compared with the control group, the skin samples with wounds were also exposed to thermal analysis employing DSC . In a nutshell, a precisely measured 3 mg of full-thickness skin-containing wound was trimmed/cut with great care and enclosed in a standard aluminum pan before being subjected to thermal analysis at temperatures ranging between 30\u2013180 \u00b0C at a heating rate of 10 \u00b0C/min, under constant pulses of nitrogen gas at a 40 mL/min flow rate. For the lipidic and protein regions, the melting temperature and enthalpy were noted. Results were averaged after at least three separate analyses of each sample.The tensile strength of the excised skin samples was measured after they were cut into strips of 5 cm in length and 2.5 cm in width . The strips were fastened between the lower and upper jaws of the tensiometer and perpendicularly pulled/strained with loads of 30 kg at test speeds of 5 mm/s and 10 mm/s, respectively. The greatest power necessary to rupture the skin sample and the breaking point were noted. Test of every skin specimen was performed thrice, and results were averaged.\u22121 and acquisition/exposure duration of 2 min. The corresponding ATR-FTIR spectra were compared to determine the degree of collagen deposition. The amide-I and amide-II absorbances, which come from the skin\u2019s protein composition, were measured for this purpose. The degree of collagen deposition was estimated using this unique technique that compared the absorbance of the treatment group with the control group. The results from three analyses of each sample were averaged.ATR-FTIR  was used to record the vibrational spectra of the dermal layer of skin samples from treated and untreated animal groups with a resolution of 16 cmAnimals were killed by cervical dislocation when needed, and the skin-containing wound was surgically removed, cleaned with normal saline, and stored at \u221220 \u00b0C until further usage. Histological testing was conducted on the newly repaired skin tissue that covered the incision. The stored skin samples were thawed at room temperature for 3 h, then fixed in a 10% aqueous solution of formalin for three days at ambient temperatures. The skin samples were then prepped by cutting, washing with regular saline, and dehydrating in ethanol. The samples of desiccated skin were cleaned with xylene before being fixed in paraffin wax. Using a microtome , 5 \u00b5m thick sections were created. They were then processed separately by Masson\u2019s trichrome and H&E (hematoxylin and eosin) stains. The slides were observed, and relevant portions were photographed employing an inverted microscope equipped with a camera (HDCCE\u2014X5N).p < 0.05, and the analysis of variance (ANOVA) followed by post hoc analysis or a Student\u2019s t-test was employed for analysis.At a minimum, three data replicates were used to calculate the mean and standard deviation. The significance level was established at An ideal wound-healing platform requires a hydrophilic extracellular matrix that can remove wound exudate and keep the wound bed moist for rapid regeneration ,92,93. TFurthermore, microwave treatment is envisaged to increase the crosslinking density between the polymers and other film ingredients such as glycerol, which has been reported to immobilize between polymer chains, resulting in increased water absorbency and enhanced water moisture adsorption ability of the film . MicrowaWVTR of a wound healing platform determines its efficiency in reducing the transepidermal water loss and facilitating the easy exchange of oxygen and carbon dioxide between the wound bed and external environment [WVTR will be capable of retaining more moisture at the wound surface since dry wounds take longer time to heal [WVTR are shown in t-test, p > 0.05), microwave-treated blends tend to have lower WVTR compared with the UB formulation, where MB-3 was found to have significantly lower WVTR compared with UB (WVP was highest for UB compared with MB-1 and MB-3 (2 and CO2) through but preventing water molecules passage due to large molecular size [The ironment , which i to heal . The res with UB . Similarand MB-3 b. More wlar size , which wlar size . Additiolar size . Furtherlar size .t-test, p > 0.05). In the case of UB, which was composed of untreated sodium alginate and Na-CMC blend, it is more likely to expose more hydrophilic surface groups due to loosening structure formation as a result of relaxed polymer chains, allowing the easy penetration of erosion media into the matrix, resulting in hastened solubility and hence quick erosion [The water uptake capacity regulates the film formulation\u2019s swelling, degradability, functionality, and stability , which a erosion . Convers erosion , thereby erosion .p > 0.05). It is believed that due to the affinity of microwaves towards polar functional groups of the polymers and/or other formulation ingredients, hydrophilic as well as hydrophobic interactions might have resulted in surface shifting of OH/NH, amide, and ester functional groups, enabling more attraction of water molecules, translating into higher water uptake [The water uptake results indicated that an increase in microwave irradiation time resulted in a higher water uptake capacity with time compared with the untreated blend. However, the difference was insignificant . The tensile strength tends to increase with microwave treatment time; significantly higher  tensile strength was observed when the blend was subjected to 3 min of microwave treatment. Higher tensile strength is believed to appear due to higher cross-linking density, which shall be optimized as higher cross-linking density translates into a reduction in percent elongation, which may make the film non-elastic [The mechanical strength of a polymeric film reflects its ability to withstand friction and stress during handling and/or application at the wound site . The ten-elastic . Microwa-elastic . In a st-elastic . Sun et -elastic . They cot-test, p < 0.05) rise in the melting transition temperature as well as corresponding enthalpies was observed for MB-3, where the sodium alginate moiety showed \u2206T value of 199.23 \u00b1 2.08 \u00b0C and \u2206H value of 2.35 \u00b1 0.02 J/g. In comparison, for Na-CMC, the \u2206T value of 260.32 \u00b1 0.58 \u00b0C and \u2206H value of 1.48 \u00b1 0.06 J/g were observed. A significant rise in melting transition, as well as the energy required to induce transition, during the thermal analysis of the microwave-treated blend film depicted that microwave treatment enabled the formation of additional interactive forces between both polymer moieties, i.e., electrostatic and/or hydrogen bonding, through both polymer polar functional groups [Thermal analysis, such as DSC, depicts the films\u2019 behavior as a function of temperature and interprets the degradation process, thermal transition, and thermal stability of the films . The DSCl groups ,110, the\u22121) and a significant increase in hydrophobic moieties  were observed, depicting rigidification of hydrophilic domains of the film and fluidization/elasticity of hydrophobic domains occurred when films were treated with a microwave for 3 min. The rigidification of hydrophilic moieties of the polymeric blend film could be attributed to the formation of additional linkages between the polar functional groups of both polymers and/or polymers and excipients, which is envisaged to translate into a delay in erosion ability. In contrast, fluidization of hydrophobic domains is predicted to increase the elasticity of the matrix, translating into higher mechanical strength when the polymer blend was treated with microwaves for 3 min.All the film formulations were subjected to vibrational spectroscopic analysis using an ATR-FTIR to elucidate the extent of hydrophilic and hydrophobic interactions between the polymers and/or polymers and excipients following microwave treatment. The results are shown in p < 0.05, WVP of the film scaffold. As stated earlier, the sodium alginate-sodium CMC film scaffold showed an optimal range of WVP; thus, wound therapy in a wet environment positively influenced re-epithelization; hence, it encourages healing with no scar development [p < 0.05) as compared with that found for the untreated control group (58%). Among both test groups, the polymeric film proved significantly efficient in healing diabetic wounds.The in vivo wound healing ability of the microwave-treated polymer film group and the untreated control group was tested in the diabetic rat model. The pictographs of wound morphology are shown in elopment . The polRe-epithelialization, a characteristic hallmark of cutaneous wound contraction, is governed by the restoration of skin tissues to form a granulation barricade on the open wound . The wout-test, p > 0.05), where the untreated skin samples showed \u2206T = 66.86 \u00b1 1.08 \u00b0C, with corresponding \u2206H = 0.89 \u00b1 0.4 J/g, while in the film-treated group, it appeared to be 69.32 \u00b1 1.20 \u00b0C, with corresponding enthalpy \u2206H = 1.25 \u00b1 0.2 J/g. In contrast, in the case of proteinous domains, a significant increase in the melting transition temperatures and enthalpies was observed in samples harvested from the film-treated group compared with the untreated group. The transition temperature significantly increased from 159.54 \u00b1 1.78 \u00b0C to 176.42 \u00b1 1.18 \u00b0C , with a significant rise in enthalpy . The rise in melting transition and enthalpies of skin protein domains in the film-treated group advocates forming a compact and cross-linked protein structure at the wound site. Sodium alginate and Na-CMC are already reported to possess wound-healing properties [The thermal analysis results of skin samples harvested on the 14th day of post-wounding from both animal groups are presented in operties ,62,86. Mt-test, p < 0.05). The increased tensile strength indicates a compact and dense arrangement of collagen protein in skin structure due to the rigidification of the dermal layer\u2019s hydrophilic moieties , depicting the development of a more dense skin structure [Uniform and greater extents of collagen deposition are expected to significantly increase the mechanical strength of the newly regenerated skin tissue at the wound site. Therefore, the skin samples harvested on the 14th day of wounding were subjected to tensile strength analysis, and the results are shown in tructure ,115.\u22121, C=O stretching in O=C\u2013N\u2013H), while amide-II bands  have been reported to arise from peptide linkages of collagen [t-test, p < 0.05, 3344.3 cm\u22121 to 3327.5 cm\u22121) compared with the untreated control group , where the absorbance band underwent a significant shift to a lower wavenumber region of 1638.8 from 1643.1 cm\u22121, with similar changes observed with the amide-II . To further strengthen this claim, a ratio of absorbance values of corresponding bands of untreated to film-treated skin was calculated, which was found to be significantly higher for film-treated groups , showing more rigidity of hydrophilic moieties in the dermis compared with the untreated group (1.20 \u00b1 0.04). The high wavenumbers and absorbance ratios indicated the rigidity of hydrophilic OH/NH parts of the dermal layer, describing the development of a denser skin structure [The ATR-FTIR spectra of the dermal layer of skin samples from the untreated control group and polymeric film-treated animal groups are demonstrated in collagen ,117. As ol group a. Similatructure  and a grtructure .Histological analysis results of the H&E and Masson trichrome staining are shown in Similarly, as evident in the results of Masson trichrome, film-treated samples showed a higher amount of collagen accumulation along with proper orientation at the wound site . Wound cThe present study investigated the effectiveness of microwaves in physically cross-linking two natural polymer blends to improve the resulting film\u2019s physicochemical properties from the perspective of wound healing application. The results demonstrated that treating sodium alginate and Na-CMC blend with a fixed frequency of 2450 MHz microwave at a fixed power for 3 min improved the physicochemical properties of individual polymers, thus customizing polymer properties in the form of increased moisture adsorption, low water vapor permeability and water vapor transmission rate, delayed erosion, high water uptake, increased mechanical strength, and homogeneous and uniform surface morphology. These properties were achieved due to tailored pore size and enhanced interaction and compatibility between polymers, facilitating the exchange of oxygen and carbon dioxide between the wound bed and the external environment, preventing enhanced water loss from the wound, which is envisaged to promote healing. Moreover, in the in vivo study, the microwave-modified (MB-3) blend film hastened the skin tissue regeneration, with rapid wound closure, increased collagen deposition, and higher percent re-epithelization compared with the untreated group. Combining sodium alginate and sodium CMC with microwave treatment in film formulation may open new horizons in skin tissue regeneration applications in diabetic wound treatment."}
+{"text": "Pexophagy occurs via distinct NIX- and USP30/NBR1-dependent pathways. Peroxisomes are organelles with key roles in metabolism including long-chain fatty acid production. Their metabolic functions overlap and interconnect with those of mitochondria, with which they share an overlapping but distinct proteome. Both organelles are degraded by selective autophagy processes termed pexophagy and mitophagy. Although mitophagy has received intense attention, the pathways linked to pexophagy and associated tools are less well developed. We have identified the neddylation inhibitor MLN4924 as a potent activator of pexophagy and show that this is mediated by the HIF1\u03b1-dependent up-regulation of BNIP3L/NIX, a known adaptor for mitophagy. We show that this pathway is distinct from pexophagy induced by the USP30 deubiquitylase inhibitor CMPD-39, for which we identify the adaptor NBR1 as a central player. Our work suggests a level of complexity to the regulation of peroxisome turnover that includes the capacity to coordinate with mitophagy, via NIX, which acts as a rheostat for both processes. Selective autophagy requires that an organelle destined for elimination is marked and then linked to the phagophore membrane. This normally occurs via a LC3-interacting region (LIR) that links to lipid-modified LC3 , 2. In mInherited mutations in peroxisomal genes can lead to debilitating peroxisomal disorders. Many of these have been linked to peroxisome biogenesis, but it is now apparent that the other arm of peroxisome homeostasis, namely pexophagy, is also critical , 18. For2O2)  cell line, expressing a peroxisomal matrix targeting signal joined with a mKeima fluorophore (Keima-SKL). This fluorophore reports on lysosomal delivery of peroxisomes. The lower pH of the lysosome leads to a change in the excitation spectrum properties of the reporter and the resultant pexolysosomes are represented in red pseudocolour  22, 24), 2422, 22O2) , 26, 27.All agents led to increases in pexophagy indices of similar magnitudes . Both DFWe next showed that MLN4924-induced pexophagy requires the canonical autophagy machinery as the number of pexolysosomes is sensitive to depletion of the key autophagy orchestrator ATG7 . It is iAlthough NIX is known to directly insert into the mitochondrial membrane , its assPreprint). However, the actual pexophagy event does not require organelle coating with ubiquitin, owing to its direct insertion into membranes. Moreover, CMPD-39 does not increase NIX levels  or target-specific siRNA oligonucleotides (Dharmacon On-target plus smart pool), using Lipofectamine RNAi-MAX  according to the manufacturer\u2019s instructions. For plasmid transfections, Lipofectamine 3000  was used according to the manufacturer\u2019s instructions.hTERT-RPE1, hTERT-RPE1-Keima-SKL, and hTERT-RPE1-YFP-Parkin cells were routinely cultured in Dulbecco\u2019s Modified Eagle\u2019s medium DMEM/F12  supplemented with 10% FBS , 1% non-essential amino acids , and 1% penicillin/streptomycin at 37\u00b0C and 5% COPreprint) were transfected with pCDNA3.1-mKeima-SKL-BlastR (derived from pCDNA3.1-mKeima-SKL-Neo (hTERT-RPE1-Cas9i-PuroS cells Preprint-SKL-Neo ), using The following ON-Target Plus Smart Pool siRNAs were obtained from Dharmacon: BNIP3 , BNIP3L/NIX , ATG7 , ACBD5 , NBR1 . DsRed-BNIP3L/NIX plasmid was obtained from Addgene (100763). DsRed-N1 plasmid was a gift from Francis Barr (University of Oxford).Antibodies and other reagents used were as follows: anti-BNIP3L , anti-BNIP3 , anti-TOM20 , anti-TOM20 , anti-PMP70 , mouse anti\u2010actin , anti-Cullin-2 , anti-HIF1\u237a , anti-ATG7 , anti-NBR1 , anti-LC3 , MLN4924 , deferiprone , 4-PBA , clofibrate , hydrogen peroxide , oligomycin A , antimycin A , Mitotracker Deep Red FM .Cultured cells were lysed with RIPA buffer  and routinely supplemented with mammalian protease inhibitor cocktail  and PhosSTOP . Proteins were resolved using SDS\u2013PAGE , transferred to the nitrocellulose membrane , blocked in 5% milk (Marvel) or 5% BSA  in TBS , supplemented with Tween\u201020 , and probed with primary antibodies overnight. Visualization and quantification of Western blots were performed using IRdye 800CW and 680LT-coupled secondary antibodies and an Odyssey infrared scanner (LI\u2010COR Biosciences).As previously described , hTERT-Rg for 2 min at 4\u00b0C. Cell pellets were resuspended in HIM buffer  and centrifuged again at 1,000g for 5 min at 4\u00b0C. The cell pellet was resuspended in HIM buffer, supplemented with 50 mM 2-chloroacetamide, mammalian protease inhibitor cocktail , and PhosSTOP , before cells were mechanically disrupted by shearing with a 23-gauge needle. The cell homogenate was then centrifuged at 600g for 10 min to obtain the postnuclear supernatant. Heavy membranes enriched in mitochondria were removed by centrifugation at 7,000g for 15 min, and the supernatant was centrifuged at 100,000g for 30 min to generate the light membrane pellet fraction (LM). Equal amount of protein from both postnuclear supernatant and LM (10 \u03bcg/lane) was resolved by SDS\u2013PAGE .hTERT-RPE1-YFP-Parkin cells were washed twice with ice-cold PBS, followed by centrifugation at 1,000Cells were fixed using 4% paraformaldehyde in PBS, permeabilised with 0.2% Triton X-100 in PBS, stained with AlexaFluor-405, -488, or -594\u2013coupled secondary antibodies, and imaged using a Zeiss LSM900 with Airyscan . The images were processed using Adobe Photoshop 2022 and Fiji v2.9.0 software. For colocalisation analysis, single confocal z-planes were analysed with the JaCoP plugin in Fiji v2.9.0 to derive the Mander\u2019s overlap coefficient M1. Quantification of colocalisation was performed from three independent experiments analysing >25 cells per experiment.5)  2 d before image acquisition using a 3i Marianas spinning disk confocal microscope . Live cells were imaged sequentially (Ex445/Em600 then Ex561/Em600). Images were processed using Adobe Photoshop 2022 and Fiji v2.9.0 softwares. Analysis of pexophagy levels in hTERT-RPE1-Keima-SKL was performed using the semi-automated \u201cmito-QC Counter\u201d plugin implemented in Fiji v2.9.0 software as previously described (For live cell imaging, hTERT-RPE1-Keima-SKL cells were seeded onto an IBIDI \u03bc-Dish (2 \u00d7 10escribed . The anaP-values are indicated as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 and derived by one-way ANOVA and Bonferroni\u2019s multiple comparisons post hoc test. All statistical analyses were conducted using GraphPad Prism 9."}
+{"text": "The development of mental health disorders is common in the university population, and mindfulness-based interventions (MBIs) seem to be effective in addressing them in different contexts. Thus, this study investigated the impact of an 8-week MBI adapted to university students from the Mindfulness-Based Relapse Prevention (MBSR) on different symptoms related to mental health problems, specifically symptoms of anxiety, depression, stress and insomnia.University students (n\u2009=\u2009136) were randomized into MBI group (n\u2009=\u200971) or wait-list group (n\u2009=\u200965). All participants completed self-administered questionnaires before and after the intervention, and the experimental group answered questionnaires weekly during intervention. Generalized mixed models were used to assess the effects of the intervention.There were improvements in the symptoms of stress , depression  and insomnia  from the beginning of the intervention to the final assessment when it was compared to the control group. No effect was found in respect of trait anxiety. The MBI was found to be effective in reducing important symptoms related to university students\u2019 mental health, possibly grounding further research on the intervention\u2019s potential of preventing the development of mental disorders.The research was registered in the Brazilian Registry of Clinical Trials (ReBEC) - number RBR-63qsqx, approved at 09/16/2019. The mental health of university students has been a topic of growing interest for researchers and psychologists worldwide. High rates of mental disorders have been reported in this population , 2, leadIn Brazil, according to a study by the National Association of Directors of Federal Institutions of Higher Education (Andifes) in 2018, about 30% of undergraduate students in federal institutions had sought psychological care in the previous two years and more than 10% took psychiatric medication . In addiImpaired sleep is another aspect of this group\u2019s mental health that deserves attention , 10, espThus, it becomes clear the association among these symptoms, as well as the high need to find methods to promote students\u2019 well-being and improve their mental health. In this regard, mindfulness-based interventions may be an important tool to be better explored, based on recent years reports of positive results in different settings. Mindfulness can be defined as the ability to \u201cpay attention at the present moment in an intentional and non-judgmental way\u201d  and has Mindfulness-based interventions (MBIs) have been proven to be effective in a wide range of problems , such asWhen it comes to its effects in university students, literature already provides some very important benefits brought by MBIs, especially when targeting those with an already diagnosed mental disorder . SimilarHowever, more than being used as an adjunct treatment, the techniques could serve a greater purpose if also offered as a possibility of psychoeducation and self-care for those with or without mental disorders\u2019 symptoms, aiming at preventing the emergence or aggravation of symptoms and future development of mental disorders. This approach of MBIs is not commonly reported in literature but might be a promising aspect to be evaluated. Therefore, the present study aimed at analysing the impacts of MBI on symptoms of anxiety, depression, stress and insomnia in university students and explore the path of stress, depression and insomnia throughout the intervention. Our initial hypothesis was that MBIs could improve mental health, represented by a decrease in the scores of instruments used to measure the cited symptoms.This is a randomized controlled clinical trial undertaken in person in two Brazilian universities. It is part of a larger study entitled \u2018Risk prevention based on mindfulness for young populations in educational contexts\u2019 (CNPQ (n\u00ba426092/2018-0) and was registered in the Brazilian Registry of Clinical Trials (ReBEC), registration number RBR-63qsqx \u2013 submission date: 20/02/2019.The inclusion criteria were: Undergraduate or graduate students aged over 18 enrolled at any Brazilian university, interested in the objectives of the study and who agreed to participate in the research. The exclusion criteria were: the presence of self-reported psychotic disorder, severe cognitive impairment or having already undertaken mindfulness therapy.A total of 136 participants were recruited and randomized, of whom 76 finished the study (55.8%) taking part on the second application of questionnaires (T1). Of those, 37 were from the experimental group and 39 from the control group. Students from any university were accepted as long as they could participate in the mindfulness sessions at one of the two public universities where the study was developed. There were no exclusions of volunteers from the research and reasons for dropout were not collected. Dropouts from the experimental group did not complete more than 4 sessions out of 8 or did not answer the questionnaires by the end of the research. Dropouts from control group did not take part in the second application of questionnaires but were also offered the opportunity to participate in the MBI offered after.Volunteers were recruited through social media and an initial presentation was made in person to clarify the research procedures and objectives. After signing an Informed Consent Form in person, data collection began with the participants completing the instruments individually, supervised by a member of the research team. The data collected at this point was considered baseline (T0). The participants were then randomly assigned to either the experimental group or a wait-list control group through computer-based generation of numbers in blocks, considering the 12 different groups that were conducted throughout the year. In the following week, the experimental group started the MBI 8-weeks protocol, during which they filled in the instruments PHQ-9, ISI and PSS-10 weekly, at the beginning of each session. After eight weeks, all volunteers were asked to complete all questionnaires again (T1). The STAI questionnaire was not applied weekly due to its stability characteristic, therefore anxiety was only measured at the beginning and by the end of the intervention. The flowchart of the study procedures is described in Fig.\u00a0a questionnaire inquiring age, sex, psychological disorders, education, and parents\u2019 education. This questionnaire was answered only once, at the beginning of the participation, by each volunteer.A 9-item questionnaire evaluating the frequency of the participants\u2019 depressive symptoms in the past week, scored using a Likert scale ranging from 0 (none) to 3  validated in Brazil , 35. ParA 10-item scale with responses based on a Likert Scale ranging from 0 (never) to 4  that evaluates the frequency of stressful situations in the past month. The Brazilian validated version (36) was used. Participants who were in the intervention group answered it weekly, as well as at T0 and T1 alike the control group.A 7-item questionnaire evaluating insomnia symptoms and its influence on daily life, measured by responses on a Likert scale ranging from 0 to 4, validated in Brazil . ParticiA 20-item instrument that evaluates anxiety symptoms using a 4-point Likert scale, from 0 - \u201calmost never,\u201d to 4 - \u201calmost always\u201d. In this study, only the trait subscale was used, since it is less sensitive to environmental change. Higher scores represent more symptoms, and the scale used had already been validated in Brazil . The queAn adaptation of the MBRP (Mindfulness Based Relapse Prevention) protocol was used, following the parameters given by Crane (2016) . In thisThe intervention happened weekly for 8 weeks in groups composed by 3 to 10 participants each, all facilitators had a professional qualification in mindfulness and had previous experience conducting the protocol. Following the same pattern as the original protocol, sessions had as main theme, in order: (session 1) mindfulness in daily life and autopilot; (session 2) awareness of challenges, sensations, thoughts and reactions; (session 3) mindfulness in daily life; (session 4) mindfulness in challenging situations; (session 5) acceptance and skilful action; (session 6) seeing thoughts as thoughts; (session 7) self-care and balanced lifestyle; and (session 8) social support and continuation of practice. Also, participants were encouraged to practice mindfulness at home, with both the formal and informal techniques discussed throughout the sessions.The analyses were performed using RStudio software version 1.2.5019, considering a significance level of 5%. Packages used were glmmTMB  and siPlGeneralized mixed models were used to assess the effects of MBI on the outcomes . As the outcomes were measured from the sum of items evaluated on Likert scales, three possible discrete distributions were tested for the dependent variables: normal, Poisson and negative binomial. The choice of the best distribution was based on adherence criteria, using the lower Akaike Information Criteria (AIC) .All models considered as fixed effects group, time and the interaction between both. The control variables were sex, age and whether or not students were in exam periods. To consider the effect of dependence between temporal measures, the subjects\u2019 id was included as a random effect, allowing variation of the intercept between individuals. The analyses were conducted among the subjects who did not have missing values in the variables used in each model . The interaction effects were represented in line graphs, containing the average of the outcome weighted by covariate levels.Among those who completed the questionnaires at T1, 61 were women (80.3%) and 15 men (19.7%), with an average age of 25 years (\u00b1\u20096.2 SD), ranging from 18 to 41 years. Most of the participants were undertaking undergraduate courses (61.3%), rather than postgraduate courses (38.7%), and 84.2% of participants studied at public universities. The description of all randomized participants can be found in Table\u00a0The symptoms analysed weekly throughout the intervention , when compared to baseline, showed a significant decrease in all sessions Table\u00a0. The proThe effect of the mindfulness-based intervention over time, in respect of mental health issues, was verified for the variables stress , depression  and insomnia . No effect was found for anxiety symptoms Table\u00a0; Fig.\u00a04.Contrast tests between pairs showed that there were no differences between groups at baseline in any of the parameters. For stress, significant differences occurred between the mindfulness group at baseline  and T1  ; control at baseline  and mindfulness at follow up ; and control  and mindfulness at T1 . This means that those in the mindfulness group presented a significant difference at T1 both in respect of initial scores and of the second scores from control group.For depression, the results were similar; differences were found in the pairs: mindfulness at baseline  and T1  ; control at baseline  and mindfulness at follow up ; control  and mindfulness at T1 . Regarding insomnia, the mindfulness group showed a significant reduction from baseline  to T1  .Reduced symptoms of depression, stress and insomnia were observed in university students after the application of an adapted version of the MBRP, when compared to the control group. However, it did not modify symptoms of anxiety, differing from what was expected. These results add to the literature in respect of the impacts that this type of intervention can have on the mental health of university students.Also, during protocol, there were statistically significant differences in the symptoms of stress, depression and insomnia ever since the first section. Although it has not happened consistently and progressively, in general, there was a reduction in these symptoms presented by university students throughout the entire protocol. It is important to highlight that these trends may not be the result of each session directly and all measures were compared to the baseline, but it can be important to explore ways in which the sessions could have influenced the observed results.A continuous decrease in these symptoms in the first three sessions could be seen. In general, these sessions were about the presentation of the concept and relationship of mindfulness to what is experienced in everyday life. A slight increase in symptoms of depression and insomnia was seen after the fourth session, when challenging situations are discussed and how mindfulness can be used to deal with them. In addition to the effect of the session itself, situations outside the context of the practices may also have contributed to the result found, but the changes were slight. The average of both symptoms dropped again in the following week, after the fifth session.After the sixth session, in which thoughts and our reactions to them are worked on, there was an increase in stress and insomnia symptoms, while depression symptoms remained stable when compared to the previous week. It is possible that, when turning the attention to the thoughts in an attempt to disengage, but still without the consolidated skill, observation brings discomfort and stress, which can affect sleep.According to the Monitoring and Acceptance Theory (MAT), awareness of experiences, that is, the ability to monitor attention, can explain the improvement presented with regard to the cognitive functioning of individuals who undergo mindfulness protocols, but this same skill can increase affective reactivity , which cFinally, after the seventh session, when the focus is on self-care, all symptoms showed a visible decrease, which remained until the end of the protocol. One previous study that assessed the weekly change in stress symptoms in patients at a medical centre was found, where a statistically significant difference was observed in relation to the baseline only after the fourth session . AlthougA previous study evaluated the effect of a brief mindfulness intervention performed in just three sessions in an adult audience without major psychological symptoms and observed a decrease in the Depression, Anxiety and Stress Scale (DASS) score and an improvement in life satisfaction . Four-sePracticing mindfulness between sessions is already known as an important factor for the number of positive consequences observed , 53. HowThus, the need for further studies to explore the moment when changes happen is highlighted, in order to better understand what the possible modifications are in the protocols. This information could help both cultural and specific adaptations for different populations, as well as changes in the number of sessions of interventions.Corroborating the initial hypotheses, symptoms of depression presented by the students decreased significantly after the 8-week MBI protocol. This result is in line with the literature, where improvements in these symptoms have already been reported through MBIs in several populations , 57 and Although none of the participants declared a sleep disorder, the insomnia severity questionnaire pointed to a low quality and quantity of sleep at baseline. This may not have been perceived by students as a problem or been identified as a sleep disorder. Following the intervention, there was an improvement in insomnia symptoms, which might indicate the impact that mindfulness can have on the lives of its practitioners. A lower quality and quantity of sleep have already been shown to be associated with lower academic performance and quality of life, decreased cognitive functions and an increased propensity to develop mental disorders \u201364. AlsoThe protocol used did not specifically focus on stress, but students also reported a decrease in perceived stress after the intervention. Stress is a significant health challenge in the 21st century, with many people developing disorders and having a reduced quality of life due to this instinctive response proving to be maladaptive in some modern contexts. The effect of MBIs on stress has been reported for decades in different populations \u201367. A reHowever, contrary to our expectations, the students\u2019 anxiety symptoms did not improve after the mindfulness protocol when compared to the control group. In fact, there is no consensus in the literature on the effect of mindfulness on anxiety, with some studies pointing to improvements , 69 and One important observation made when conducting the research was that students were very interested in taking part in mindfulness practices. Despite this initial interest, there were a high number of dropouts from the groups. Future researches are necessary in order to better explore the reasons why students present such a high rate of dropout.The study has limitations that should be considered. The use of self-administered questionnaires limits objectivity, making it necessary to use multiple methods to overcome this issue. The high number of dropouts during the protocol, despite being common and within expectations, affects the results of the study and needs to be taken into account. A longer follow-up time after the intervention would be beneficial in investigating more long-term effects. The research sample contained a large disproportion between genders, with 80% being female, and therefore limiting the generalization of the results to men, although previous studies have suggested that the relationship between mindfulness and mental health is similar between genders , 73. MorDespite these limitations, the present study makes an important contribution to the literature on interventions to improve the mental health of university students, adding longitudinal data from a population that is still little explored in Brazil. In addition, results from an increasing diversity of populations, settings and cultures can enhance the understanding of the benefits and limits of mindfulness practices. This study also makes it possible to analyse the symptoms throughout the intervention, enabling a better understanding of when and how the changes occur. Finally, this study makes an important contribution to the understanding of how a relatively brief approach (8-weeks protocol) can impact a population that contains a high number of individuals who may suffer with mental health issues.It is important to emphasize that further studies with a longer T1 time are required to observe the protocol\u2019s long-term effects. Moreover, it could be of interest to evaluate whether MBIs have an impact on seeking psychological support, and the viability of implementing MBIs\u2019 protocols in universities as a prevention program in order to prevent the development of mental disorders.It was observed a decrease in the symptoms of mental health disorders in university students after its participation in a MBI intervention focusing on reactivity and health promotion, an adaptation of MBRP protocol. Addressing mental health problems before the development of disorders, and even in the presence of it, in university students is an important step towards a healthier and more productive higher educated population. Mindfulness practices could be further investigated and used as a potential investment in this area, enabling health education, health promotion and mental disorders prevention among university students, regardless of the chosen protocol, as the improvements related to symptoms of mental health problems seem to be transversal to all."}
+{"text": "Above\u2009~\u200980\u00a0\u00b0C, a more substantial variation in these thermal properties was registered: at 94\u00a0\u00b0C, \u03b1 and k respectively experienced a 2.3- and 1.5- fold increase compared to their nominal values, showing average values of 0.346\u00a0mm2/s and 0.828\u00a0W/(m\u00b7K), accordingly. Conversely, Cv was almost constant until 55\u00a0\u00b0C and decreased afterward  at 94\u00a0\u00b0C). Concerning the lung tissue, both its \u03b1 and k were characterized by an exponential increase with temperature, showing a marked increment at supraphysiological and ablative temperatures , respectively, i.e., 13.7- and 13.1-fold higher compared to their baseline values). Regression analysis was performed to attain the best-fit curves interpolating the measured data, thus providing models of the temperature dependence of the investigated properties. These models can be useful for increasing the accuracy of simulation-based preplanning frameworks of interventional thermal procedures, and the realization of tissue-mimicking materials.This work proposes the characterization of the temperature dependence of the thermal properties of heart and lung tissues from room temperature up to\u2009>\u200990\u00a0\u00b0C. The thermal diffusivity ( Indeed, tissue thermal properties are widely employed in simulations of hyperthermal and thermal ablation procedures for cancer treatment,44 analysis of the heat transfer between implantable devices, ablation catheters and the body,60 modeling of the heat transport during physiological mechanisms54 and to realize tissue-mimicking phantoms.6The characterization of thermo-physical properties of biological tissues has a key role in several branches of medical, biological, and bioengineering applications.52 afterward, a decrease in blood flow has been observed in several tissues.12 In this range, also the metabolism of the cells is modified, indeed, cells require a higher amount of energy to guarantee basic conditions, such as ion gradients across cell membranes, and to preserve structural and physical properties.42 For temperatures higher than 45\u201350\u00a0\u00b0C, the damage starts to be irreversible.18 To obtain long-term benefits, it is required to exceed 60\u00a0\u00b0C, leading to the denaturation of proteins and instantaneous cell death. For temperatures above 80\u00a0\u00b0C, physical changes in the tissue occur due to phenomena such as the evaporation of water. All these phenomena are accompanied by quantitative temperature-dependent variations of the macroscopical properties of the tissues.The effects of the temperature on the tissue depend upon the range of temperature variation and the permanence of the medium at a specific temperature. In general, we can categorize the temperature effects on the biological tissues as follows. When the tissue temperature is increased until 42\u00a0\u00b0C to 45\u00a0\u00b0C for around 30\u00a0min, the blood flow and vasodilation are usually augmented, thus dispersing heat to protect cells from thermal damage;2 Thermal ablation therapies include high-intensity focused ultrasound, radiofrequency, microwave, and laser ablation, which are the typical thermal ablation strategies used for localized tumor treatment.53 They can be performed through minimally invasive approaches, and have shown to be effective in treating malignant cells from a large group of cancers and in various biological tissues such as liver, kidney, and lungs.59While with implantable devices it is important that body temperature does not exceed the limit of almost 42\u00a0\u00b0C to avoid unwanted damage, during thermal therapies the tissue temperature has to increase in the range of 50\u2013100\u00a0\u00b0C, until the required therapeutic levels are met.37 Radiofrequency and microwave ablation are the most used thermal ablation techniques for inoperable lung cancer.Thermal therapies for localized malignancies are mainly introduced to treat not operable patients. In the specific case of lung, 70\u201380% of the patients cannot undergo surgery, and image-guided thermal ablation can represent a good minimally invasive alternative to treat patients with early-stage primary lung cancer, oligometastasis, or local recurrence.5 At ablative temperatures, the target tissue undergoes irreversible coagulation necrosis and then evolves into nonconducting myocardial scar tissue, being this therapy definitive compared to drug therapy.57Ablation is also effective in cases of arrhythmias or atrial fibrillation because the cells responsible for the propagation of the dysrhythmia are destroyed.23Cardiac ablation is nowadays a routine procedure that provides patients with arrhythmias relief from symptoms and results in an improvement in quality of life.In both these cases, the aim is to destroy pathological cells and to preserve, at the same time, the surrounding healthy tissue and anatomical structures. The limited experimentation in this field, mostly motivated by ethical reasons, and the poor standardization in the clinical practice make the role of heat transfer modeling and tissue-mimicking phantoms strategic for the analysis of the problems and the optimization of the procedures.k) [W/(m\u00b7K)], thermal diffusivity (\u03b1) [mm2/s], and volumetric heat capacity (Cv) [(MJ/(m3\u00b7K)], which govern the heat transfer, and also vary with the local temperature.12 The k describes the energy transfer by conduction in tissue in the steady state. The \u03b1 is instead related to the nonsteady state and refers to the capability of the biological media to conduct thermal energy with regard to reserve heat.46Cv is defined as the ratio between k and \u03b1, and it represents the amount of energy (heat) that must be added to cause an increase of one unit in temperature, per unit volume. The relationship among these properties is defined by the following equation:\u03c1 and c the tissue density and the specific heat of tissue, respectively.46The knowledge of the thermal properties of target tissues and their values as a function of temperature is required for the accurate prediction of the thermal effects in all the mentioned scenarios. Indeed, the amount of delivered heat and damage are strictly related to the temperature distribution in the biological medium. The way the temperature distributes within the tissue is influenced by the tissue's thermal properties, such as thermal conductivity  versus temperature change.19A few other studies have been carried out to estimate the thermal properties of relevant organs, such as heart and lung, and in most cases, the temperature range did not cover higher temperature values 48 which are reached during localized thermal therapies on these organs.51 Hence, motivated by the limited data on heart and lung, and by the huge demand for accurate models for describing the heat transfer in these organs, this paper focuses on the measurement of the thermal properties of these tissues, from room temperature up to 94\u00a0\u00b0C. The thermal conductivity, thermal diffusivity, and volumetric heat capacity of ex vivo porcine heart and lung were measured with a dual-needle sensor technique, which has been previously validated by our group on other organs.43 The behavior of the measured thermal properties of the heart and lung tissues as a function of temperature has been described by tissue-specific curves, and the analysis of the measurement repeatability has been carried out.Over the last 40\u00a0years, many studies have been conducted to analyze the thermal properties of biological tissue.a water thermal bath  used to control the water temperature in the range of interest (from room temperature to\u2009>\u200990\u00a0\u00b0C);a thermally conductive metallic container filled with the sample, suitably sealed to avoid direct contact between the specimen and the water of the thermal bath;four thermocouples  for water and sample monitoring;TEMPOS thermal properties analyzer with an SH-3 dual-needle sensor probe;a metallic needle instrumented with 10 temperature sensors based on Fiber Bragg Grating (FBG) technology.The experimental setup employed to measure the thermal properties of the deflated lungs and heart tissues consists of Fig.\u00a0a:a water24 For these reasons, each heart was divided into two portions, discarding not homogeneous parts of the sample like valves and blood clots. The lung samples were prepared by removing the trachea and avoiding bronchi, bronchioles, and alveoli.For both the heart and lung tissue specimens, some precautions were taken to guarantee an adequate volume of tissue to fill the metal container, ensuring, in particular, that the needles of the dual-needle probe were surrounded by at least 15\u00a0mm of tissue.27 with an SH-3 dual-needle sensor. This sensor, which operates in a range of temperatures between \u2212\u00a050 and 150\u00a0\u00b0C, is connected to the analyzer of thermal properties and can measure k, Cv, and \u03b1 in solid and granular materials. The sensor is composed of two needles, the heating needle and the measuring one, both 30\u00a0mm in length, placed 6\u00a0mm away from each other and with a diameter of 1.3\u00a0mm.Each sample was arranged inside the metal container covered with a silicone lid previously fixed with parafilm to prevent direct contact between the tissue and the water. The container was inserted into the thermal bath to allow the tissue to heat up and reach the desired temperature, maintaining it throughout the experimental procedure. The lid was drilled with six holes: two for the SH-3 dual-needle sensor, one for the 10-temperature-sensors metallic needle based on FBG technology, and three for the thermocouples. The thermal properties were measured using a commercial analyzer 2/s, and 2.03\u00a0MJ/(m3\u00b7K) for k, \u03b1, and Cv, respectively; we measured values equal to 0.386\u00a0W/(m\u00b7K), 0.193\u00a0mm2/s, and 2.00\u00a0MJ/(m3\u00b7K) for k, \u03b1, and Cv, accordingly . Hence, the utilized system was demonstrated to operate properly and in absence of event flaws.The thermal properties of a reference material  were measured to validate the accuracy of the used measurement system and assure that it was functioning in accordance with the specifications. At room temperature , the thermal properties of the standard material provided by the manufacturer are 0.384\u00a0W/(m\u00b7K), 0.189 mmth) and an additional time of 90\u00a0s is required by the other needle to measure the temperature variation of the tissue caused by the transfer of heat from the heating needle to the adjacent tissue, so each measurement takes an overall time of 2\u00a0min.\u00a0The obtained data are processed by subtracting the drift rate from the ambient temperature.The operating mechanism of the instrument used for measuring the tissue thermal properties relies on the initial verification of the thermal drift of the tissue collecting data for 30\u00a0s: if the temperature drift is below a specific threshold (>\u20090.002\u00a0\u00b0C/s), the instrument can start the measurement process. The heating needle takes 30\u00a0s to warm up  at the beginning of the heating period; the temperature recorded by the monitoring needle (T) is subtracted from T0 to determine the temperature variation needed to solve the Eqs.\u00a0::T0) at tq, r, which are dependent on the SH-3 dual needle sensor probe characteristics, and t and th, the k (W/m\u00b7K) and the \u03b1 (m2/s) can be obtained from the Eqs.\u00a0(T is the temperature rise at the measuring needle (the heating needle increases the tissue temperature by about\u2009<\u20091\u00a0\u00b0C above the baseline temperature), q is defined as the amount of heat released per unit of length (W/m) which derives from the imposition of a certain current at the heating needle for the determined time th, i.e., 30\u00a0s, in order to locally increase the temperature of the sample, r is the distance from the heated needle to the measuring needle which is equal to 6\u00a0mm, t is time (s). Ei(\u2212x) is the exponential integral function, which is approximated, in case of small arguments, that is in case of small distances and prolonged observations periods, as the sum of ln(x), i.e., the natural logarithm of the argument, and the Euler\u2013Mascheroni constant, i.e., \u03b3\u00a0\u2248\u00a00.5772.39 The Cv is then calculated with the following formula:Knowing the values of physical parameters such as the Eqs.\u00a0 using a Syx value. This dimensionless parameter is a measure of the quality of fit as it indicates how well a theoretical heating curve, used as a model, may fit the recorded heating curve. Hence, lower values of Syx can be used as a metric to assess the goodness of the fit, i.e., a higher similarity of the theoretical and the actual curves, albeit even at higher Syx results may still be accurate . Details on the accuracy of the TEMPOS thermal properties analyzer are provided in the manual of the instrument.27Along with the values of the estimated thermal properties, attained through the least squares method, the thermal properties analyzer also provides, at the end of the measurement, a so-called \u03b1, Cv, and k  in the range from room temperature to ablative temperatures, led to favoring the storage protocol used.The largest experimental campaign  was performed on samples that, immediately after the collection of fresh organs  from a local butcher, were stored at \u2212\u00a020\u00a0\u00b0C, removed 12\u00a0h before the experiment, and placed in a refrigerator at 4\u00a0\u00b0C to allow the specimen to gradually thaw. The decision to adopt this experimental procedure was due, first, to the fact that an attempt was made to standardize the measurement protocol by keeping the organs always in the same conditions and minimizing the potential effects of deterioration.ex vivo hearts for a total of 9 heart tissue samples and 6 ex vivo lungs for a total of 9 lung tissue samples for the so-called full analysis and intra-sample repeatability analysis.For each experimental trial from room to ablative temperatures, we used 3 tissue samples. The experimental trial was repeated 3 times for each tissue type. Overall, we used 5 ex vivo porcine cardiac and lung tissues  at five selected temperatures in the range from room to ablative temperatures. This was performed in order to make a comparison with the thermal properties of frozen-thawed tissues, subjected to the previously described experimental storage procedure, at the same temperatures. For these experimental trials, measurements were repeated three times consecutively (10\u00a0min apart between one measurement and the next one) for each temperature value.Moreover, we have carried out a preliminary analysis by evaluating the thermal properties of fresh The temperatures of the samples and the thermal bath were measured with two different and complementary approaches.k-type thermocouples (0.1\u00a0\u00b0C accuracy) were used to measure the absolute temperature of the tissue samples and water bath. The first thermocouple was placed in the tissue, at the same depth and close to the dual needle sensor and was used to further verify the temperature of the tissue measured by the SH-3 dual-needle sensor connected to the thermal analyzer (the TEMPOS system is able to resolve temperature to \u00b1\u20090.001\u00a0\u00b0C); the second thermocouple was placed on the superficial zone of the sample; the third thermocouple was positioned at around 4\u00a0cm depth in the tissue to verify that the temperature of the tissue was homogenous through the whole sample  and the optical information was acquired with the ENLIGHT Sensing Analysis Software . The information provided by the 10 sensors was useful to assess the distribution of the temperature in the tissue and the time required for the sample to reach thermal equilibrium while heated with the thermal bath.The temperature distribution within the tissue was measured by a 10-sensors metallic needle based on Fiber Bragg Grating (FBG) technology .Ts are expressed in terms of mean value and measurement uncertainty, calculated using the following Eq.\u00a0 represents the temperature-dependent thermal property, i.e., k, \u03b1, or CV, and T is the temperature recorded by SH-3 dual-needle sensor connected to the thermal analyzer. The values a, b, c, and m, q are the coefficients of the equation in the best data fitting and were derived using MATLAB\u00ae software  employing the least squares method.39 The coefficient, R2, and the analysis of the residuals were used to evaluate the goodness of the fitting and the chosen models. In the analysis of the residuals, the residuals were calculated as the difference between the measured property and the value calculated with , , y(T) reThe intra-sample repeatability analysis has been performed for each sample and each set temperature. A total of 3 consecutive measurements of the thermal properties were performed, with a time-lapse of 10\u00a0min between each acquisition. The mean value and the standard deviation of the three repetitions were calculated to assess the repeatability of the measurement.The temperature distribution measured with FBGs during single experiments and at different sample depths indicate that the tissue reached equilibrium after about 50\u00a0min Fig.\u00a0. For thi\u03b1, k, and Cv and the associated standard deviations for the three consecutive measurement repetitions, at each temperature, for heart tissue. Besides, Fig.\u00a0The results of the preliminary analysis conducted to investigate the thermal properties of fresh and frozen-thawed tissue samples, from room up to ablative temperatures, are depicted in Fig.\u00a0ex vivo porcine heart tissue were measured over a temperature range of 21\u201394\u00a0\u00b0C. Figure\u00a0x-axis against which the variance of the thermal properties is estimated is defined by the temperature measured by the SH-3 dual-needle sensor connected to the thermal analyzer.The thermal properties of the \u03b1 associated with the heart tissue showed an approximately constant behavior up to\u2009~\u200970\u00a0\u00b0C followed by a gradual increase until about 80\u00a0\u00b0C and a more substantial increase up to 94\u00a0\u00b0C. Increments of 27% and 131% with respect to the initial value  were assessed at temperatures of 79\u00a0\u00b0C and 94\u00a0\u00b0C, respectively. Besides, the observed trend for k was almost constant until around 80\u00a0\u00b0C, then k slightly increased  at 86\u00a0\u00b0C), and it reached its maximum at 94\u00a0\u00b0C with a value of 0.828\u00a0W/(m\u00b7K), corresponding to an increase of 55% from the nominal value at 21\u00a0\u00b0C. Regarding Cv, its behavior was rather constant until 55\u00a0\u00b0C, afterwards, Cv subsided until 94\u00a0\u00b0C, showing, at the latter temperature, a decrease of 32% compared to its baseline value at 21\u00a0\u00b0C.The 2, and coefficients of equation  the trend of the experimentally measured properties over temperature were derived using equation . Regressequation  used to k and Cv, the curves . Likewise, the value of k was subject to an exponential increase with temperature. From the nominal value of 0.207\u00a0W/(m\u00b7K) measured at 22\u00a0\u00b0C, k rose by 6.3, 9.9, and 13.1 times, at 83, 89, and 91\u00a0\u00b0C, respectively; thus, reaching a maximum value of 2.721\u00a0W/(m\u00b7K).As it is possible to observe, the Cv showed no substantial fluctuations within the investigated temperature range, registering values of 1.33\u00a0MJ/(m3\u00b7K) and 1.30\u00a0MJ/(m3\u00b7K) at room temperature and at 91\u00a0\u00b0C, accordingly.Differently from the other two thermal properties, 2 parameter, \u03b1 and k show a value close to 1, so the exponential model can be considered an accurate model for the approximation of the experimental data attained for these thermal properties. In the case of CV, the curve which most accurately approximates the experimental measurements at various temperatures is given by the linear equation  specimens.3 These results may be useful for the improvement of laboratory protocols regarding tissue conservation for better accountability of experimental measurements. Then, the full analysis to evaluate inter samples variability and the intra-sample repeatability analysis were performed.We first performed a preliminary analysis on the thermal properties of fresh and frozen-thawed tissue, at different selected temperatures, which highlighted no substantial differences between the tissues undergoing these different conservation protocols. The attained results are in accordance with previous experiments on different biological media  which showed, differently from other physical properties such as optical properties\u03b1, k, and Cv in a temperature interval between 21 and 94\u00a0\u00b0C, thus expanding the temperature range considered in previous studies.55 We first validated our measurement approach by comparing the results attained at ambient temperature with the values reported in previous works which employed self-heated thermistor probes for the thermal properties characterization , 3.57\u00a0MJ/(m3\u00b7K), respectively, which are in accordance with the values reported by Bhavaraju and Valvano for porcine myocardium at 25\u00a0\u00b0C , and 3.39\u00a0MJ/(m3\u00b7K))10 and by Valvano et al. at 23\u00a0\u00b0C for porcine myocardium , and 3.71\u00a0MJ/(m3\u00b7K)) and human myocardium , and 3.70\u00a0MJ/(m3\u00b7K)).55 Subsequently, we assessed the temperature dependence of the heart tissue thermal properties in the hyperthermic and ablative temperature range. We observed that the values of \u03b1 remained almost constant until around 70\u00a0\u00b0C and gradually changed until\u2009~\u200980\u00a0\u00b0C. Above\u2009~\u200980\u00a0\u00b0C, a more substantial variation in this thermal property was registered. Similarly, the k remained almost constant up to\u2009~\u200980\u00a0\u00b0C, then, above this temperature, it gradually increased up to 86\u00a0\u00b0C and was subjected to a steeper rise at 94\u00a0\u00b0C. The attained results are in line with the observations reported by Bhavaraju and Valvano, who investigated the \u03b1, k, and density in ex vivo porcine myocardial tissue at 25, 37, 50, 62, and 76\u00a0\u00b0C.10 In this temperature interval, they observed a decrease of k as a function of temperature, whose average values were 0.515\u00a0W/(m\u00b7K) at 25\u00a0\u00b0C and 0.476\u00a0W/(m\u00b7K) at 76\u00a0\u00b0C. However, the variations of k and \u03b1 were not significant (according to a variance of the data at a 5% level of significance), in the mentioned range, despite a significant loss in the water content due to temperature increase over 50\u00a0\u00b0C .20 However, at higher temperatures (>\u200990\u00a0\u00b0C), which can be easily reached close to the energy-delivery applicator during thermal ablation procedures, the tissue \u03b1 and k experience a steep rise which may in turn affect the heat distribution within the biological tissue during treatment. Differently from the profiles of \u03b1 and k, Cv was almost constant until 55\u00a0\u00b0C and gradually decreased until\u2009~\u200980\u00a0\u00b0C; above 80\u00a0\u00b0C the decrease became more marked.It is worth noticing that according to our results, the cardiac tissue ex vivo pulmonary tissue, we investigated their temperature dependence up to 91\u00a0\u00b0C, hence providing a first comprehensive analysis of the thermal dependence of \u03b1, k and Cv associated with lung tissue, up to ablative temperatures. The results attained at room temperature  were 0.155\u00a0mm2/s, 0.207\u00a0W/(m\u00b7K), 1.33\u00a0MJ/(m3\u00b7K), for \u03b1, k, and Cv, accordingly. These measurements are in line with the values registered by Silva et al. on ex vivo lung samples at 22.11\u00a0\u00b0C , Cv\u2009=\u20091.84\u00a0MJ/(m3\u00b7K))  tissues.48 Conversely, the k and Cv associated with lung tissue were substantially lower .Regarding the thermal properties of \u03b1 and k of lung tissue were characterized by an exponential increase with temperature. Conversely, no substantial variations were observed for the Cv, whose minimum and maximum values were 1.20\u00a0MJ/(m3\u00b7K) and 1.58\u00a0MJ/(m3\u00b7K), correspondingly. It is worth mentioning that, differently from what was observed for cardiac tissue, already at a temperature close to those that mark instantaneous thermal damage in tissue , \u03b1 and k of lung tissue were found to vary about 3 times from the nominal value at room temperature. The marked change in \u03b1 and k due to temperature elevation is emphasized by the fact that, at temperatures\u2009>\u200990\u00a0\u00b0C, \u03b1 and k associated with lung are greater than those associated with cardiac tissue, showing respectively 6.1-fold and 3.3-fold higher values compared to heart tissue at 94\u00a0\u00b0C. The observed increase in \u03b1 and k with temperature for lung tissue was higher compared to also other organs such as liver, kidney, and brain, which were subject of previous studies.50 For both the heart and lung tissues, the measurement uncertainty related to \u03b1 and k was found to increase at higher temperatures, which is also in accordance with previous experimental investigations.49Both the 10 Approaching the water phase transition temperature, the water evaporation phenomenon initiates, together with vapor diffusion and condensation in other districts, characterized by lower local pressure, of the same tissue.39 These phenomena may alter the overall tissue capability to transfer and retain heat compared to nominal conditions. As observed in other studies for liver tissue,39 also in our investigation, the most prominent variations in \u03b1 and k were attained at temperatures\u2009>\u200991\u00a0\u00b0C. The marked variation of the \u03b1 and k for the lung tissue with temperature, in comparison to the heart, and other tissues,47 may be due to the peculiar porous structure and characteristics of the lung.41 As a matter of fact, at baseline conditions, studies have shown a similar water content for lung tissue compared to other organs, while its density is around half of the value determined for the other tissues .48The mechanisms underlying the temperature-dependent variation of thermal properties in biological media are yet to be fully enlightened, however, the observed change in thermal properties in heart and lung tissue may be ascribable to the cellular and subcellular modifications in tissue due to temperature change. Indeed, the temperature increment involves the escape of the extracellular water content from tissue, the modification of the protein structures due to the breaking of hydrogen bonds, and the alteration of cell membranes allowing water to leak out into the extra-cellular space.40 An accurate estimation of the delivered thermal dose could in turn be beneficial for the minimization of procedural downsides, such as steam pops and hematic clots formation during catheter-mediated ablation for the treatment of cardiac arrhythmias,56 and the precise thermal eradication of lung neoplasms without excessive damage to the surrounding healthy tissue.7 Furthermore, the reported thermal properties could be useful for the refinement and validation of medical devices that must interface with heart and lung tissue, and for the realization of phantoms mimicking the behavior of these tissues.In conclusion, in this work, we devised the analysis of the temperature dependence of thermal properties of heart and lung tissues. The best-fit regression curves based on the experimentally measured data are proposed to be utilized in predictive mathematical frameworks toward optimized preplanning of hyperthermic and ablative thermal procedures involving heart and lung tissues. Indeed, the inclusion of temperature-dependent properties in computational models of the bioheat transfer may help enhance the accuracy of the prediction in terms of estimation of temperature distribution and final tissue thermally coagulated zone.in vivo lungs, as the thermal properties of inflated lungs might be decreased by the presence of the air,58 thus affecting the heat transfer throughout the tissue. Moreover, although porcine tissue exhibits high morphological and histological similarity with human tissue,31 the evaluation of the temperature sensitivity of tissue thermal properties should be also expanded to human healthy and tumorous tissues. Finally, the temperature dependence of thermal properties should be assessed during in vivo trials, to include the effect of blood perfusion and other metabolism-related contributions on the recovered measurements.Future studies should be envisaged to further enlighten the mechanisms of heat transfer in tissue and the temperature-induced variation of the thermal properties in the biological media in the hyperthermia and ablative temperature intervals. Future works should also consider including in the experimental procedure more physiological-like conditions. An example is given by the air normally present in the"}
+{"text": "The estimation of measurement uncertainty was performed along with the assessment of regression models describing the trend of measured quantities as a function of temperature to be used in simulations involving heat transfer in kidney tissue. A direct comparison of the thermal properties of the same tissue from two different species, i.e., porcine and bovine kidney tissues, with the same experimental transient hot-wire technique, was conducted to provide indications on the possible inter-species variabilities of k and \u03b1 at different selected temperatures. Exponential fitting curves were selected to interpolate the measured values for both porcine and bovine kidney tissues, for both k and \u03b1. The results show that the k and \u03b1 values of the tissues remained rather constant from room temperature up to the onset of water evaporation, and a more marked increase was observed afterward. Indeed, at the highest investigated temperatures, i.e., 90.0\u201392.8 \u00b0C, the average k values were subject to 1.2- and 1.3-fold increases, compared to their nominal values at room temperature, in porcine and bovine kidney tissue, respectively. Moreover, at 90.0\u201392.8 \u00b0C, 1.4- and 1.2-fold increases in the average values of \u03b1, compared to baseline values, were observed for porcine and bovine kidney tissue, respectively. No statistically significant differences were found between the thermal properties of porcine and bovine kidney tissues at the same selected tissue temperatures despite their anatomical and structural differences. The provided quantitative values and best-fit regression models can be used to enhance the accuracy of the prediction capability of numerical models of thermal therapies. Furthermore, this study may provide insights into the refinement of protocols for the realization of tissue-mimicking phantoms and the choice of tissue models for bioheat transfer studies in experimental laboratories.This experimental study aimed to characterize the thermal properties of ex vivo porcine and bovine kidney tissues in steady-state heat transfer conditions in a wider thermal interval (23.2\u201392.8 \u00b0C) compared to previous investigations limited to 45 \u00b0C. Thermal properties, namely thermal conductivity ( Moreover, the thermal diffusivity (\u03b1) of a tissue quantitatively represents its capability to transfer heat in relation to storing thermal energy , k is thermal conductivity [W/(m\u00b7K)], r is the distance between the two needles (6 mm), \u03b1 is thermal diffusivity [mm2/s], t is time [s], ht is heating time [s] (30 s), and x) is the exponential integral, approximated using polynomials: in particular, for small arguments, it is approximated as the sum between the Euler\u2013Mascheroni constant (\u03b3 \u2248 0.5772) and the natural logarithm of the argument, ln(x) [The container was placed into the water thermal bath to reach the desired temperature, which was kept constant during the measurement of thermal properties. The thermal bath was set to specific temperatures for each sample, and measurements of the thermal properties were performed in steady-state heat transfer conditions, i.e., only when the sample reached thermal equilibrium. The temperature range of interest set in the thermal bath was ~22 \u00b0C to >90 \u00b0C, and was divided into 10 steps for porcine kidneys and 9 steps for bovine kidneys. The TEMPOS thermal properties analyzer equipped with an SH-3 dual-needle sensor was used to measure e method ,37,38,39e method ,57. Indee method . In the t, ln(x) ,37,59.EU) were calculated. Each experimental trial concerning the measurement of the thermal properties from room to ablative temperatures was repeated three times for each tissue type, i.e., three experimental trials from room to ablative temperatures were performed for the measurement of the thermal properties of porcine kidney. Three experimental trials from baseline to ablative temperatures were also conducted for the measurement of the thermal properties of bovine tissue.For each thermal parameter, the mean value EU is defined by s, given in Equation (4), multiplied by the coverage factor fk. This coverage factor is obtained considering a confidence level of 95% for a Student\u2019s t-distribution. Thus, having 3 measurements (2 degrees of freedom), fk = 4.30 [n is the number of experiments for each temperature (n = 3), y is the measured thermal property and The f = 4.30 .(4)s=\u2211ik and \u03b1 is represented by an exponential curve as follows:In order to obtain the trend of the studied properties as a function of the temperature, the best-fit model was obtained for each thermal parameter. Concerning the modeling of thermal properties, the best-fit model for \u00ae . Finally, the coefficient of determination  to ensure the measurement system was accurate and functioned correctly without any flaws. At room temperature, i.e., 22.3\u201326.0 \u00b0C, the results of our measurements were k = 0.379 W/(m\u2219K) and \u03b1 = 0.189 mm2/s, while the values reported on the Certificate of Quality Assurance are k = 0.384 W/(m\u2219K) and \u03b1 = 0.189 mm2/s. The difference of <1.5% between our measurement results on the standard material and the values reported by the manufacturer indicated the accurate reading capability of our system [Prior to proceeding with actual measurements of thermal properties in ex vivo biological tissues, measurements were conducted on a reference material provided by the manufacturer  are reported in The thermal properties of ex vivo porcine kidney tissue were measured in steady-state heat transfer conditions at different tissue temperatures, i.e., from nominal conditions at room temperature (23.9 \u00b0C) to approximate body temperature (35.4 \u00b0C), and up to hyperthermia and ablative temperatures . The attained results, in terms of the average values of k and \u03b1, reported in In order to visualize the trend of the thermal properties with increasing tissue temperatures, the data of k of porcine kidney tissue, its nominal value at room temperature was equal to 0.549 W/(m\u00b7K). A gradual increase from 23.9 \u00b0C to 46.2 \u00b0C, followed by a slight decline from 60.0 \u00b0C to 82.3 \u00b0C, can be observed. Finally, an exponential growth up to 92.8 \u00b0C was registered, with a value of 0.648 W/(m\u00b7K) at 92.8 \u00b0C. Comparing the value of k at the initial value and last value of the temperature interval  an overall increase of 18% was observed at 92.8 \u00b0C with respect to the baseline value measured at room temperature.Concerning the k values, which are represented by an exponential curve defined using Equation (6). The regression coefficients are reported in 2. The results of the residuals analysis are reported in \u03b1 at different temperatures, \u03b1 remained almost constant from room temperature up to 60.0 \u00b0C, showing values in the interval of 23.9\u201360 \u00b0C, ranging from a minimum of 0.155 mm2/s (observed at 23.9 \u00b0C) to a maximum of 0.163 mm2/s (at 46.2 \u00b0C). After 60.0 \u00b0C, the value \u03b1 increased with temperature, reaching values of 0.178 mm2/s at 82.3 \u00b0C, 0.194 mm2/s at 86.6 \u00b0C, and 0.216 mm2/s at 92.8 \u00b0C, corresponding to increases, compared to the baseline values, of 14%, 25%, and 39%, respectively. \u03b1 at the different temperatures along with the best-fit curve interpolating the experimental measurements. The trend of \u03b1 as a function of tissue temperature was modeled using an exponential curve (Equation (6)), whose regression coefficients are reported in Concerning the measurement of k and \u03b1 of ex vivo bovine tissues as a function of temperature from 23.2 \u00b0C up to 90.0 \u00b0C. The same experimental setup employed for the evaluation of the temperature dependence of the thermal properties of porcine kidney tissue was utilized for the measurement of k of bovine kidney tissue, attained using the dual-needle technique. As is observed, the value of k exponentially increased when temperatures of 90 \u00b0C were reached. At nominal conditions, i.e., room temperature, k was equal to 0.528 W/(m\u00b7K); at body temperature , the average value of k was 0.551 W/(m\u00b7K). At the maximum investigated temperature value, i.e., 90.0 \u00b0C, k reached a value of 0.703 W/(m\u00b7K), showing increases of 33% and 28% compared to the baseline values at room and body temperatures, respectively. In k is also shown. The exponential model (Equation (6)), similar to what was observed for the k of porcine kidney tissue, can be considered an accurate approximation of measured thermal property values at the different temperatures. 2R, which is equal to 0.805. The analysis of the residuals over temperature  was registered at 56.4 \u00b0C. Moreover, an increment in the tissue temperature up to 90.0 \u00b0C led to an increase in \u03b1 of 21% compared to the nominal value at ambient temperature. The exponential model, for which the regression coefficients are shown in \u03b1 values . Concerning the characterization of the \u03b1 values a and desk attained at temperatures of 23.2\u201323.9 \u00b0C, 35.4\u201336.9 \u00b0C, 40.9\u201341.5 \u00b0C, 46.2\u201348.8 \u00b0C, 56.4\u201356.7 \u00b0C, 69.5\u201370.1 \u00b0C, 75.8\u201376.4 \u00b0C, and 90.0\u201392.8 \u00b0C for both porcine and bovine samples, along with the associated measurement uncertainty (estimated with a 95% confidence level). It is possible to notice that, for instance, at room temperature, i.e., 23.2\u201323.9 \u00b0C, the difference between the average values of k associated with porcine kidney tissue and those related to bovine tissue was 4%. Likewise, at the highest investigated temperature (90.0\u201392.8 \u00b0C), the difference in terms of the average values k between the porcine and bovine tissue was <9%. Overall, at the same tissue temperature, the results of the k of porcine tissue were not statistically significantly different from the values of k measured for the bovine kidney . This was observed for all the investigated temperatures. k data for both porcine and bovine kidney samples. For the sake of comparison between the values of \u03b1 measured for porcine and bovine tissues, the bar plots showing the average values attained at different increasing temperatures are presented in k, considering the same tissue temperature, no statistically significant difference was found between the values of \u03b1 measured for porcine tissue and the values recovered for bovine tissue . The exponential curves which best fit the measured values of \u03b1 are shown in In The objective of the present experimental study was threefold. We devised the characterization of the thermal properties of ex vivo porcine and bovine kidney tissues from room up to supraphysiological  temperatures in order to provide quantitative values of these properties in a wider thermal interval, compared to previous investigations that were limited to 45 \u00b0C . Furtherk and \u03b1, of biological media was performed in a temperature-controlled environment by means of a dual-needle probe, capable of measuring tissue temperature and delivering a specific thermal energy to the sample. The probe was connected to a commercial thermal property analyzer designed in accordance with ISO 2008 standards and in compliance with ASTM 5334 and IEEE 442 [The measurement of the thermal properties, i.e., IEEE 442 . The samIEEE 442 ,40,62. Mk, our measurements are in agreement with values registered at room, body, and hyperthermic temperatures for ex vivo porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex  using self-heating thermistors [k for different kidney tissues investigated in the present study and other studies that used various tissue temperatures. At room temperature, our measurements resulted in average values of 0.549 W/(m\u00b7K) for porcine kidney tissue at 23.9 \u00b0C and 0.528 W/(m\u00b7K) for bovine tissue at 23.2 \u00b0C, whilst the values measured by Valvano et al. (at 23 \u00b0C) were 0.524, 0.525, 0.524, 0.525, and 0.529 W/(m\u00b7K) for ex vivo porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex, respectively [k were 0.559 W/(m\u00b7K)  and 0.551 W/(m\u00b7K) , showing a maximum percentage difference of lower than 4% compared to the values reported at 37 \u00b0C by Valvano et al. in porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex. Concerning higher temperatures, i.e., close to 45 \u00b0C, we attained average values of k = 0.573 W/(m\u00b7K)  and k = 0.541 W/(m\u00b7K) . Hence, in this case, the maximum difference between the values reported by Valvano et al. (45 \u00b0C) [k remained rather constant until the onset of water vaporization, i.e., close to 90 \u00b0C [k values of porcine and bovine tissues, the best-fit regression models interpolating experimental data were represented by exponential curves. Concerning porcine kidney tissue, at 92.8 \u00b0C, a 1.2-fold increase in the average value of k compared to its baseline value at room temperature was observed. Similarly, for bovine kidney tissue, at 90.0 \u00b0C, the average value of k was 1.3 times higher with respect to its value at nominal conditions. These results are in line with the experimental observations reported for ovine kidney tissues by Silva et al. [k measured for the porcine kidney and the values attained for bovine kidney tissue.For values of rmistors . Table 5ectively . Thus, aa et al. . Indeed,\u03b1 of porcine and bovine kidney tissues, as a function of temperature , we noticed an exponential trend with increasing temperature, as also observed for the values of k. The obtained average values at room temperature were 0.155 mm2/s (at 23.9 \u00b0C) and 0.151 mm2/s (at 23.2 \u00b0C) for porcine and bovine tissues, accordingly. At room temperature, a maximum percentage difference of 14% was observed in comparison with the values measured by Valvano et al. (at 23 \u00b0C) in porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex using self-heating thermistor probes [\u03b1 obtained in our investigation along with the results of other experimental trials performed on ex vivo ovine kidney, porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex [\u03b1 attained in this study are slightly higher compared with the average values measured by Valvano et al. at room temperature, body temperature, and at 45 \u00b0C in porcine renal cortex, rabbit kidney, human renal pelvis, human renal medulla, and human renal cortex [\u03b1 values for both porcine and bovine kidney tissues. At the highest investigated temperatures, i.e., 90.0\u201392.8 \u00b0C, the average \u03b1 values were subject to a 1.4- and 1.2-fold increase for porcine and bovine kidney tissues, correspondingly. Also in this case, the results are in accordance with the measurements reported in ovine kidney tissue [\u03b1 at 93 \u00b0C was shown compared to its nominal value at room temperature , no statistically significant differences between porcine and bovine kidney tissue were reported, despite the anatomical and structural differences between the two tissue types. This observation may be insightful for the proper selection of the tissue models for the optimization of different procedures involving heat transfer in kidney tissue. Indeed, porcine and bovine kidney tissues have been advocated as experimental models for training and medical research, also involving the optimization of thermal therapies [Concerning the comparison of the herapies . Howeverk and \u03b1 of human tissue as a function of temperature, considering both diseased and healthy kidney tissues for a thorough characterization of their thermal features.In conclusion, this study presents the characterization of the thermal properties of porcine and bovine kidney tissues as a function of temperature, ranging from room up to supraphysiological thermal ranges. The provided quantitative values, as well as the best-fit regression models describing the trends of thermal properties, can be used as inputs for numerical models of thermal therapies. Indeed, mathematical equations concerning the trends of the thermal properties of kidney tissue as a function of temperature can be easily incorporated into in silico models for a more reliable description of the thermal behavior of biological tissue. Accurate information on the thermal behavior of kidneys is required to enhance the prediction capability of computational and mathematical tools toward more accurate therapy pre-planning. Moreover, the measured properties may be used for the realization of tissue-like materials for testing, validation, and the improvement of preclinical devices. Thus, the measured properties may be useful for the design of new investigations aiming to improve thermal-energy-based therapies such as catheter-based renal denervation and thermal ablation procedures for unhealthy tissue treatment. Furthermore, the inter-species variabilities analysis of the thermal properties of porcine and bovine tissue may provide insights into the refinement of protocols and choice of tissue models for bioheat transfer studies in experimental laboratories. Additionally, species-dependent specific data on the thermal properties of tissues can help realize more accurate simulation models with respect to the type of tissues chosen for experimental validation. The principal limitation of the present study is the use of ex vivo healthy animal tissue models. Thus, future in vivo studies should be conducted considering the influence of physiological phenomena such as metabolic heat and perfusion on the variance of thermal properties with temperature. In addition, in the present study, measurements were performed with the aim of preserving the structure of the kidneys and penetrating both the cortex and medulla with a dual-needle probe. Hence, the measurement of the thermal properties refers to the thermal characteristics of both these tissue structures, considered as a whole. It may be of interest, also considering possible implications for improving thermal ablation treatments, to evaluate, in future studies, possible variations in thermal properties for the different regions of kidney tissue. Furthermore, additional investigations should also be devised for the measurement of the"}
+{"text": "Small extracellular vesicles (sEVs) are an important intercellular communicator, participating in all stages of cancer metastasis, immunity, and therapeutic resistance. Therefore, protein cargoes within sEVs are considered as a superior source for breast cancer (BC) biomarker discovery. Our study aimed to optimise the approach for sEV isolation and sEV proteomic analysis to identify potential sEV protein biomarkers for BC diagnosis. sEVs derived from BC cell lines, BC patients\u2019 plasma, and non-cancer controls were isolated using ultracentrifugation (UC), a Total Exosome Isolation kit (TEI), and a combined approach named UCT. In BC cell lines, the UC isolates showed a higher sEV purity and marker expression, as well as a higher number of sEV proteins. In BC plasma samples, the UCT isolates showed the highest proportion of sEV-related proteins and the lowest percentage of lipoprotein-related proteins. Our data suggest that the assessment of both the quantity and quality of sEV isolation methods is important in selecting the optimal approach for the specific sEV research purpose, depending on the sample types and downstream analysis. According to cancer statistics, breast cancer (BC) accounts for 31% of female cancers in the United States in 2023, and it ranks among the top causes of mortality in women, with an estimated 43,170 deaths [Extracellular vesicles (EVs) are a heterogenous population found in all biofluids, including small EVs (sEVs) ranging from 50 to 150 nm; 100\u20131000 nm large EVs (lEVs); and the largest vesicle, known as apoptotic bodies, which range from 500 to 5000 nm . Among vThe ideal standardised isolation method for sEV research should have a simple and fast process; high throughputs, purities, and recovery rates; and low cost. Ultracentrifugation (UC) is the standard method for sEV isolation. However, UC is associated with several drawbacks, including long processing times, low throughputs, and variable purity levels, and it may lead to sEV aggregation, which can affect the reliability and reproducibility of the downstream analysis . PolymerAccording to a recent study, the combination of two or more isolation techniques may be an effective approach to overcome the limitations of individual methods, depending on the sample types . A hybriIn this study, we utilised a combined approach of a half-cycle of UC and Total Exosome Isolation kit (TEI) isolation methods, referred to as UCT. Our hypothesis is that UCT could potentially achieve a greater yield of sEVs with improved purity, resulting in better outcomes in BC protein biomarker discovery through proteomic analysis. We compared three different isolation methods, including UC, TEI, and a combination approach (UCT), to assess each method based on various parameters, including the sEV yield, purity, morphology, size, protein contamination, and proteomic statistical analysis. To the best of our knowledge, this study is the first to compare three isolation methods for determining the optimal technique in sEV proteomic analysis. The evaluation includes a comparison of these methods using both cell-conditioned medium and plasma samples.First, to examine the size distribution and particle concentration, NTA was performed on isolates from 60 mL of cell medium supernatants of three different MDA-MB-231 batches obtained through three different methods, including TEI, UC, and UCT. The size distribution of the TEI-derived isolates exhibited the broadest range, from approximately 45 to 535 nm. The UCT isolates had a size range from 55 to 385 nm, while the UC isolates ranged from 45 to 335 nm. Among the three methods, the UCT isolates demonstrated the smallest mean mode size, measuring 140 nm, which was significantly smaller than the mode size of the TEI isolates, at 197 nm A,B. The To investigate the expression of the sEVs obtained through the TEI, UC, and UCT methods, sEVs were captured in an equal number of particles, as determined by NTA using mixed CD9 and CD81 beads. The captured sEVs were then assessed using flow cytometry, employing a hybridised fluorescence detection of common sEV markers CD9, CD63, and CD81. Notably, the signal intensities of CD9, CD63, and CD81 in the UCT isolates were higher compared to the signal intensities of those in the other isolates, suggesting that UCT yielded the highest amount of sEVs in an equivalent number of particles F. ConverThe presence of sEVs in each isolate was verified through TEM analysis, which also included a morphological structure examination . The sizTo determine the optimal isolation method for sEV protein research, the label-free quantification was analysed for the total proteome in sEVs isolated using TEI, UC, and UCT. PCA  score plot data were used to visualise consistent spectral patterns in each MD-MBA-231 cell-medium supernatant A. The thTo identify, based on MS data, the number of sEV-related proteins in the isolates from the different isolation methods, a Venn diagram was constructed. The UC isolates obtained the highest number of sEV proteins, followed by the UCT isolates B. SpecifThe sEV isolate proteins identified from the supernatant of the MDA-MB-231 cell medium were annotated using GO analysis using DAVID. Overall, in terms of analysing the particle number in the MDA-MD-231 cell-derived sEV isolates, the UCT method demonstrated the highest purity and the presence of smaller particles. However, when specifically examining sEVs, the UC method exhibited higher sEV marker expression and purity. Moving onto the analysis of the entire proteome within the isolates, consistent patterns were observed across the replicates for the TEI and UCT isolates. In contrast, the UC isolates displayed variable patterns in terms of identified proteins across the different batches. When comparing the sEV proteins, the UC isolates showed a significantly higher number of sEV proteins compared to the other proteins, with minimal lipoprotein contamination.These findings emphasise the importance of employing diverse characterisation methods to assess the quality and quantity of sEVs as well as the significance of selecting an appropriate isolation method, considering the advantages and disadvantages of each method, based on the downstream analysis requirements. Given that the aim of our study is to identify potential sEV protein biomarkers in BC, we have chosen to use the UC isolation method for the following experiments with cell lines.The UC method was used to isolate sEVs from three BC cell lines, namely MDA-MB-231, MCF-7, and SK-BR-3, as well as a normal breast cell line, MCF-10A, to identify potential sEV biomarkers by LC-MS/MS proteomics. Through the analysis, we identified 1463 significantly upregulated and 118 downregulated proteins in the MDA-MB-231 cell-line-derived sEVs, followed by 711 upregulated and 122 downregulated proteins in the MCF-7 cell-line-derived sEVs, and 295 upregulated and 192 downregulated proteins in the SK-BR-3 cell-line-derived sEVs D,E. ThesAdditionally, we observed the involvement of the Notch signalling pathway, a known cancer-related function, as well as the complement and coagulation cascades, which are associated with the early immune response. It is important to note that while 158 sEV proteins exhibited significantly different expression levels in BC cell lines, some proteins did not appear in the STRING analysis owing to the absence of protein\u2013protein interactions or significant annotations. In addition, 10 sEV proteins were identified, showing the most significantly different expression in the BC cell lines compared to the normal breast cell line . DownregThe size distributions of the sEV particles derived from 300 \u00b5L of three BC patients\u2019 plasma and three non-cancer individuals\u2019 control plasma were analysed using NTA. Consistent with the findings from the cell-line experiments, the size distribution of the TEI isolates exhibited greater variability and a broader range compared to those of the other isolates A. The TEThe expressions of CD9, CD63, and CD81 in human plasma samples were detected using flow cytometry. The expression level in the UC isolates was higher compared to that in the TEI isolates. Although the expression level in the UCT isolates was slightly higher than that in the TEI isolates, the difference was not statistically significant F. The raIn the TEM images, it was evident that all the isolates obtained from the three different methods exhibited distinct cup-shaped sEVs ranging in size from 100 nm to 200 nm . Notablyn = 3) through proteomic analysis. Following protein isolation from each of the three different isolation methods, which included TEI, UC, and UCT, PCA was conducted on the identified proteins from each isolation method, which demonstrated that the proteins from each method clustered together   A,B. Usine \u00b1 1.5) . In the e \u00b1 1.5) . Our STRe \u00b1 1.5) . It is i+ BC and guide the appropriate treatment (NCT04288141). Another clinical trial aims to validate the glycosylation of sEVs as a diagnostic biomarker for early BC diagnosis (NCT05417048). Two other studies focus on proteomic profiling within BC plasma-derived sEVs to identify a diagnostic biomarker for BC (NCT05798338) and a prognostic biomarker for neoadjuvant chemotherapy in BC treatment (NCT05831397). However, the lack of standardised methods for isolating sEVs has led to significant variability in both the quality and quantity of isolated sEVs, depending on the isolation methods and sample type. Consequently, the choice of the isolation method has a profound impact on the downstream analysis, leading to divergent outcomes. In this study, we provided a comprehensive qualitative and quantitative analysis comparing the isolated sEVs obtained through the TEI, UC, and UCT isolation methods. Our findings offer valuable insights and guidance for selecting the most suitable isolation method, particularly for proteomic analysis purposes.sEVs are stable and abundant in various body fluids and are considered as a potential biomarker source for cancer diagnosis and monitoring cancer progression in real time. Currently, blood-derived sEV proteins hold promise as a prospective reservoir of novel biomarkers. Given that a blood sample is a complex mixture of various components and that blood-derived sEVs contain highly heterogenous information across patients, it is imperative to initially conduct a pilot study. There are a few ongoing clinical trials for sEV-based BC biomarker research. One study measures the expression of the HER2-HER3 dimer in plasma-derived sEVs to diagnose HERIn the conditioned medium of MBA-MB-231 cells, the UCT isolates demonstrated the most consistent particle isolation within the sEV size range. Consequently, the mode size of the particles obtained through UCT isolation was smaller than those of the particles obtained through the other methods, and the particle concentration was improved compared to the concentration of particles obtained from UC isolation alone. In terms of the particle number and protein contamination ratio, the UCT isolates exhibited higher particle numbers and similar protein contamination levels compared to the UC isolates, indicating the purest isolates in terms of the particle number and protein contamination ratio. However, it is important to note that these results do not directly indicate the purity and concentration of sEVs. The particle number per millilitre of the MDA-MB-231 cell-derived sEV isolates corresponded to the observations from the TEM images. The UCT isolates had a higher number of non-sEV vesicles compared to the UC isolates. In the flow cytometry analysis, the strongest expressions for CD9, CD63, and CD81 were detected in the UCT isolates, while western blotting showed that the set of sEV markers was the strongest in the UC isolates. During the flow cytometry process, the exclusive capture of sEVs by CD9- and CD81-conjugated beads could be the underlying reason for the potential loss of sEVs in the UC isolates that bear sEV surface markers different from those in CD9 and CD81. As a result, this loss of sEVs in UC isolates in the flow cytometry results may account for discrepancies in the overall expression of sEV markers observed in the western blotting results. It is important to emphasise that the UC isolates contained the highest number of sEV proteins compared to the other two isolates, even though the percentage of genes related to sEVs was the lowest. This is likely due to UC isolates containing the highest number of total proteins, which may also be related to the wider UC cluster observed in the principal component analysis. The proteins identified in the UC isolates exhibited more variability compared to those identified in the other isolates, resulting in a higher number of identified proteins.No research has been conducted thus far comparing the proteomes of sEVs isolated through TEI, UC, and UCT across various BC cell lines. To the best of our knowledge, there is only one paper available in the literature that examined the difference between UC and TEI specifically in MDA-MB-231 cells, utilising western blotting to visualise the expression of sEV markers . InteresThe overlapped potential sEV biomarkers across the BC cell lines were associated with several functions shown in the STRING network. Generally, cancer cells release a higher number of sEVs into the TME for intercellular communication, which transports various biological molecules, such as proteins and nucleic acids . In the In BC cell lines, a total of 152 sEV proteins were found to be upregulated, while 16 sEV proteins were downregulated. Upon analysis, we successfully identified the top 10 sEV proteins that displayed the most significant differences in their expression levels. The significantly downregulated sEV proteins in BC cell lines were BDH2, insulin (INS), LAMA3, and TPX2. INS is a well-known factor related to high-risk BC patients who have obesity ,40. TissIn the human plasma samples, the characterisation of sEV particles yielded diverse results compared to those of the BC cell lines, likely due to the complexity of human samples. Notably, the TEI and UCT isolates exhibited various peaks in the size distribution. The UCT isolate had the largest portion of total particles within the largest size range. Paradoxically, the TEI isolates, which contained the highest abundance of protein contamination, exhibited the smallest particle size in the highest peak. The protein contamination in the UCT isolates from the plasma samples was higher than that in the UC isolates, while the BC cell results showed similar amounts between UC and UCT. Owing to the variability in the values across the plasma samples, the ratio of the particle number to the protein amount did not show any significant differences among the isolation methods. In contrast to the isolates from the BC cells, the UC isolates from the human plasma demonstrated noticeably higher purity than those of the isolates obtained using the other methods. Western blotting revealed weak signals for all the sEV marker proteins in the plasma isolates. This could be attributed to the higher protein contamination observed in the plasma isolates, as shown in the TEM results and GO analysis, making it challenging to detect specific sEV marker proteins in only 20 \u00b5g of proteins.The proteomic analysis in this study provides additional evidence of distinct proteomic profiles in sEVs, depending on the sample type and isolation methods ,19,47. TIn a previous study, there was no report for validating UCT methods for isolating sEVs from human plasma samples. Only one paper demonstrated that UC isolates in human serum detected using western blotting showed more intense sEV marker expression compared to that of UCT isolates . The innThe STING network analysis demonstrated that sEV biomarker candidates isolated using UC from BC plasma were associated with the clotting cascade, while those isolated using UCT were associated with the complement and coagulation cascades. The complement and coagulation cascades are involved in innate immune responses, which are a more plausible function for BC biomarkers. Interestingly, the potential biomarker cluster identified in both UC and UCT isolates had functions related to lipoprotein particles. One study suggested that the direct interaction between lipoproteins and EVs is an important factor in transferring biological information and EV uptake pathways . MoreoveIn BC plasma, the expressions of the sEV ALB, COL1A1, CSN1S1, EEF1A2, and RPL3 were significantly upregulated, while the expressions of C1S, C5, C7, and CFHR2 were downregulated, and these dysregulated proteins are considered as biomarker candidates for BC diagnosis. Although ALB has normally been known as contamination within sEV isolates, like lipoprotein, in sEV research , sEV ALBThe sEV proteins that have been identified in BC cell lines and in human BC plasma hold promise as markers in BC diagnosis, including APOA4, C3, C7, CLU, F2, and SERPINC1. These proteins are related to innate immune responses and the regulation of the insulin-like growth factor (IGF) transport and uptake by IGF-binding proteins. In a previous study, sEVs derived from BC cells were found to stimulate IGF-1 to induce transcription factors related to EMT in neighbouring cells , suggestRecently, many advanced isolation methods have been developed. One of the techniques is asymmetrical-flow field-flow fractionation (AF4) . The com\u00aeCRL-1435TM) was maintained in IMDM medium  supplemented with 10% (v/v) foetal bovine serum (FBS) and 1% (v/v) antibiotics (50 U/mL of penicillin and 50 \u00b5g/mL of streptomycin). The SK-BR-3 HER+ BC cell line  was cultured in McCoy\u2019s 5a medium  supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. The MCF-7 ER/PR+ BC cell line  was cultured in RPMI-1640 medium  supplemented with 10% (v/v) FBS and 1% (v/v) antibiotics. The MCF-10A human mammary epithelial cell line (ATCC\u00aeHTB-81TM) was cultured using MEGM\u2122 BULLETKIT\u2122 . All the cell lines that were used were negative for mycoplasma testing and were authenticated within the last three years through Short Tandem Repeat profiling by employing the PowerPlexR 18D System .The MDA-MB-231 triple-negative breast cancer (TNBC) cell line  at 37 \u00b0C for 2 min. Two volumes of complete growth media were added, and the suspension was then transferred to a 15 mL Falcon tube and centrifuged at 1000 rpm for 5 min at room temperature (RT). The cells were resuspended in complete growth media for passaging.All the cell lines were cultured in a cell incubator  with 5% COv/v) exosome-depleted FBS  and 1% (v/v) antibiotics (50 U/mL of penicillin and 50 \u00b5g/mL of streptomycin).Exosome-depleted cell media were appropriate cell media mixed with 10% .The BC cell lines were cultured until they reached a confluency of 60\u201370%. The cells were gently washed with DPBS twice and incubated with the exosome-depleted medium for 48 h (H). The medium was collected after 48 h to isolate the sEVs. The supernatant was collected and pre-purified by centrifugation to remove any cell debris. The supernatant was first centrifuged at 300\u00d7 n = 3) and 2 mL plasma samples from non-cancer controls (n = 3). Human ethics approval was obtained from UNSW Australia Human Research Ethics Advisory Panels (2022/HC220456) for using human plasma samples to conduct the EV analysis. The plasma samples were first centrifuged at 2000\u00d7 g for 20 min at 4 \u00b0C followed by centrifugation at 10,000\u00d7 g for 20 min at 4 \u00b0C. The final supernatant was filtered through a 0.22 \u03bcm filter and stored at \u221280 \u00b0C for downstream experiments.All the plasma samples were obtained from the Health Precincts Biobank, which is a part of UNSW Biospecimen Services. A total of six individual plasma samples were used in this study, including 3 mL plasma samples from BC patients , 4478359 for the cell culture media and 4484450 for the plasma), UC, and a combination of UC and TEI (UCT) were used according to the manufacturers\u2019 instructions.g for 1 H from 2 \u00b0C to 8 \u00b0C. The resulting pellet was collected and resuspended in filtered PBS. Similarly, for the pre-purified plasma, the sample was mixed with a 0.5 volume of filtered PBS and a 0.2 volume of the reagent and incubated at RT for 10 min. After incubation, the sample was centrifuged at 10,000\u00d7 g for 5 min at RT, and the pellet was collected in filtered PBS.TEI: The pre-purified cell supernatant was mixed with a 0.5 volume of the reagent and incubated overnight (o/n) from 2 \u00b0C to 8 \u00b0C. Following incubation, the sample was centrifuged at 10,000\u00d7 g for 70 min twice, and the pellet was collected in PBS. The pre-purified plasma was ultracentrifuged at 120,000\u00d7 g for 2 h twice before the pellet was collected in filtered PBS.UC: UC was performed using a Beckman Optima XPN-100 and an L-100 XP  equipped with Type 70 Ti and SW 55 Ti rotors. The pre-purified cell supernatant was ultracentrifuged at 100,000\u00d7 g for 70 min using a Beckman Optima XPN-100, and the pellet was resuspended in 500 \u03bcL of filtered PBS. The redissolved pellet was mixed with a 0.5 volume of the TEI reagent and incubated o/n from 2 \u00b0C to 8 \u00b0C. The sample was centrifuged at 10,000\u00d7 g for 1 h from 2 \u00b0C to 8 \u00b0C. For the plasma, samples were ultracentrifuged once at 120,000\u00d7 g for 2 h using a Beckman Optima L-100 XP. The pellet was redissolved in 200 \u03bcL of filtered PBS, mixed with a 0.2 volume of the TEI reagent, and incubated for 10 min at RT. The sEVs were obtained as a pellet after centrifugation at 10,000\u00d7 g for 5 min. For the sEV characterisation, the pellet was resuspended in filtered PBS.UCT combination method: The pre-purified cell supernatant was ultracentrifuged once at 100,000\u00d7 A Qubit Protein Assay  was used to quantify the protein concentration following the manufacturer\u2019s instructions. In brief, 10 \u03bcL of the isolates were combined with 190 \u03bcL of the Qubit working solution to yield a total volume of 200 \u03bcL. The mixture was then incubated at RT for 15 min, and the resulting solution was read using a Qubit fluorometer.To quantify the protein content in the sEV isolates, a Pierce BCA protein assay kit  was employed following the manufacturer\u2019s instructions. To create a standard curve spanning a range of 0\u2013500 \u00b5g/mL, nine points of serial dilution with BSA were prepared. The sample was diluted 1:10 in distilled water to yield a total volume of 150 \u03bcL, which was then mixed with 150 \u03bcL of the BCA working solution. All the samples and standard points were in replicates and incubated at 37 \u00b0C for 2 h. The absorbance of each sample was measured using a Synergy HT microplate reader  at a wavelength of 562 nm, and the obtained absorbance values were converted to micrograms per millilitre using the standard curve. The final concentration was multiplied by the dilution factor to obtain the actual protein content in the sEV isolates.Ten or twenty micrograms of protein were separated on a 4\u201312% NuPAGE Bis-Tris gel  and blotted onto 0.45 \u03bcm polyvinylidene fluoride membranes . The membranes were incubated with 5% BSA for 1 H at RT for blocking. After blocking, the membranes were incubated with primary antibodies at 4 \u00b0C o/n and washed three times with TBST. The washed membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 H at RT and washed three times. The membranes were then imaged using an enhanced chemiluminescence (ECL) substrate  and ImageQuant LAS 4000 system . Anti-rabbit  and anti-mouse  secondary antibodies were used. Calnexin , CD63 , CD9 , CD81 , Flotillin-1 , HSP70 , Syntenin , and TSG101  were purchased from Abcam. All the antibodies were diluted at a ratio of 1:2000 except for CD81, which was diluted at 1:500.NTA was performed using a NanoSight NS300 system  equipped with a 532 nm wavelength green laser and NTA software . The isolated EV samples from the cell supernatant were diluted with freshly filtered PBS (0.22 \u03bcm) and loaded into the detection chamber by a 1 mL syringe. The same settings were applied to all the samples: camera level: 9, detection threshold: 7, capture: 60 s, number of captured images: 5, and temperature: 25 \u00b0C. The original particle concentrations were calculated based on the measured concentrations and the dilution factor.TEM was performed using a JEOL 1400 with a magnification scale ranging from 100 to 200 nm and a voltage of 100 kV. A two-hundred mesh carbon-coated copper grid  was made hydrophilic by a glow discharge. Seven microlitres (\u03bcL) of each sample were absorbed in a grid for 10 min at RT, followed by negative staining with filtered 2% aqueous uranyl acetate.7/mL, Thermo Fisher Scientific, 10620D) and anti-CD81 magnetic beads  in 200 \u03bcL of PBS with 2% BSA o/n at 4 \u00b0C. The EV-captured beads were washed twice with PBS and 2% BSA and separated during the washing step using a MagJET separation rack (Thermo Fisher Scientific). The EVs captured by the magnetic beads were stained with the following antibodies: a mixture of APC-conjugated anti-CD9 ; anti-CD81 ; and anti-CD63  for hybridised EV detection. The isotype control was APC-conjugated anti-mouse IgG1  and was incubated at 4 \u00b0C in the dark for 30 min. The beads were washed twice, and 500 \u03bcL of PBS was added. The sample was analysed using BD LSR Fortessa X-20 flow cytometry . The data were processed using FlowJo software (Version 10). We used the ratio between the geometric means of the fluorescence intensity (gMFI) from each marker and the IgG isotype control to quantify each marker level.Immunomagnetic bead capturing was applied for accurate EV quantification in flow cytometry. The same number of particles in the EV samples measured by NTA were incubated with the following antibody-conjugated magnetic beads: 10 \u03bcL of anti-CD9 magnetic beads  enzyme-to-protein ratio for o/n digestion. The digested peptides were purified using Pierce detergent removal spin columns, 125 \u03bcL, 25 columns  and C18 tips  and extracted in 80% ACN and 0.1% TFA.The digestion was performed with RIPA buffer  containing a proteinase and phosphatase inhibitor cocktail (100\u00d7) . The proteins were precipitated using acetone precipitation. The proteins were reduced with 20 mM dithiothreitol and 20 mM ammonium bicarbonate at 37 \u00b0C for 30 min and alkylated with 40 mM iodoacetamide solution for 15 min at RT. Trypsin  was added to a final 1:100  in line with a fritless nano-column (75 \u03bcm \u00d7 20 cm) containing reverse-phase C18 media . The samples were eluted using a 120 min linear gradient of H2O:CH3CN  to H2O:CH3CN  at a flow rate of 200 \u03bcL/min. All the MS/MS spectra were obtained in data-dependent acquisition (DDA) mode from m/z 350 to 1750 at a resolution of 70,000 at m/z 200 and an accumulation target value of 106 ions. Up to ten of the most abundant ions  with charge states of \u2265+2 and \u2264+6 were fragmented by high-energy collision dissociation.The digested peptides were solubilised in 10 \u00b5L of 0.1% formic acid and analysed by LC-MSMS using a Q Exactive Plus mass spectrometer (Thermo Scientific). The samples were loaded onto a micro-C18 precolumn  with HHomo sapiens database . The peptides in the raw data file were merged and identified with SEQUEST HT. The fixed modification was cysteine carboamidomethylation, and the variable modification was the oxidation of methionine. The digestion enzyme was trypsin with two missed tryptic cleavages. A 10 ppm peptide mass tolerance and a 0.6 Da fragment ion mass tolerance were used. The minimum peptide and maximum peptide lengths were 6 and 144, respectively. The false discovery rate (FDR) was less than 1%, as calculated using the Percolator algorithm.For the total proteomic analysis of the raw data, the data were analysed using Proteome Discoverer 2.4.1.15 with ion-based label-free quantification and compared against a p value was considered as statistically significant. The statistically significant p values were indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.One-way ANOVA with multiple comparisons was used for the statistical analysis. The data were plotted as means \u00b1 standard deviations (SDs) and generated by GraphPad Prism . The https://david.ncifcrf.gov/ (accessed on 20 April 2023) was used. The sEV-related proteins were identified using Funrich 3.1.4 [Homo sapiens. Venn diagrams were drawn using Funrich 3.1.4 and molbiotools (https://molbiotools.com/ (accessed on 2 May 2023). The biological role and intercellular interaction of the upregulated and downregulated sEV proteins in the BC cell lines and plasma were analysed using STRING (version 11.5) [The principal component analysis (PCA) was performed using the \u201cstatistical analysis\u201d tool of Metaboanalyst . For thech 3.1.4  with theon 11.5)  of Cytoson 11.5)  with theon 11.5) . The intThe two most frequently used sEV isolation methods, UC and TEI, are highly impacted by limitations, such as lower purities and sEV concentrations. In addition, there is currently no standardised method for sEV isolation specifically for proteomic analysis. In our current study, we thoroughly evaluated the combined approach compared to the sole UC and TEI methods, taking into account both the quantity and quality of the isolated sEVs, with a focus on suitability for proteomic investigations. Our findings provide valuable guidance for selecting the suitable approach among three isolation methods depending on the specific research objectives, sample types, and downstream analysis requirements. Regarding the exploration of sEV biomarkers for BC in clinical settings, our study has uncovered a solution to these problems through the implementation of a combined approach, which involves a half-cycle of UC followed by the TEI kit. This combination not only enhances the concentration of sEVs and efficiency of the sEV isolation process compared to traditional techniques but also identifies increased numbers of potential sEV protein biomarkers in BC plasma samples. These significant findings collectively demonstrate that the integration of UC and TEI isolation techniques significantly enhances the proteomic analysis of sEV-derived proteins in the context of plasma research. Although there have been many advanced isolation techniques, UCT can be easily implemented without the need for specialised equipment. To conclude, our study provides compelling evidence that the combination of UC and TEI isolation techniques is a promising method for isolating sEVs from human plasma samples and studying their potential protein biomarkers in BC. Moreover, in contrast to previous studies that demonstrated UCT\u2019s efficiency using a straightforward approach, this paper stands alone in its comprehensive exploration of UCT isolation efficiency across multiple criteria, including size, morphology, concentration, and purity, specifically for proteomic analysis in human samples. Future studies are warranted to further optimise this method and validate its effectiveness in a large set of clinical cohorts."}
+{"text": "Furthermore, higher corrosion resistance is also achieved by the Ni/binary-SiC coatings.In order to increase the hardness, wear resistance and corrosion resistance of nickel-based coatings, pure nickel is often co-electrodeposited with silicon carbide (SiC) particles. However, SiC particles tend to agglomerate and precipitate in the bath, which reduces the amounts of nanoparticles and causes nonuniformity. Herein, we solve these problems by using binary non-ionic surfactants (Span 80 and Tween 60) to effectively disperse SiC particles (binary-SiC) in the bath, which suppresses nanoparticles agglomeration and leads to uniformly distributed SiC particles in the composite coatings. In comparison to the Ni/SiC coatings electrodeposited from the commonly used SDS-modified SiC, the coatings prepared with binary-SiC (Ni/binary-SiC) show finer crystallization and a smoother surface. In addition, the Ni/binary-SiC coatings exhibit higher hardness (556 Hv) and wear resistance (2.95 mg cm Electroplated nickel is an extensively implemented industrial protective coating that protects various components from corrosion and wear. However, due to its low strength and hardness, pure nickel cannot effectively protect parts. Silicon carbide (SiC) is a potential functional material with high hardness, high thermal conductivity and high stability, and has been widely used in optical devices, nanotechnology and nuclear material science ,2,3. To To solve the above problems, surfactants are usually added to the bath to change the surface hydrophobicity of particles . For exaBy far, many typical surfactants have been used to disperse nanoparticles, including SDS , hexadec\u22122). Furthermore, higher corrosion resistance is also achieved by the Ni/binary-SiC coatings.Herein, we made an attempt to use binary non-ionic surfactants (Span 80 and Tween 60) to disperse SiC particles in a Ni bath, and the Ni/SiC composite coatings with uniformly distributed SiC particles were electrodeposited. In comparison to the Ni/SiC coatings electrodeposited from the commonly used SDS-modified SiC, the coatings prepared with binary-SiC (Ni/binary-SiC) show finer crystallization and a smoother surface. In addition, the Ni/binary-SiC coatings exhibit higher hardness (556 Hv) and wear resistance  and binary surfactants (binary-SiC) could be detected, which could be indexed to the -CH2- and -CH3 groups [\u22121 corresponds to the sulfate group, which illustrates that SiC particles are successfully modified by SDS. In binary-SiC, the absorption peak at 1100 cm\u22121 is related to the unique functional groups of C-O-C of Tween 60. Meanwhile, the significant transmittance differences in the range of 400\u2013800 cm\u22121 in binary-SiC may be attributed to the absorption of cis C=C groups (665\u2013770 cm\u22121) for Span 80, which illustrates the adsorption of Span 80 on the SiC particle surface [In order to evaluate the absorption of surfactants on SiC particles, Fourier-transform infrared (FTIR) spectra were collected. As shown in i-C bond . Two abs3 groups . In SDS- surface .\u2212 and H+ ions in an aqueous solution, which results in a negative zeta potential for blank SiC [As presented in lank SiC . For SDSlank SiC ,36. Genelank SiC . Non-ionlank SiC ,38. The lank SiC ,39.Ihkl)( is the peak intensity obtained from the sample and Ihkl)0( is the corresponding peak intensity of the standard Ni pdf card (JCPDS no. 87-0712). As indicated, the preferentially oriented crystal plane is (200) for the Ni/SDS-SiC coating, while it changes to (111) for the Ni/binary-SiC coating. As proved in the literature, the composite coatings with the preferentially oriented crystal plane of (200) usually show lower hardness and higher ductility than that of (111) [D is the average thickness of the grain perpendicular to the crystal plane, B is the width of the half peak height of the diffraction peak of the measured sample, \u03b8 is the Bragg angle, \u03bb is the X-ray wavelength (1.5406 \u00c5) and K is the Scherrer constant. As shown in XRD and XPS measurements were further conducted to evaluate the phase structure and composition of different coatings. As shown in of (111) . The graength 1.56 \u00c5 and K3/2 region of metallic Ni, while the two peaks at 856.7 eV and 861.9 eV correspond to oxidized Ni and a shakeup satellite (Sat.) peaks, respectively. The existence of the oxidized Ni might be attributed to the partial oxidization of Ni at the surface [XPS spectra in  surface . The Si TEM images were taken to observe the microstructure and crystal morphology of the Ni/SiC composite coatings. As illustrated in \u22122 , sorbitan oleate , polyoxyethylene sorbitan monostearate , sodium dodecyl sulfate  and sodium hydroxide (NaOH) were purchased from Shanghai Macklin Biochemical Co., Ltd. SiC powder , nickel chloride (NiCl2\u00b76H2O), boric acid (H3BO3), sodium carbonate (Na2CO3) and trisodium phosphate (Na3PO4\u00b712H2O) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. Sulfuric acid (H2SO4) was purchased from Modern Oriental (Beijing) Technology Development Co., Ltd. High purity nickel plate (99.99%) was purchased from Qinghe Shenghang Metal Materials Co., Ltd.Nickel sulfate  were added, and ultrasonic treatment was applied for 10 min. Finally, the dispersed SiC suspensions were slowly added to the Watts plating solution, stirred for 2 h and dispersed by ultrasonic treatment for 10 min. The Watts plating solution contains 300 g L\u22121 NiSO4\u00b76H2O, 45 g L\u22121 NiCl2\u00b76H2O and 35 g L\u22121 H3BO3.The SiC powder was pre-treated before being added to the bath. First, SiC powder was degreased with absolute ethanol. Then acid washing was carried out with 15% diluted sulfuric acid for activation for 1 h. After filtration and separation, the SiC powder was washed with deionized water to neutralize. Then, 0.2 g L2 was degreased in an alkaline chemical solution containing 90 g L\u22121 NaOH, 40 g L\u22121 Na2CO3 and 40 g L\u22121 Na3PO4\u00b712H2O, then washed with 15% diluted sulfuric acid to remove oxides before plating. (2) A carbon steel plate and a 99.99% high-purity nickel plate with a larger size of 60 \u00d7 70 mm2 were used as the cathode and anode, respectively. (3) The pH of the bath was adjusted with an appropriate amount of diluted sulfuric acid to about 4 and the stirring rate was controlled at 400 r/min. The co-electrodeposition was implemented by using a direct current power device at a current density of 2.0, 4.0, 6.0 and 8.0 A dm\u22122. During co-electrodeposition, the temperature and duration were set at 40 \u00b0C and 15\u201360 min, respectively.The co-electrodeposition of the Ni/SiC composite coatings was conducted as follows: (1) A carbon steel plate with a size of 20 \u00d7 50 mmSize distribution data and zeta potential distribution data were collected on a Zetasizer particle size analyzer . Due to the influence of the refractive index of the plating bath, measurements are carried out in a 1:1 diluted composite plating solution to obtain accurate results. Fourier-transform infrared (FT-IR) spectra were measured on a Nicolet IS10 infrared spectrometer to evaluate the modification effect of binary surfactants.X-ray diffraction (XRD) patterns were obtained on a D/max-2500 diffractometer at 40 kV and 200 mA with Cu K\u03b1 (\u03bb = 1.5406 \u00c5) radiation. The morphology was observed via scanning electron microscopy . XPS  was used to determine chemical compositions. An atomic force microscope  was employed to characterize the variation in the fluctuation of the composite coatings. A transmission electron microscope  was used to investigate the microstructure of the composite coatings through high-resolution TEM images and selected area electron diffraction patterns.2. The mass loss was calculated by measuring the mass of the composite coatings before and after friction. Tafel plots and EIS spectra (vs. SCE) were obtained in a 3.5 wt% NaCl solution on an electrochemical workstation  by using a three-electrode cell.The hardness of the composite coatings was measured on the surface by a Micro Vickers Hardness Tester  at 100 g for 15 s. The abrasive resistance of the composite coatings was characterized by an dry abrasion machine  with a 500 g normal load and a wear area of about 5.73 cm\u22122) than the Ni/SDS-SiC coating. In addition, the Ni/binary-SiC coating exhibited better corrosion resistance. Compared with previous reports, we provided a new insight into the homogeneous dispersion of SiC nanoparticles and the preparation of uniform and high-quality Ni/SiC composite coatings by electroplating.We have used Span 80 and Tween 60 to disperse SiC particles in the Ni bath, and a composite coating (a Ni/binary-SiC coating) was successfully prepared by electrodeposition. Compared with the Ni/SDS-SiC coating, more SiC particles (10.8 wt%) were incorporated in the Ni/binary-SiC coating, and the crystallization was more refined (average size = 15 nm) with a smoother and denser morphology. Moreover, the Ni/binary-SiC coating presents obviously higher hardness (556 Hv) and higher wear resistance (2.95 mg cm"}
+{"text": "To investigate the changes in 30\u00b0 and 60\u00b0 position sense in patients with anterior cruciate ligament (ACL) injury at different time points after injury and reconstruction.Patients were divided into six groups according to time after ACL injury and reconstruction: group A (ACL injury 1.5\u20136\u00a0months), group B (ACL injury 6\u201312\u00a0months), group C (ACL injury\u2009>\u200912\u00a0months), group D (postoperative ACL reconstruction 1\u20136\u00a0months), group E (postoperative ACL reconstruction\u2009>\u20096\u00a0months), and group F consisting of 14 healthy adults (control group). The ability of the affected leg to reproduce the same joint position during knee flexion was tested using active joint position sense assays to assess proprioception in both the lower extremities of the patient or between groups.P\u2009=\u20090.03; Group B: 2.90  vs 8.30 , P\u2009=\u20090.001; Group E: 6.25  vs 9.60 , P\u2009=\u20090.009). However, no significant differences were detected for a double lower limb contrast of 60\u00b0  vs 3.00 , P\u2009=\u20090.044). Finally, the affected side of patients in groups C, D and E had significant differences in position perception at 30\u00b0 compared with healthy subjects (P\u2009<\u20090.01), and the affected side of patients in groups C and E had significant differences in position sense at 60\u00b0 compared with healthy subjects (P\u2009<\u20090.01).Proprioception decreased rapidly during the early stages of ACL injury. Significant difference in the affected side at 30\u00b0 compared to the healthy side  vs 4.15 , ACL injury had a greater impact on the patient's 30\u00b0 position sense, with only a small impact for 60\u00b0. Further, the early and middle proprioception recovery stages after ACL injury were the best before surgery. Finally, proprioception recovery training should be performed soon after injury. The anterior cruciate ligament (ACL) plays a major role in maintaining knee-joint stability by contributing to both the functionality and mechanical congruence of the lateral and medial tibiofemoral joints . ACL injIn addition to its function as a knee stabilizer, the ACL also has proprioceptive functions , 7. PropACL injury leads to knee dysfunction, primarily because the number of proprioceptors on the ACL decreases, tissue destruction around the knee joint causes abnormal proprioceptive afferents, and the knee joint cannot perceive joint location and transmit motor sensation signals . As ligaHohmann et al.  believedEighty-four adults with unilateral ACL injuries, ranging in age from 18 to 45\u00a0years, were recruited for the study between October 1, 2021, and August 1, 2022. Patients were divided into five groups based on the time since injury: group A , group B , group C , group D (postoperative ACL 1\u20136\u00a0months), and group E (postoperative ACL\u2009>\u20096\u00a0months). Group F consisted of 14 healthy adults (average age: 23\u00a0years) and was regarded as the control group (Table The inclusion criteria were as follows: (1) MRI showing simple ACL injury with good ligament tissue structure; (2) no meniscal injury; (3) no presence of internal tumors, infection, fracture; (4) no mental illness; (5) no unstable vital signs in major organs such as the heart, brain, and kidney; (6) no ACL injury secondary to immune and metabolic diseases; (7) no severe osteoporosis; (8) no venous thrombosis; (9) not pregnant or lactating women; (10) informed consent and voluntary cooperation of patients; (11) unilateral knee injury.The exclusion criteria were as follows: (1) Limited joint movement prevents accurate testing; (2) patients unable to complete evaluation and rehabilitation training as required.Joint angle error testing is a viable method to assess clinical joint proprioception , which cThe specific operations were as follows: The knee was moved from a 90\u00b0 flexion starting position passively to each of the target angles of 30\u00b0 and 60\u00b0. We have reminded patients to close their eyes before collecting data. Please patient hold the leg in the 30\u00b0 knee extension and 60\u00b0 knee extension positions for 10s to allow the patient to memorize the position, and was then returned to 90\u00b0 knee flexion. After a pause of 10\u00a0s, the patient, with the memory of the active knee flexion, moves the lower limb in the same way by active contractions and stops when the patient perceives that the target angle has been reached. The mean values of the six trials were obtained for each patient at each angle and used to calculate the difference between the actual angle achieved and the target angle . The smaThe sample size was estimated using the G-power 3.1  calculator. Considering a 95% significance level and 80% power, we calculated the sample size to be 78. However, taking into account the potential dropouts of some patients or unavailability of data, a total of 90 samples we were tested and 84 samples were finally selected. Data were processed using SPSS 22.0. Comparisons between the patients\u2019 lower limbs were performed using the paired t test for normally distributed quantitative variables, while the Wilcoxon signed-rank sum test was used for non-normally distributed quantitative variables. To compare the data between groups, two independent sample t tests were used for those with normal distribution and the Wilcoxon rank sum test was used for those without normal distribution. Data plots were obtained using GraphPad Prism 8 software.P\u2009<\u20090.05) 1.5\u201312\u00a0months (Group A and B) after injury, as well as 6\u00a0months (Group E) after reconstruction, Group A has poorer position perception on the affected side than on the unaffected side, while Group B and Group E have better position perception on the affected side than on the unaffected side (Table P\u2009<\u20090.05) only between 1.5 and 6\u00a0months (Group A) after ACL injury, and the affected side of group A is worse than the unaffected side , and Group B has a better sense of position than Group C. As was the comparison between the groups 6\u201312\u00a0months after ACL injury and 0\u20136\u00a0months after reconstruction (Group B and D), and Group B has a better sense of position than Group D (P\u2009<\u20090.05). Statistically significant (P\u2009<\u20090.05) in the ACL injury over 12\u00a0months group compared to healthy individuals (Group C and F), and Group C has a worse sense of position than Group F. Further, there was a statistically significant difference (P\u2009<\u20090.05) between the healthy group and the ACL reconstruction group more than 6\u00a0months after surgery (Group E and F), and Group E has a worse sense of position than Group F. A significant difference (P\u2009<\u20090.01) was also found between the healthy group and the ACL reconstruction group 1\u20136\u00a0months after surgery (Group D and F), and Group D has a worse sense of position than Group F  (P\u2009<\u20090.01) between the uninjured side and the healthy person (Group F) in the three groups with ACL injury greater than 12\u00a0months (Group C), 0\u20136\u00a0months after ACL reconstruction (Group D), and ACL reconstruction greater than 6\u00a0months (Group E), and the proprioception of group C, D and E was worse than that of group F  compared with healthy person (Group F), and the results showed that groups C and E had better proprioception than group F  and ACLR longer than 6\u00a0months (Group E) were statistically significant (P\u2009<\u20090.05) was found between healthy and uninjured patients with ACL injury greater than 12\u00a0months (Group C and F), and the results showed that groups C had better proprioception than group F  and improved briefly between 6 and 12\u00a0months (group B) with professional rehabilitation. However, after more than one year of injury (group C), the position sense decreases again. In terms of overall trends, both the affected and unaffected side position sense are in a state of decline. Even after ACLR there was no significant improvement, and a continuous deterioration on the unaffected side. In contrast, 60\u00b0 position sense only decreases at the beginning of the injury. This indicated that ACL injury has more influence on patients' sense of position at 30\u00b0, and less influence on patients' sense of position at 60\u00b0. Indeed, Zhang et al. showed that the perception of 30\u00b0 position on the affected side decreased in the early stage of ACL injury, and this phenomenon exacerbated over time . This maMoreover, we found that the proprioception of the affected side was significantly worse than that of the unaffected side in the same individual in this study. This agrees with a study by Hu , which fAlthough several studies have demonstrated better proprioception on the healthy side than on the affected side after ACL injury or reconstruction, it has also been shown that ACL injury not only decreases proprioception on the affected side, but also affects proprioception on the healthy side. Li et al. similarly found that the unaffected side would decline with the decrease in proprioception on the affected side one year after ACL injury . Other sWe found that positional awareness improved after ACLR compared to preoperative, but did not fully recover. And in the long term, the positional sense on the affected side gradually improved, while the positional sense on the unaffected side gradually deteriorated. This suggests that although ACLR is one of the main treatments for ACL injuries, proprioception is not fully restored after ACLR, which is consistent with the findings of several previous studies. Young et al.  argued tFinally, it is worth noting that in our comparison between patients with ACL and healthy subjects, we found that patients with ACL injury had a better 60\u00b0 position perception than healthy subjects on the same side of the lower limb. Although previous studies have shown poor 30\u00b0 position in patients with ACL injury, our current data show enhanced 60\u00b0 position perception. We believe that this may have to do with the body's compensatory mechanisms. When the 30\u00b0 position sensory area deteriorated, the 60\u00b0 position sensory area was compensated and enhanced. But this idea has not been proven yet, and further tests are needed to confirm it.In this study, we only studied the position perception at 30\u00b0 and 60\u00b0 after ACL injury, and future studies can explore the position perception changes at other angles. Moreover, our study results show that patients' 30\u00b0 position sense decreases significantly, while patients' 60\u00b0 position sense is better than healthy people, and the reasons for this need to be further explored in future studies.This study had several limitations which should be addressed. Firstly, we therefore need a large amount of data and long-term studies to further confirm the findings of our study. Moreover, the angle we tested was only a reference to previous studies and did not further explore other angles of proprioceptive change, which will need to be refined in subsequent studies.In conclusion, this study showed that both the proprioception of the affected and control sides decreased after ACL injury, indicating that it is difficult to return to the pre-injury state even after ACLR."
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