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+{"text": "Fatty liver (FL) is the most frequent liver disease in Western countries. We used data from the Dionysos Nutrition & Liver Study to develop a simple algorithm for the prediction of FL in the general population.216 subjects with and 280 without suspected liver disease were studied. FL was diagnosed by ultrasonography and alcohol intake was assessed using a 7-day diary. Bootstrapped stepwise logistic regression was used to identify potential predictors of FL among 13 variables of interest . Potential predictors were entered into stepwise logistic regression models with the aim of obtaining the most simple and accurate algorithm for the prediction of FL.An algorithm based on BMI, waist circumference, triglycerides and GGT had an accuracy of 0.84 (95%CI 0.81\u20130.87) in detecting FL. We used this algorithm to develop the \"fatty liver index\" (FLI), which varies between 0 and 100. A FLI < 30 (negative likelihood ratio = 0.2) rules out and a FLI \u2265 60 (positive likelihood ratio = 4.3) rules in fatty liver.FLI is simple to obtain and may help physicians select subjects for liver ultrasonography and intensified lifestyle counseling, and researchers to select patients for epidemiologic studies. Validation of FLI in external populations is needed before it can be employed for these purposes. The logits of the other predictors  were linear.Continuous variables are given as medians and interquartile ranges (IQR) because of skewed distributions. Comparisons of continuous variables between subjects with and without FL were performed with the Mann-Whitney test and those of nominal variables with the Fisher's exact test. To identify candidate predictors of FL, we performed a stepwise logistic regression analysis on 1000 bootstrap samples of 496 subjects (probability to enter = 0.05 and probability to remove = 0.1) . All varCandidate predictors identified at bootstrap analysis were evaluated using three stepwise logistic models before obtaining a final prediction model . The goodness of fit of the models was evaluated using the Hosmer-Lemeshow statistic and their accuracy was assessed by calculating the non-parametric area (AUC) under the receiver-operating curve (ROC) with 95% confidence intervals (95%CI) ,24. The vs. 34%). Age, ethanol intake and cholesterol did not differ between subjects with and without FL. On the contrary, ALT, AST, GGT, BMI, waist circumference, the sum of 4 skinfolds, glucose, insulin and triglycerides were significantly higher in subjects with than in those without FL.Table p = 0.0766; model not shown). The model based on the remaining 5 predictors fitted well  and had a ROC-AUC of 0.85 .Figure p = 0.0780; model not shown). The model based on the 5 remaining predictors fitted well  and had a ROC-AUC of 0.85 .Since insulin is not routinely measured, we tested whether its removal from the model would decrease the accuracy of the estimate. After exclusion of insulin, the predictors most frequently identified were triglycerides 100%), GGT (80%), BMI (79%), ALT (70%), the sum of 4 skinfolds (68%) and gender (67%) . The model based on the remaining 4 predictors fitted well  and had a ROC-AUC of 0.85 .Since skinfolds are not routinely measured, we tested whether their removal from the model would decrease the accuracy of the estimate. After exclusion of the sum of 4 skinfolds, the predictors identified most frequently were triglycerides 100%), BMI (95%), ALT (77%), GGT (73%) and waist circumference (58%)  and 3 (p = 0.1038) vs. Model 1 revealed no difference so that we choose Model 3 for further analysis. The bootstrapped regression coefficients of Model 3 are given in Table A comparison of the ROC-AUCs of Models 2 (0.953*loge (triglycerides) + 0.139*BMI + 0.718*loge (ggt) + 0.053*waist circumference - 15.745) /   (1 + e 0.953*loge (triglycerides) + 0.139*BMI + 0.718*loge (ggt) + 0.053*waist circumference - 15.745) * 100  FLI = . Thus, we confirm that insulin is an independent risk factor for FL in the general population . It is oThe main limitations of the Dionysos Nutrition & Liver Study are the suboptimal respondent rate (58%) and the fact that ultrasonography cannot detect steatohepatitis (SH) . This laThe \"fatty Liver index\" (FLI) we developed is accurate and easy to employ as BMI, waist circumference, triglycerides and GGT are routine measurements in clinical practice ,29,30. I7DD = 7-day diary95%CI = 95% confidence intervalsALT = Alanine transaminaseAnti-HCV = Antibodies against hepatitis C virusAST = Aspartate transaminaseAUC = Area under the curveBMI = Body mass indexFL = Fatty liverFLI = Fatty liver indexGGT = Gamma-glutamyl-transferaseHbsAg = Hepatitis B surface antigenHBV = Hepatitis B virusHCV = Hepatitis C virusHCV-RNA = Hepatitis C ribonucleic acidIQR = Interquartile rangeLR+ = Positive likelihood ratioLR- = Negative likelihood ratioNAFLD = Non-alcoholic fatty liver diseaseROC = Receiver-operating curveSH = SteatohepatitisSN = SensitivitySP = SpecificitySLD = Suspected liver diseaseThe authors declare that they have no competing interests.GB co-designed the Dionysos Nutrition & Liver Study, analyzed the data and wrote the manuscript; SB and CT co-designed the Dionysos Nutrition & Liver Study and contributed to the manuscript; LM, FM and AC performed the medical evaluation of subjects; MP performed the nutritional assessment of subjects. All authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:"}
+{"text": "To make lignocellulosic fuel ethanol economically competitive with fossil fuels, it is necessary to reduce the production cost. One way to achieve this is by increasing the substrate concentration in the production process, and thus reduce the energy demand in the final distillation of the fermentation broth. However, increased substrate concentration in simultaneous saccharification and fermentation (SSF) processes has been shown to result in reduced ethanol yields and severe stirring problems. Because the SSF medium is being continuously hydrolyzed, running the process in fed-batch mode could potentially reduce the stirring problems and lead to increased ethanol yields in high-solids SSF. Different enzyme feeding strategies, with the enzymes either present in the reactor from start-up or fed into the reactor together with the substrate, have been studied, along with the influence of the enzyme feeding strategy on the final ethanol yield and productivity.In the present study, SSF was run successfully with 10% and 14% water-insoluble solids (WIS) in batch and fed-batch mode. The mixing of the material in the reactor was significantly better in fed-batch than batch mode, and similarly high or higher ethanol yields were achieved in fed-batch mode compared with batch SSF in some cases. No general trend in the dependence of ethanol yield on enzyme feeding strategy was found.The optimum enzyme feeding strategy appears to depend on the conditions during SSF, such as the WIS concentration and the concentration of inhibitory compounds in the SSF medium. Replacing fossil fuels with so-called biofuels, such as bioethanol, is one way of reducing greenhouse gas emissions from the transport sector, which is responsible for a considerable proportion of total COmissions . CurrentBioethanol can be produced from lignocellulosic material by hydrolysis of the cellulose and hemicellulose to monomeric sugars, followed by fermentation of these sugars to ethanol ,3. Perfo2 as a catalyst has been shown to be successful for softwood and other lignocellulosic materials [Before beginning SSF, the raw material needs to be pretreated to break down the hemicellulose and make the cellulose more accessible to the enzymes used in the hydrolysis Figure . Steam eaterials -18. Thisaterials . Some ofaterials ,20-22 anaterials ,21,23-26To make ethanol an economically competitive alternative to fossil fuel, it is necessary to reduce the production cost. Recovery of ethanol from the fermentation broth by distillation is one of the most energy-intensive steps in the wood to ethanol conversion process ,8. MajorOne reason for the decrease in ethanol yield at higher DM content is difficulty in stirring , which cTo our knowledge, the effects on overall ethanol yield and enzyme consumption of different methods of adding the enzymes to fed-batch SSF have not been studied. The aim of this study was therefore to investigate the effects of different enzyme feeding strategies to optimize the SSF process for ethanol production.Spruce was kindly provided by a sawmill in Southern Sweden . The wood was chipped at a knife mill  and sieved to obtain a chip size of 2-10 mm. The chips were stored in a plastic bag at 4\u00b0C before use. The same batch of raw material was used for all experiments.2 (2% w/w moisture) for 20 min at room temperature, in tightly sealed plastic bags. The amount of SO2 absorbed was determined by weighing the plastic bags and their contents before and after impregnation.The softwood chips were impregnated with SOet al. [The impregnated softwood was pretreated in a steam pretreatment unit equipped with a 10 L reactor, as previously described by Palmqvist et al. . All steet al. . When thSaccharomyces cerevisiae), purified from compressed baker's yeast . The cells were added to a 300 mL Erlenmeyer flask together with 70 mL of an aqueous solution containing 23.8 g/L glucose, 10.8 g/L (NH4)2SO4, 5.0 g/L KH2PO4 and 1.1 g/L MgSO4 7H2O. The solution also contained 14.4 g/L trace metal solution and 1.4 g/L vitamin solution, prepared as described by Taherzadeh et al. [The inoculum culture was prepared on an agar plate containing pure baker's yeast 2SO4, 10.5 g/L KH2PO4 and 2.2 g/L MgSO4 7H2O, 60 g/L trace metal solution and 6.0 g/L vitamin solution. Cultivation was started by adding 60 ml inoculum. Batch cultivation was performed at a stirrer speed of 700 rpm. The fermentor was aerated, and the air flow was adjusted to ensure a concentration of dissolved oxygen of > 5% at all times.The working volume for batch cultivation was 500 mL, and the medium contained 20.0 g/L glucose, 22.5 g/L (NH4)2SO4, 5.3 g/L KH2PO4 and 1.1 g/L MgSO4 7H2O, was added over a period of 16-24 hours. The final concentration of hydrolysate in the fermentor was equivalent to that which would have been obtained if the slurry from pretreatment had been diluted to 7.5% WIS. Fed-batch cultivation was performed in the aerated fermentor at a stirrer speed of 1000 rpm.Once the concentration of dissolved oxygen increased rapidly, indicating that all the ethanol produced during batch cultivation had been depleted, batch cultivation was changed to fed-batch cultivation. This occurred 21-22 hours after start of the aerobic batch cultivation in the various cultivation batches. Fed-batch cultivation was performed with hydrolysate from the pretreatment step. A total volume of 1 L feed containing hydrolysate supplemented with glucose and salt solution, to give a feed concentrations of 80 g/L glucose, 11.3 g/L . The time elapsed between cell harvest and the addition of the cells to SSF was < 2 h.2)2HPO4, 0.025 g/L MgSO4 7H2O and 1.0 g/L yeast extract. The SSF experiments were performed with a yeast cell concentration of 5 g dry yeast cells/kg final working weight. A commercial cellulase mixture was used, consisting of a cellulase derived from Trichoderma reesei  (57.8 filter-paper units (FPU)/g and 38 IU/g) supplemented with a \u03b2-glucosidase preparation  (503 \u03b2-glucosidase IU/g). The level of enzymes added corresponded to a total cellulase activity of 5 FPU/g WIS and a total \u03b2-glucosidase activity of 8 IU/g WIS.All SSF experiments were performed in 2 L fermentors  for 120 hours, with a working weight of 1.3 kg. The temperature in the reactor was maintained at 37\u00b0C, and the pH was continuously adjusted to 5 with 2.5 M NaOH. In the batch experiments, the diluted slurry was autoclaved at 121\u00b0C for 20 min. In the fed-batch experiments, the slurry in the fermentor at start-up was autoclaved in the same way, whereas the substrate feed was not autoclaved. The substrate intended for the feed was pressed  to the WIS concentrations given in Table The following enzyme feeding strategies were investigated.(A) Batch SSF (reference) Figure .(B) Fed-batch SSF with all enzymes added to the fermentor at start-up. To make this comparable with feeding strategies (C) and (D), water equal to the amount of enzyme solution added with the feed in (C) and (D) was mixed with the substrate feed Figure .(C) Fed-batch SSF with enzymes divided between the batch and substrate feed according to the WIS content in these. The enzymes were mixed with the substrate feed at start-up Figure .(D) As in (C) above, but with the difference that the enzymes were added at the same time as the substrate feed but were not mixed with the substrate before addition to the reactor Figure .In fed-batch SSF, the feed was added manually in four equally sized portions at 4, 5.5, 7 and 8.5 hours after start-up, as early addition of substrate has been shown to give better results than late addition ,39. ExpeAll analyses were performed in duplicate. DM content was determined by drying the samples in an oven at 105\u00b0C until a constant weight was obtained. The composition of the spruce and of the washed solids from the pretreated slurry was determined according to the National Renewable Energy Laboratory (NREL) procedure for the determination of structural carbohydrates and lignin in biomass . The solSamples from analysis of the raw material and the washed solids, the hydrolysate of the pretreated slurry, and samples from SSF were analyzed for their content of monomeric sugars. All samples were filtered through a 0.2 \u03bcm filter to remove particles before analysis. Analyses were carried out using a high-performance anion-exchange chromatograph (HPAEC) coupled with pulsed amperometric detection (PAD) and an electrochemical detector . A gradient pump (GP40), an autosampler (AS50), a guard column (Carbo Pac PA1) and an analytical column (PA10)  were used. The eluent was 2 mM NaOH at a flow rate of 1 mL/min, and the injection volume was 10 \u03bcL.2SO4 as eluent at a flow rate of 0.5 mL/min.The samples taken from the SSF experiments were also analyzed for their byproduct content  and ethanol using high-performance liquid chromatography (HPLC), a refractive index detector  and strong cation exchange resin column  at 65\u00b0C with 5 mM HUnless otherwise stated, the ethanol yield is given as the ethanol yield from the SSF step, and is expressed as a percentage of the theoretical yield, based on the contents of glucose and mannose in the pretreated material.The DM content of the wood chips before pretreatment was 48%. After pretreatment, the slurries had a DM content of 13.3% (batch 1) and 16.0% batch 2) WIS. The compositions of the raw material and the two pretreated batches are presented in Table  WIS. TheBatch SSF with 10% WIS resulted in an ethanol yield of 77.4%. Fed-batch SSF at this fiber concentration with all enzymes added in the batch phase at start-up Table .Experiments with a final WIS content of 14% using whole pretreated slurry in SSF  and a slightly lower ethanol yield for fed-batch SSF when all the enzymes were added to the fermentor at start-up Figure . In thisSSF at a final WIS content of 14% and washed slurry resulted in the formation of lactic acid, starting between 10 and 24 hours after start-up in all cases Table .The initial ethanol productivity was equal or slightly higher in fed-batch SSF experiments than in batch SSF Figure .Running SSF in fed-batch instead of batch mode greatly increased the mixing in the reactor, especially at the higher WIS concentration studied. Furthermore, in all cases, the enzyme feeding strategy had an effect on the ethanol yield in SSF. However, the enzyme feeding strategy resulting in the highest ethanol yield was different not only for the different substrate concentrations studied, but also for different inhibitor concentrations. One explanation of this could be the difference in the degree of deactivation of the enzymes in the different cases. In the experiments with hydrolysate present in the fermentor Table , SSF 4B.When adding part of the enzyme mix together with the substrate feed, it appears to be advantageous to mix the enzymes with the substrate before addition to the reactor. This indicates that the hydrolysis of the substrate feed has already started before addition to the fermentor at room temperature, or that some of the enzymes added with the substrate feed adsorb to material already in the reactor, rather than to the fed substrate if they are added separate from the fed substrate.Previous studies on fed-batch SSF have given different results. In some cases, fed-batch SSF resulted in higher ethanol yields than batch SSF ,43, wheret al. [et al. [Rudolf et al.  reported [et al. . AnotherRunning SSF in fed-batch mode does not necessarily give higher ethanol yields than running in batch mode, but when the enzyme-adding method is suitable, similar or slightly higher ethanol yields could be obtained in fed-batch SSF compared with batch mode. The appropriate feeding strategy for the enzymes in fed-batch SSF appears to depend on the conditions during SSF; for example, the WIS and inhibitor concentrations. In the present study, the dependence of ethanol yield on enzyme feeding strategy differed not only with WIS content, but also with inhibitor concentration in experiments with the same WIS content.The authors declare that they have no competing interests.KH planned and carried out the experiments, analyzed the results and wrote the paper. GZ participated in the design of the study, helped analyzing the results and contributed to the draft of the manuscript. MG helped to draft the manuscript. All authors read and approved the final manuscript."}
+{"text": "This landmark transformation would formally result in the 1,2-bisalkylation of nonactivated alkenes! In this Thematic Series, you will find excellent contributions tackling various problems of carbometallation reactions, indicating a lively and rapidly moving field, and I have no doubt that more elegant transformations of C\u2013C unsaturated bonds will continue to appear, including the enantioselective carbometallation reaction of substituted nonactivated alkenes. I would like to warmly thank all the contributors of this Thematic Series that have beautifully highlighted the state of the art of the field.Following the pioneering Ziegler addition of nucleophiles to nonactivated unsaturated carbon\u2013carbon bonds, the controlled carbometallation reaction has emerged. Since then, reactions that result in the addition of a carbon\u2013metal bond of an organometallic across a carbon\u2013carbon unsaturated system, leading to a new organometallic in which the newly formed carbon\u2013metal bond can be used for further synthetic transformations, are called carbometallation reactions. In the past few decades, the intra- as well as intermolecular additions of various organometallic species to a large variety of alkynes, alkenes and allenes have been successfully reported. Although the carbometallation reaction on alkynes is generally a well-controlled and predictable transformation, leading to large variety of substituted stereocontrolled alkenyl metals, the addition of organometallic species to acyclic nonactivated alkenes still represents a formidable and yet unresolved synthetic challenge. Particularly stimulating would be the regio-, stereo- and enantioselective addition of organometallic species on \u03b1,\u03b2-disubstituted double bonds, leading to a configurationally stable spI have tremendously enjoyed reading this Thematic Series and I am convinced that you will all share in this pleasure!Ilan MarekHaifa, January 2013"}
+{"text": "P < 0.05) and 20\u2009mg/kg b.wt.  has anticonvulsant effects. Thus, our data suggest that diazoxide as ATP-sensitive potassium channels opener has anticonvulsant activity against dichlorvas-induced seizure.Dichlorvos, a synthetic organophosphate toxin, is used as pesticides. These toxins can be used as pesticides in farming and medicine for the devastation and/or elimination of ectoparasites of animals. Reports have shown that Dichlorvos generate seizure effects in various animals. Potassium channel opener is extensively used for medication of cardiovascular and other diseases. Studies have shown that potassium channel opener has anticonvulsant effects in different animal models. The goal of this study was to evaluate the effect of dizoxide on Dichlorvos-induced seizures in mice. In this research, the animals received different doses of Diazoxide  intraperitoneally 30\u2009min before intraperitoneal injection of Dichlorvos (50\u2009mg/kg b.w.t). After Dichlorvos injection, latency of clones, severity of seizure, and finally death as the fate were investigated. Results showed that Diazoxide dose-dependently decreased the severity of Dichlorvos-induced seizures, so that Diazoxide at a dose of 5\u2009mg (the lowest,  So, further investigation is needed to evaluate the efficacy of this agent in AChE inhibition-induced seizure.In summary, this study showed that Diazoxide (K"}
+{"text": "The Nef protein of human immunodeficiency virus (HIV) promotes viral replication and progression to AIDS. Besides its well-studied effects on intracellular signaling, Nef also functions through its secretion in exosomes, which are nanovesicles containing proteins, microRNAs, and mRNAs and are important for intercellular communication. Nef expression enhances exosome secretion and these exosomes can enter uninfected CD4 T cells leading to apoptotic death. We have recently reported the first miRNome analysis of exosomes secreted from Nef-expressing U937monocytic cells. Here we show genome-wide transcriptome analysis of Nef-expressing U937 cells and their exosomes. We identified four key mRNAs preferentially retained in Nef-expressing cells; these code for MECP2, HMOX1, AARSD1, and ATF2 and are important for chromatin modification and gene expression. Interestingly, their target miRNAs are exported out in exosomes. We also identified three key mRNAs selectively secreted in exosomes from Nef-expressing U937 cells and their corresponding miRNAs being preferentially retained in cells. These are AATK, SLC27A1, and CDKAL and are important in apoptosis and fatty acid transport. Thus, our study identifies selectively expressed mRNAs in Nef-expressing U937 cells and their exosomes and supports a new mode on intercellular regulation by the HIV-1 Nef protein. The human immunodeficiency virus expresses the prototypic retroviral Gag (capsid), Pol (polymerase), and Env (envelop) proteins. Additionally, it also expresses two regulatory  and four accessory  proteins . Of thesExosomes are 30\u2013100\u2009nm vesicles that are formed by the inward invagination of MVB membranes and are released in the extracellular medium when MVBs fuse with the plasma membrane . These vThe first report indicating exosomal Nef secretion showed vesicles secreted from HIV infected cells by confocal laser-scanning microscopy and by their sedimentation behaviour . Later,  eyfp and nef-eyfp genes, respectively, were cloned into BglII and HpaI sites in the pMSCV retroviral transfer plasmid. The positive clones were confirmed by restriction digestion and analyzed for EYFP or Nef-EYFP expression by transient transfection in HEK293T cells and western blotting with anti-GFP antibody. Retroviruses expressing Nef-EYFP or EYFP were generated by cotransfection of HEK293T cells in a T25 flask with 2\u2009\u00b5g of the transfer plasmid, 1\u2009\u00b5g of pGag-Pol, and 0.5\u2009\u00b5g of pVSVg using the calcium phosphate method. The culture supernatants were collected after 36\u2009hr and used as the source of recombinant retroviruses. Human monocytic U937 cells were washed with RPMI, starved for 90\u2009min without serum, and then transduced with 500\u2009\u00b5L of culture supernatants per 1 \u00d7 106 cells. After 4\u2009hrs of virus adsorption, the cells were washed and kept in complete medium for 48\u2009hr prior to the addition of 350\u2009ng/mL puromycin. The cells were split every 48\u2009hr and those surviving after 5 passages were used for the analysis. The clones were sorted for the EYFP positive population using a Becton Dickinson Aria Cell Sorter in the Central Facility of the National Institute of Immunology, New Delhi, India. The sorted clones were cultured for 4-5 passages and checked for purity and YFP expression using Cyan-ADP flow cytometer (Beckman Coulter). Data was analyzed using Summit 4.3 software.This has been described in detail elsewhere . BrieflyThe procedural details of RNA extraction from cells and exosomes followed by the estimation of its yield and purity using spectrophotometric protocols and its RIN score using the Agilent 2100 Bioanalyzer  have been described elsewhere . The micBroadly the two datasets are cellular RNA and exosomal RNA. In the cellular mRNA dataset, there are three samples from U937 cells expressing Nef-EYFP  and two control samples from U937 cells expressing EYFP . The third control sample mYC was an outlier and was not considered in the analysis. The exosomal mRNA dataset has three samples for U937 cells expressing Nef-EYFP  and three control samples from U937 cells expressing EYFP . There are 50,238\u2009mRNAs in each dataset. The zero-mean normalization method  was usedk-means algorithm [k cluster means using an iterative procedure that minimizes the sum of squared error criterion. To obtain partitioning of the data, each gene is assigned to the mean to which it is closest. Here Euclidean distance was used as a measure of the distance between two genes. As the number of samples in the mRNA data is very small while the number of genes is large, direct application of any statistical analysis on the dataset was difficult. Therefore, clustering was first applied and the resulting clusters were analyzed to identify those that represent upregulated and downregulated genes in the experimental (Nef-EYFP) samples with respect to the control (EYFP) samples for both cellular and exosomal mRNA.Variable string length genetic algorithm (VGA) clustering  is a genlgorithm . It is ut-test, and permuted t-test on the normalized data to finally identify the upregulated and downregulated genes. From a statistical perspective, the t-test is a standard tool for differential expression analysis when the number of samples is large. Permuted t-test is normally useful when there is no information on distribution of the data. For small sample sizes, application of t-test and permuted t-test may not be appropriate. Therefore, we also used SAM, a very powerful tool for small sample size of the data. Finally, we took the intersection of upregulated cellular mRNAs (referred to as mNCup) and the intersection of downregulated cellular mRNAs (referred to as mNCdown) obtained from the t-test, permuted t-test, and SAM for the mNC versus mYC cellular dataset. Again, for the mNE versus mYE exosomal dataset, we followed the same procedure to obtain the upregulated mRNAs (referred to as mNEup) and downregulated mRNAs (referred to as mNEdown). We refer to the nondifferentially expressed mRNAs in Nef cells and exosomes as mNCnon and mNEnon, respectively.After using clustering to identify potential differentially expressed genes, we utilized three statistical tests , which isel while those that are selectively retained in Nef-expressing cells are referred to as mNCsel. The set mNEsel was obtained by the intersection of mNCdown and mNEup. Similarly, the set mNCsel was obtained by the intersection of mNCup and mNEdown.After statistical analysis, the mRNAs that are selectively secreted from Nef-expressing cells to exosomes were identified. These are referred to as mNEsel and the corresponding miRNAs that target them  were considered. In the second network, the genes of mNEsel and the corresponding miRNAs were studied. The status of the miRNA targets of significant mRNAs was verified in Nef-expressing cells/exosomes as reported in our previous study [The miRNA-gene network analysis was carried out and two networks were constructed. In the first network, the genes of mNCus study .Data acquisition was carried out as described in up; n = 8522) and those in cluster 5 were downregulated  in Nef-expressing cells. For the normalized exosome mRNA datasets (mNE versus mYE), VGA clustering was unable to identify any cluster. We therefore performed the less stringent k-means clustering and found four clusters on the exosomal (mNE versus mYE) dataset  and mRNAs in cluster 4 to be downregulated  in exosomes purified from Nef-expressing cells.The mRNA data was normalized using zero-mean normalization followed by VGA clustering. The analysis on normalized data (mNC versus mYC) identified six clusters . Among t dataset . This clt-test, and permuted t-test on different gene clusters in each group and determined the common genes from the three statistical tests. This provided 1889 upregulated (mNCup) and 2380 downregulated (mNCdown) mRNAs in Nef-expressing cells (up) and 2328 downregulated (mNEdown) mRNAs in exosomes from Nef-expressing cells (up and mNEdown groups (mNCup\u2229mNEdown) to identify 81\u2009mRNAs that are selectively retained in Nef-expressing cells and are not secreted in exosomes despite their high intracellular levels  to identify 7\u2009mRNAs that are preferentially secreted in Nef exosomes despite their low expression levels in Nef-expressing cells  and the miRNAs, which target these. The top four mRNAs identified are MECP2, HMOX1, AARSD1, and ATF2 ( trans to edit the amino acid moiety from incorrectly charged tRNA(Ala) and thus prevents mistranslation of proteins [Next, we carried out network analysis of mRNAs selectively retained in Nef-expressing cells or secreted in exosomes from these cells, and the miRNAs that target them. In the first network , we consand ATF2 . Among tand ATF2 . MECP2 eand ATF2 , which iand ATF2 . The hemand ATF2 . AARSD1 and ATF2 . It is aproteins . ATF2 isproteins . Thus thsel) and the validated miRNAs that target their genes. We identified three mRNAs in this group, which are targeted by miRNAs that are selectively retained in Nef-expressing cells. These are AATK (apoptosis associated tyrosine kinase) and SLC27A1 (solute carrier family 27 fatty acid transporter member 1), which are targeted by miR32 and mir93*, and CDKAL1 (CDK5 regulatory subunit associated protein 1-like 1), which is targeted by miR32  and influence Hsp27 promoter activation . LEDGF iexosomes . It is nexosomes . We showexosomes , 37. It exosomes . We show*) to be retained in these cells. Recent studies show AATK to promote apoptosis in melanoma cells and to be regulated by the Src kinase [ in silico analysis here shows that the mRNA for fatty acid transporter protein is selectively packaged in Nef exosomes. This may lead to altered SLC27A1 production in Nef-expressing cells and can potentially increase the levels of this protein in cells receiving these exosomes. Thus, exosomal packaging of SLC27A1 mRNA could be a mechanism for regulating free fatty acid levels during HIV infection. The third mRNA is for CDKAL1, which is a member of methylthiotransferase family. While its exact function is not known, genes in this family are involved in posttranscriptional modifications of tRNA [We also identified the mRNAs for three proteins, AATK, SLC27A1, and CDKAL1, to be preferentially secreted out in exosomes from Nef-expressing cells and their targeting miRNAs (miR32 and miR93c kinase , 38 and c kinase , 39. It c kinase . It was c kinase . The SLCc kinase , which ic kinase , 44. Recc kinase . Our in  of tRNA . in silico analysis of the transcriptomic data of Nef-expressing monocytic cells and their exosomes and identifies key mRNAs that are exclusively retained in cells or secreted in exosomes. The mRNAs preferentially retained in cells are essentially involved in chromatin modification and transcriptional regulation. These pathways are critical for active gene expression as well as latent viral reservoirs. The mRNAs selectively packaged in exosomes are involved in apoptosis and fatty acid transport. These are critical for lipid metabolism and bystander cell activation and death. Thus, our in silico analysis has identified key intracellular and extracellular signalling pathways targeted by HIV in monocytic cells. Our study further supports the paradigm that HIV utilizes the exosomal intercellular communication network to optimize its spread in infected hosts [Overall, this study employsed hosts , 48. FurThis study presents transcriptomic and network analysis of human monocytic cells expressing the HIV-1 Nef protein and their exosomes. We identified four mRNAs that are exclusively retained in Nef-expressing cells while their targeting miRNAs are exported out in exosomes. These are involved in chromatin modification, transcriptional regulation, and stress response. We also identified three mRNAs that are preferentially secreted in exosomes and whose targeting miRNAs are retained in Nef-expressing monocytes. These are involved in apoptosis induction and fatty acid transport. Monocytes are important latent viral reservoirs, and apoptosis of bystander cells and dysregulation of fatty acid metabolism are important events in HIV pathogenesis. Thus, our findings have important implications in understanding HIV pathogenesis from the triangular axis of mRNAs, miRNAs, and exosomes, which has remained poorly studied."}
+{"text": "Toxoplasma gondii can invade virtually any nucleated cell and infect almost all warm-blooded vertebrates, whereas Eimeria tenella infects only chickens and is restricted in its growth to epithelial cells of the caecum. Proteins released from the microneme secretory organelles (MICs) are critical for apicomplexan invasion of host cells and allow parasites to bind a diverse range of host cell oligosaccharide epitopes. MICs bear modular arrangements of sequences with adhesive proteins and interestingly the sialic-acid binding MAR (microneme adhesive repeat) domain containing proteins (MCPs) are suggested to make significant contributions to the different host and tissue tropisms of T. gondii and E. tenella.The phylum Apicomplexa comprises a wide variety of parasites of significant medical and economic relevance. These parasites have extremely different host and tissue tropisms; for example E. tenella MCPs. Variants of the previously described HxT motif were analysed showing that HxT and VxT variants bind, whereas HxS and YxE variants did not. One of these MCP containing a single MAR (EtMCP2) showed an apical localization when expressed as a fusion with the fluorescent reporter mCherry in transgenic populations and a similar pattern of transcripts per zoite during endogenous development in vitro as the well-characterised microneme protein EtMIC2.In this study, we evaluated the binding capacity of Type I MAR domains from novel Eimeria parasites as a rapid tool for the study of endogenous protein localization by fusion with a fluorescent reporter.Variation in the binding properties of the MAR of different EtMCPs was confirmed and their ability to bind a wider range of sialic acids and terminal linkages should be studied. In addition, transgenesis technology has been used for first time in The online version of this article (10.1186/s13071-017-2454-4) contains supplementary material, which is available to authorized users. Eimeria  cause chicken coccidiosis, a disease with a huge economic impact in the poultry industry. Disease pathology is characterised by diarrhoea, malabsorption and for some species haemorrhage, and has a severe impact on animal welfare, efficiency of feed conversion and weight gain. Eimeria parasites disseminate readily through flocks via the oral-faecal route and are highly prevalent throughout the world [Seven species of the genus he world . Coccidihe world .Eimeria parasites in order to elucidate the functional significance and immunogenicity of parasite sub-cellular structures aids discovery of antigens for new types of vaccines. A substantial amount of data from high-throughput technologies is now available [Toxoplasma gondii [Eimeria spp. are less developed, however, efficient random integration of transgenes is well established [Understanding the biology of vailable \u20135. Howeva gondii , and area gondii , 8 and ta gondii , 10. Revablished , 12 for ablished \u201317.E. tenella protein EtMIC3) make increased contacts to the sialyl saccharide leading to a higher level of specificity in binding, and precluding interaction with N-glycolyl sialic acids [Proteins secreted from apicomplexan microneme organelles (MICs) play important roles in parasite adhesion and invasion of host cells . Some MIic acids .Toxoplasma gondii and Neospora caninum possess proteins with both Type I and Type II MAR. However, to date all MAR domains identified in Eimeria species are exclusively Type I, suggesting that a lack of Type II MAR may contribute to the exquisite host and site specificity displayed by these parasites [Eimeria tenella, EtMIC3 containing seven Type I MAR binds a restricted range of sialyl glycans with a strong preference for \u02512,3-linkages that are predominant in the chicken gut [E. tenella genome [arasites , 22. Forcken gut . Four ada genome   and reverse-transcription qPCR (RT-qPCR) that EtMCP2 transcript/genome ratio during sporozoite invasion and intracellular schizogony is similar to that of the known microneme proteins, EtMIC2 and EtMIC5. Thus, despite an inability to bind sialyl moieties, the Type I MAR protein, EtMCP2, appears to be expressed within the microneme organelles of E. tenella.In this paper, we examine the ability of EtMCP Type I MARs with variant HxT motifs to bind in vitro to fetuin-agarose or to cultured cells. To explore the subcellular location of an MCP with a variant, non-binding motif (EtMCP2), we generated a transgenic population of Eimeria tenella Wisconsin strain [6 cells/well) at a multiplicity of infection of 1:1 and incubated at 41\u00a0\u00b0C / 5% CO2 for 4, 24, 48 and 72\u00a0h.n strain  was propn strain , and spon strain  were carNcoI and NotI restriction sites into the plasmid pET32b (+) . The recombinant proteins were expressed as soluble polyhistidine (His6) and thioredoxin fusions in BL21 (DE3) pLysS E. coli  and were purified and quantified as described previously [Complementary DNA (cDNA) sequences corresponding to protein repeats containing MAR with varying HxT motifs Table  were ampeviously .Fetuin agarose saline suspension  was packed in mini filter columns and equilibrated with phosphate buffer . Recombinant protein quantified by Bradford (Sigma-Aldrich) was suspended in phosphate buffer (200\u00a0\u03bcg), loaded to the top of the affinity column and maintained at 4\u00a0\u00b0C for 3\u00a0h for complete binding . The col6 cells/well) and left to settle into a monolayer for 4\u00a0h, washed gently in phosphate buffered saline (PBS), blocked in 1% bovine serum albumin (BSA) for 30\u00a0min, and washed again in PBS. 500\u00a0\u03bcg of recombinant protein was added per well and incubated for 1\u00a0h at 4\u00a0\u00b0C. Supernatants were removed and the cells were washed several times with PBS. MDBK monolayers were removed from wells with trypsin (Sigma-Aldrich), pelleted and stored at -20\u00a0\u00b0C until further analysis.MDBK cells were seeded into 24-well plates (0.3\u00a0\u00d7\u00a010w/v) non-fat milk (Bio-Rad) overnight. Membranes were incubated for 1\u00a0h with mouse anti-histidine tag antibody , then washed three times with tris buffered saline-Tween20 (TBS-Tween) 0.05% (v/v) and incubated for 1\u00a0h with goat anti-mouse IgG antibody HRP conjugate . Membranes were washed three times with TBS-Tween 0.05% (v/v), once with TBS and finally with distilled water before adding Luminata substrate (Merck Millipore). Chemiluminiscence was visualised in a G:BOX  and images were taken with GeneSnap 7.12 software (Syngene).Eluted fetuin-binding fraction (50\u00a0\u03bcg in 50\u00a0\u03bcg of 2\u00d7 Laemmli loading buffer (Sigma-Aldrich)) and MDBK-bound protein fractions (resuspended in 100\u00a0\u03bcg 1\u00d7 Laemmli) were electrophoresed in NuPAGE 4\u201312% Bis-Tris 10-well gels  and stained with Coomassie blue . Proteins were transferred from gels to nitrocellulose membranes  by semi-dry blotting following the manufacturer\u2019s protocols (Invitrogen), and membranes were blocked in 5%  following protocols described previously [6 transfected sporozoites were used to infect two chickens via the cloacal route. Shed fluorescent oocysts were selected by FACS (BD FACS Aria\u2122 III) for subsequent in vivo passages to amplify the proportion of transgenic parasites within the populations.Restriction enzyme-mediated integration (REMI) transfection into eviously . After 2Infected monolayers and oocysts of transgenic populations were observed under UV fluorescence in a Leica DMI3000B microscope and photographed with a Leica DCF365FX camera. Image processing was performed using the LAS AF (Leica Microsystems).E. tenella sporozoites were fixed in 4% paraformaldehyde in PBS for 15\u00a0min and washed in PBS. Fixed monolayers were blocked by incubating in 3% BSA, 0.25% Triton \u00d7100 in PBS for 30\u00a0min then in rabbit anti-EtMIC2 serum (1/500) for 1\u00a0h. After three washes in PBS, monolayers were incubated with goat anti-rabbit IgG conjugated to Alexa Fluor 488 , washed in PBS, then observed under UV fluorescence using a Leica DMI3000B microscope and photographed with a Leica DCF365FX camera. Image processing was performed using the LAS AF (Leica Microsystems).MDBK monolayers infected for 4\u00a0h with E. tenella sporozoites following the manufacturer\u2019s protocols . cDNA was synthesized from total RNA using SuperScript II\u00ae reverse transcriptase (Invitrogen) as described previously [Total DNA and RNA were extracted from an in vitro time course of parasite development in MDBK cells infected with wild-type eviously .Eimeria spp. 5S rDNA [EtMIC2, EtMIC5, EtMCP2 and EtActin . Standard curves were prepared for all genes from 107 to 102 sporozoites or plasmid copies, against which the total number of parasites or transcript copy numbers were quantified, respectively. The transcription levels along the course of intracellular schizont development were normalised to the number of parasite genomes. Data were analysed by a one-way ANOVA with post-hoc Bonferroni test .Real time qPCR was performed in a CFX96 Touch\u00ae Real-Time PCR Detection System (Bio-Rad) using DNA-binding dye SsoFastTM EvaGreen\u00ae Supermix (Bio-Rad) as described previously . The num 5S rDNA  and a stE. tenella were aligned in order to predict their potential binding properties  as well as MAR3 of EtMIC3 (recMIC3.3) were expressed as soluble recombinant fusion proteins in EtMIC2 or EtMCP2 fused directly to mCherry  of either Et-MIC2-mChe and Et-MCP2-mChe) by two consecutive passages through chickens with fluorescence-activated cell sorting (FACS) in between to enrich the populations in fluorescent oocysts. Within oocysts . In free sporozoites, EtMIC2 transcripts \u00a0=\u00a022.708, P\u00a0=\u00a00.012) and slightly higher, but not significantly, than those of EtMCP2 \u00a0=\u00a022.708, P\u00a0=\u00a00.066). As schizogony progressed, values decreased significantly for all three genes. A very similar pattern was observed for EtMIC2 and EtMCP2, where number of transcripts per zoite were markedly reduced during schizogony \u00a0=\u00a084.105, P\u00a0<\u00a00.0001 and F\u00a0=\u00a028.489, P\u00a0=\u00a00.019, respectively) compared to EtMIC2 levels. As expected, EtActin was low and constant along the endogenous development.We documented the transcript numbers per zoite of d EtMIC5 , 30) andpts Fig.\u00a0 were at E. tenella the only MAR protein explored to date is microneme protein EtMIC3; this has 7 Type I MAR, is expressed by invading sporozoites, binds the surface of chicken caecal epithelial cells, and has a strong preference for terminal N-acetylneuraminic acids with \u02512,3-linkages [E. tenella genome (EtMCP2 to EtMCP5), however, until now their adhesive properties and subcellular locations were unconfirmed.Microneme proteins are characterised by the possession of adhesive domains that play roles in parasite attachment and invasion of the host cell. One of these, the MAR domain, is restricted to coccidial parasites and has been shown to bind sialylated glycans. Structural and sequence variations between and within Type I and Type II MAR family domains results in variant capacities for binding sialylated residues, contributing to differences in host and site specificities of the parasites. For linkages . SeveralE. tenella MCPs were analysed for their predicted and actual binding properties, along with MAR3 from EtMIC3. Blumenschein et al. [E. tenella MCP are able to bind a wider range of sialic acids, including N-glycolyneuraminic acids, and a wider range of terminal linkages.In this study, MAR from n et al.  reportedn et al. . It woulE. tenella. Expression of EtMCP2 fused at its C-terminus to mCherry resulted in the fused protein being expressed with an apical location within the sporozoite in both transiently transfected and stable transgenic populations. By comparing the pattern of expression to that seen with an analogous mCherry fusion with the characterised microneme protein EtMIC2, it seems highly likely that EtMCP2 is indeed localised to the microneme organelles where it presumably plays a role in binding to host cells.We aimed to determine whether EtMCP proteins are localised to the microneme organelles and chose to explore this using EtMCP2, because its small size  was most convenient for tagging and transfection into EtMCP2 and EtMIC2 exhibited a similar dynamic pattern during intracellular development of E. tenella within cultured MDBK cells. The relative numbers of transcripts continuously decreased during development until the formation of merozoites. In contrast, transcripts of another microneme encoding gene, EtMIC5, were maintained at a higher level during endogenous development.In addition to the putative micronemal localisation, the accumulation of transcripts per zoites expressed from Eimeria spp. is now routinely used and most studies have focused on the use of Eimeria as a vaccine vehicle to deliver foreign antigens [Toxoplasma [Transgenesis in antigens , 17, 31.xoplasma , 33.E. tenella MCPs appears to play an important role in the binding properties of these proteins, as has been suggested before [Eimeria as a fusion with the fluorescent reporter mCherry exhibiting an apical localisation, typical of microneme proteins . In addition, EtMCP2 showed a similar pattern of transcripts than other miconeme proteins (EtMIC2 and EtMIC5). We concluded therefore, that at least one of these newly described EtMCPs is located in the micronemes, as was expected.The threonine (T) in the HxT motif of Type I MAR domains of d before , since o"}
+{"text": "Streptococcus iniae. Here, we report both host and bacterial determinants leading to the formation of organized macrophage aggregates as part of the host inflammatory response in a subset of infected larvae. Streptococcal capsule was a required signal for aggregate formation. Macrophage aggregation coincided with NF\u03baB activity, and the formation of these aggregates is mediated by leukotriene B4 (LTB4) produced by neutrophils. Depletion, inhibition, or genetic deletion of leukotriene A4 hydrolase (Lta4h), which catalyzes the last step in LTB4 synthesis, resulted in the absence of macrophage aggregation. Larvae with impaired neutrophil function also had impaired macrophage aggregation; however, aggregate formation was partially rescued with the addition of exogenous LTB4. Neutrophil-specific expression of lta4h was sufficient to rescue macrophage aggregation in Lta4h-deficient larvae and increased host survival following infection. In summary, our findings highlight a novel innate immune response to infection in which specific bacterial products drive neutrophils that modulate macrophage behavior through eicosanoid signaling.Immune cells sense and react to a multitude of factors including both host and microbe-derived signals. Understanding how cells translate these cues into particular cellular behaviors is a complex yet critical area of study. We have previously shown that both neutrophils and macrophages are important for controlling the fish pathogen  Klebsiella pneumoniae in a pulmonary infection model [Immune cell populations communicate to carry out coordinated responses against a broad range of insults. For example, immune cell crosstalk via a positive feedback loop involving TNF\u03b1 and IL17A between inflammatory monocytes and lymphocytes enhances the clearance of on model \u20134. Immunon model ,6. Thus,Activated leukocytes release a variety of pro-inflammatory mediators to communicate with other cells, including the eicosanoid LTB4 ,8, whichNeutrophils are typically the first cells recruited to sites of bacterial infection or wounds . While nStreptococcus iniae. S. iniae is a significant fish pathogen in aquaculture, causing an estimated $100 million in annual costs worldwide, and can be an opportunistic pathogen in humans [We used a zebrafish larval model and have characterized the formation of macrophage aggregates in response to infection with n humans . In a suS. iniae and the avirulent, capsule-deficient cpsA mutant [S. iniae [cpsA insertion mutants. Since the specific role of macrophages in response to S. iniae infection has not been examined, we performed localized infection in the otic vesicle of larvae after transient depletion of macrophages using a morpholino targeting irf8. Irf8 morphants lack macrophages but have an increased number of neutrophils [S. iniae relative to control morphants  larvae that contain fluorescently labeled macrophages. Intriguingly, we observed the formation of distinct macrophage aggregates in a portion of infected larvae. S. iniae labeled with Cell Tracker Red dye could be found within macrophage aggregates  or formalin-killed (FK) WT S. iniae. Importantly, aggregates were not induced following the addition of HK cpsA, or HK or FK WT bacteria alone  larvae also interacted with macrophage aggregates  (Tg(lyz:lta4h-2a-mCherry) larvae had significantly increased survival after infection compared to WT larvae during Lta4h depletion , Tg(mpeg1:dendra2) [Tg(mpx:mCherry-2A-rac2wt) and Tg(mpx:mCherry-2a-rac2d57n) [Tg(mpeg1:mCherry-histone2b) [Tg(lyz:lta4h-2a-mCherry) . Additionally, a previously described lta4h-deficient mutant with a retroviral insertion in the seventh exon of lta4h [N-phenylthiourea . For infections and live imaging, larvae were anesthetized in E3 medium containing 0.2 mg/ml tricaine . Where indicated, E3 was supplemented with the following drugs immediately following infection and drug solutions were changed daily: 30 nM LTB4  and 0.1% ethanol, 100 \u03bcM Bestatin  and 0.1% DMSO.Zebrafish, embryos, larvae and adults were maintained in accordance and approval (protocol M005405) with the University of Wisconsin-Madison Research Animal Resources Center IACUC . For infections and live imaging, larvae were anesthetized in E3 medium containing 0.2 mg/ml tricaine . A light cycle of 10 h darkness and 14 h light was used. Wild-type AB fish were used to generate all transgenic lines, and the following transgenic lines were used in these studies: dendra2) , Tg(mpx:ac2d57n) , Tg(mpegstone2b)  and Tg(lof lta4h  was geneS. iniae wild-type strain 9117 has been previously described [S. iniae was prepared and microinjected into the otic vesicle of zebrafish aged 2\u20133 days post-fertilization (dpf) as described [S. iniae or 100 CFU equivalent of cpsA bacteria at 95\u00b0C for 30 min. Formalin-killing was achieved by resuspending in 1 ml of 4% paraformaldehyde and incubating at 37\u00b0C for 30 min.escribed ,43. S. iescribed . Where iAll morpholino oligonucleotides (MOs) were purchased from Gene Tools, LLC , resuspended in distilled water and stored at room temperature at a stock concentration of 1 mM. One-cell stage wild-type AB embryos were injected with 3 nl of morpholinos at the following concentrations: Irf8 MO, 400 \u03bcM; Lta4h (I7E8) MO, 500 \u03bcM. Comparable doses of the standard control MO were used in each experiment. The Irf8 , morphol5\u2019- CAGTCTGATCAAGAGAAAGACTCGA-3\u2019Lta4h: 5\u2019- AATGTTTCGCTTACTTTGAAAATGG-3\u2019Irf8: Tg(mpeg1:dendra2) line that has fluorescent green macrophages. Confirmation of the Lta4h morpholino was achieved by RT-PCR using mRNA extracted from 2\u20134 dpf larvae.Elimination of macrophages in Irf8 morphants was confirmed by visual examination following injection into the Lta4h primers for RT-PCR:5\u2019-TCTGAGAAGGAATATGTGGATGAA-3\u2019lta4hF: 5\u2019-CAGCAAGAGATCTGTCTCCA-3\u2019lta4hR: lta4h-2a-mCh  was PCR amplified and inserted into a backbone vector containing minimal Tol2 elements for efficient integration, the lyz promoter for neutrophil-specific expression  and an SV40 polyadenylation sequence . A viral 2A peptide linker sequence was used to facilitate production of multiple protein products from a single transgene [DNA encoding ransgene . One-cellta4h-mutants, zebrafish were fixed in formaldehyde overnight at 4\u00b0C and immunolabeled as previously described [For examining aggregate formation in the escribed  using raescribed .Anesthetized larvae were settled onto the bottom of a custom-made, glass-bottom dish. Fluorescence images were acquired with a laser scanning confocal microscope  using a numerical aperture 0.75/20X objective. Each fluorescent channel (488 nm and 543 nm) and differential interference contrast (DIC) images were acquired by sequential line scanning. Z-series were acquired using a 200\u2013300 \u03bcm pinhole and 6\u201310 \u03bcm step sizes. Z-series were stacked using the FluoView FV1000 software. Fluorescence images acquired at 40x or 63X magnification were acquired using a spinning disk confocal microscope (Yokogawa CSU-X) with a confocal scanhead on a Zeiss AxioObserver Z.1 inverted microscope . A Photometrics Evolve EMCCD camera was used to acquire the images.Graphs displayed are the combination of averages over at least three independent experiments. Where single comparisons are displayed, a two-tailed t-test was performed. Where multiple comparisons are displayed, one-way ANOVA was performed. When comparing survival curves, data from at least three independent experimental replicates were pooled and analyzed using Cox proportional hazard regression analysis where the experimental conditions were included as group variables. The survival distributions were displayed in a graphical format using Kaplan-Meier plots. Significance throughout was defined as p < 0.05. Statistical analyses were performed using GraphPad Prism, version 6, and R statistical software, version 3.S1 FigTg(mpeg:dendra2) x Tg(lyz:lta4h-2a-mCherry) larvae. Scale bar is 20 \u03bcm. (B) Average aggregate size, as measured by the peripheral area of aggregates, and (C) average number of aggregates per larvae at 24 hpi following infection with 50 CFU S. iniae in control or Lta4h morphants in a WT or Tg(lyz:lta4h) (lyz:lta4h) larvae background. Area and number were not statistically significant across all conditions.(A) Representative 63X images of macrophage aggregates in double transgenic (PDF)Click here for additional data file.S1 MovieNon-aggregate macrophages (white arrows) nearby the aggregate are still motile, while macrophages within the aggregate are remarkably non-motile. A motile macrophage (red arrow) takes a circuitous path around the periphery of the aggregate.(M4V)Click here for additional data file."}
+{"text": "The original publication  misses oKirsten Jensen Quas, Neuropsychologist, Head of Research and Development at Brain Injury Center BOMI, Roskilde, Denmark."}
+{"text": "X-ray Photoelectron Spectroscopy (XPS) results showed the opposite shifts of binding energy for Ti 2p and Ag 3d, indicating the transfer of electrons from metallic Ag to TiO2 owing to the Ag-O-TiO2 composite formation. UV-Vis absorption spectra showed the blue shifts of the surface plasma resonance peaks, and the maximum absorption peak intensity was obtained for TiO2 at 30 nm. The surface-enhanced Raman scattering (SERS) peak intensity first increased and then decreased when the TiO2 thickness changed. The observations of SERS, XPS, and UV-Vis absorption spectra were explained by the dependency of the charge-transfer process on TiO2 thickness, which was ascribed to the changing dielectric properties in the metal/semiconductor system.TiO T2 thickness changed from 0 to 40 nm. The charge-transfer (CT) process between the molecule and the TiO2/Ag substrate may play an important role in the change of SERS intensity. The degree of charge\u2013transfer is used to evaluate the contribution of the chemical mechanism (CM) to the SERS intensity [2/Ag system, the peaks at 1077 cm\u22121 and 1436 cm\u22121 were chosen for CM analysis. The band at 1077 cm\u22121 is for the C-S stretching mode (a1 mode), which is totally symmetric to the SERS signal contributions. The other peak, 1436 cm\u22121 (b2 mode) is non-totally symmetric, because the adsorption effect and the SERS effect are affected by the CT process.The SERS peak intensity first increased and then decreased when the TiOntensity ,30. In t2 thickness was 0 nm, 10 nm, 20 nm, 30 nm, and 40 nm, which indicates the charge transition from the Fermi level of the TiO2/Ag composites to the lowest unoccupied molecular orbitals (LUMO) of the PATP molecules, as shown in According to the CT mechanism, non-vibration modes such as the b2 mode, are usually enhanced by the Herzberg\u2013Teller contribution of CT, whereas the a1 model was not affected by the contribution of CT. In this case, changes in the CT process caused by various CM effects were qualitatively analyzed by PCT. The values of the degree of charge\u2013transfer first increased from 0.693, to 0.746, 0.748, and then to 0.759, followed by a decrease to 0.751 when the TiO2 layer of different thicknesses to form the structure with Ag nanoparticles embedded in the TiO2 layer. The XPS peaks of Ti and Ag moved in opposite directions, indicating the changed electron density between the TiO2 and Ag, which in turn indicates the promotion of the electron transfer from the surface of the Ag nanoparticle to the TiO2 through the formation of the Ag-O-Ti composite. When TiO2 thickness changed from 10 nm to 40 nm, the UV spectra showed the blue shift resonance peaks from 630 nm to 560 nm and the maximum absorption peak intensity was obtained for the TiO2, namely 30 nm, due the controlled electron transfer process by the surrounding materials. The obvious SERS effects were observed, and the peak intensity first increased and then decreased when the TiO2 thickness changed, and the thickness-dependent changes were evaluated by the degree of charge\u2013transfer. The observations of the XPS, UV absorption, and SERS effect were related closely to the dielectric properties of the metal-embedded structure and the interfacial electron transfer between the TiO2 semiconductor and Ag nanoparticles.In summary, a thin film of Ag was sputtered onto the TiO"}
+{"text": "EETs) and hydroxyeicosatetraenoic acids (HETEs) by cytochrome P450s. In this study, we found that 14,15\u2010EET and 20\u2010HETE\u2010enhanced NGF\u2010induced rat pheochromocytoma PC12 cell neurite outgrowth even at the concentration of 100\u00a0\u22121nmol\u00a0L. LC\u2010MS analysis revealed that 14,15\u2010EET was effectively produced from arachidonic acid by rat CYP2C11, 2C13, and 2C23, and these P450s were expressed in PC12 cells. An inhibitor of these P450s, ketoconazole, inhibited neurite outgrowth, whereas inhibition of soluble epoxide hydrolase, which hydrolyzes EETs to their corresponding diols enhanced neurite outgrowth. To determine the mechanism of neurite formation enhancement by arachidonic acid metabolites, we focused on transient receptor potential (TRP) channels expressed in PC12 cells. The TRPV4 inhibitor HC067047, but not the TRPV1 inhibitor capsazepine, inhibited the effects of 14,15\u2010EET on neurite outgrowth of PC12. Furthermore, 14,15\u2010EET increased the cytosolic calcium ion concentration and this increase was inhibited by HC067047. 14,15\u2010EET also enhanced neurite outgrowth of primary cultured neuron from rat hippocampus. This study suggests that arachidonic acid metabolites produced by P450 contribute to neurite outgrowth through calcium influx.Polyunsaturated fatty acids, such as arachidonic acid, are accumulated in brain and induce neuronal differentiation. Arachidonic acid is metabolized to epoxyeicosatrienoic acids ( EETepoxyeicosatrienoic acidHETEhydroxyeicosatetraenoic acidP450 or CYPCytochrome P450sEHsoluble epoxide hydrolaseTRPtransient receptor potential12 is important in neuronal outgrowth of mouse axons.Polyunsaturated fatty acids, such as arachidonic acid and docosahexaenoic acid (DHA), are essential for infant development, and especially effective for neuronal diseases such as Alzheimer's disease and Parkinson's disease.+\u2013K+\u2010ATPase2+\u2010activated K+ channels and transient receptor potential (TRP) channels.Cytochrome P450s (P450s) are monooxygenase and phase I drug metabolizing enzymes. P450s also oxidize arachidonic acid to epoxyeicosatrienoic acids (EETs) and hydroxy\u2010eicosatetraenoic acids (HETEs).2 in PC12 cells.In the brain, HETEs and EETs are released from neurons, astrocytes, and cerebral blood vessels, and contribute to cerebral blood flow and angiogenesis,22.1Dulbecco's modified Eagle's medium (DMEM), Dilauroyl\u2010 phosphatidylcholine, and RN\u20101747 were purchased from Wako Pure Chemicals . Fetal calf serum (FCS), penicillin\u2010 streptomycin solution, ketoconazole, and arachidonic acid sodium salt were purchased from Sigma Chemical . 5,6\u2010, 8,9\u2010, 11,12\u2010, and 14,15\u2010EET, 5\u2010, 8\u2010, 9\u2010, 11\u2010, 12\u2010, 15\u2010, 16\u2010, 17\u2010, 18\u2010, 19\u2010, and 20\u2010HETE, and capsazepine were purchased from Cayman Chemical Co. . Antibodies against CYP2C11, 2C23, 2E1, 4A2, sEH, NADPH\u2010cytochrome P450 reductase, and \u03b2\u2010actin were prepared as described previously.2.22 and 95% air. For the neurite outgrowth assay, cells were seeded at 1\u00a0\u00d7\u00a0104 cells per well in 24\u2010well plates. After 1\u00a0day, culture medium was replaced with DMEM containing 0.25% HS, 0.13% FCS, and NGF (50\u00a0ng/mL) with EETs, DHETs or HETEs at the indicated concentrations. Differentiated cells with neurites were defined as those with neurite length greater than the cell body of individual cell, and ratio of differentiated cells to total number of cells was determined. Neurite length of differentiated cells was measured using Image J.Rat pheochromocytoma (PC12) cells were obtained from RIKEN cell bank . Cells were cultured in DMEM containing 10% horse serum, 5% fetal calf serum(FCS), penicillin (100\u00a0units/mL), and streptomycin (100\u00a0\u03bcg/mL), and maintained at 37\u00b0C in 5% CO2.35 cells per cloning ring on the poly\u2010ethylene\u2010imine coated dish with Neurobasal Medium  containing 2% B27 Supplement (Thermo Fisher Scientific), penicillin (100\u00a0units/mL) (Thermo Fisher Scientific), streptomycin (100\u00a0\u03bcg/mL) (Thermo Fisher Scientific), 5\u00a0\u03bcg/mL Insulin , and 0.5\u00a0mmol\u00a0L\u22121l\u2010Glutamine.\u22121), 14,15\u2010DHET (100\u00a0nmol\u00a0L\u22121), or TRPV4 agonist RN\u20101747 (10\u00a0\u03bcmol\u00a0L\u22121) were added to the cell medium. After 24\u00a0hours, the length of neurite diffused into the free space from cells was measured. For immunofluorescence analysis, the cells exposed to 14,15\u2010EET for 48\u00a0hours were fixed with 4% PFA, following BSA blocking for 1\u00a0hour, and incubated with anti\u2010neurofilament antibody , and Dylight594\u2010conjugated secondary antibody  in Immuno\u2010enhancer solution . Image was obtained by confocal microscopy . All experiments were conducted in accordance with guidelines on the welfare of experimental animals and with the approval of the Ethics Committee on the use of animals of Kwansei Gakuin University.Neuron\u2010rich cultures were prepared from hippocampus of Wistar rats  at embryonic day 18 as described previously.2.45 was performed as described previously.5 (50\u00a0pmol), NADPH\u2010cytochrome P450 reductase (0.3 units), and dilauroylphosphatidylcholine (5\u00a0\u03bcg) was incubated with 100\u00a0\u03bcmol\u00a0L\u22121 arachidonic acid and 1\u00a0mmol\u00a0L\u22121 NADPH in 0.1\u00a0mol\u00a0L\u22121 phosphate buffer at pH 7.4 containing 10\u00a0mmol\u00a0L\u22121 MgCl2 and 150\u00a0mmol\u00a0L\u22121 KCl for 15\u00a0minute at 37\u00b0C. One unit of the NADPH\u2010cytochrome P450 reductase activity was defined as the amount of reductase catalyzing the reduction of 1\u00a0\u03bcmol of cytochrome c per min. The final volume of the reaction mixture was 0.5\u00a0mL. The reaction was stopped by adding 100\u00a0\u03bcL of acetonitrile, and the metabolites were extracted with 2\u00a0mL of ethyl acetate. After drying the organic layer under nitrogen, the resulting residue was dissolved in ethanol and analyzed by UPLC/ESI/MS. Chromatography was performed with a C18 reversed phase column  and the UPLC system . Mobile phase A (20% methanol and 0.1% acetic acid) and mobile phase B  were used, and chromatography was done at a flow rate of 0.2\u00a0mL/minute by gradient elution as follows: a linear gradient from 100% A to 60% B at 0\u201018\u00a0minute, 60% B at 18\u201023\u00a0minute a linear gradient from 60% B to 100% B at 23\u201025\u00a0minute, and 100% B at 25\u201026\u00a0minute. Mass spectrometry was carried out using a Nanofrontier LD mass spectrometer  and then ionized by electrospray ionization (ESI). ESI was accomplished in the negative ion mode with a spray potential of 3200\u00a0V. The analytes were detected by a tandem TOF monitored for total ions at m/z 319.2 for HETEs or EETs. The amount of produced HETEs and EETs was determined by a calibration curve prepared with authentic metabolites.Purification of rat P450 , NADPH\u2010cytochrome P450 reductase, and cytochrome b2.5l\u2010lysine\u2010coated dishes. After incubation for 24\u00a0hours, cells were treated with 50\u00a0ng/mL NGF and cultured for 2\u00a0days. Cells were washed with PBS and incubated with 5\u00a0\u03bcg/mL Fura\u20102 AM in Recording medium  for 1\u00a0hour at 37\u00b0C. After washing with PBS, Recording medium was added to the dishes. Cells were stimulated with EET or DHET, and the ratio of fluorescence intensity was monitored at 340/510\u00a0nm and 380/510\u00a0nm (excitation/emission) every 0.5\u00a0second for 1\u00a0minute by an EnVision 2104 Multilabel Reader . Rat neuronal cells were isolated and seeded on the poly\u2010l\u2010lysine\u2010coated dishes. After 3\u00a0days in culture, cells were incubated with 7.5\u00a0\u03bcg/mL Fluo\u20104AM in cell culture medium for 1\u00a0hour at 37\u00b0C. After washing with PBS, Recording medium was added to the dishes. Cells were stimulated with 14,15\u2010EET and/or HC067047, and the fluorescence intensity was monitored at 485/535\u00a0nm (excitation/emission) every 0.5\u00a0second for 1\u00a0minute by an EnVision 2104 Multilabel Reader.PC12 cells were seeded in poly\u20102.6P\u00a0<\u00a00.05 was considered statistically significant.The differential significance of the results obtained was determined by One\u2010way ANOVA followed by a Bonferroni/Dunn post hoc test, and 33.1\u22121 14,15\u2010EET efficiently enhanced NGF\u2010induced cell differentiation by 240% and neurite extension by 140% compared with control, although effects of 100\u00a0nmol\u00a0L\u22121 arachidonic acid on them were small  inhibited neurite outgrowth of PC12 cells  inhibited production of 14, 15\u2010EETs by CYP2C11, 2C13 and 2C23 from arachidonic acid . Pozzi et\u00a0al also have shown that 1\u00a0\u03bcmol\u00a0L\u22121 ketoconazole efficiently inhibited EET synthase, but not 1\u00a0\u03bcmol\u00a0L\u22121 MS\u2010PPOH.To investigate the contribution of 14,15\u2010EET\u2010producing P450 to PC12 cell neurite outgrowth, cells were treated with P450 inhibitors. First, we chose a general P450 inhibitor, SKF525A, but it was toxic at the working concentration. We used ketoconazole, which is an inhibitor for CYP3A P450s, but it can inhibit CYP2C P450s and EETs synthesis at much lower concentrations than SKF525A.3.5\u22121), but not the TRPV1 inhibitor capsazepine (500\u00a0nmol\u00a0L\u22121), inhibited the neurite outgrowth enhanced by 100\u00a0nmol\u00a0L\u22121 14,15\u2010EET . 5\u00a0\u03bcmol\u00a0L\u22121 RN\u20101747 efficiently enhanced NGF\u2010induced cell differentiation and neurite extension  was added, and cultured for additional 24\u00a0hours. As a result, 14,15\u2010EET enhanced neurite length of cells by 150% compared with control, but 14,15\u2010DHET did not  14,15\u2010EET would be a physiological concentration because total hypothalamic EET concentration was estimated to be 120\u00a0ng/g wet tissue in rat.Arachidonic acid, which is abundant in the brain has been shown to be involved in neurite outgrowth, and essential for infant growth and development. It has been reported that 200\u00a0\u03bcmol\u00a0L14,15\u2010EET was mainly produced by rat CYP2C11, 2C13, and 2C23 from arachidonic acid. CYP2C13 reveals relatively low activity toward drugs and testosterone.In this study, we detected 14,15\u2010EET\u2010 producing P450s  in PC12 cells, and their inhibitor, ketoconazole, abrogated NGF\u2010enhanced neurite outgrowth, suggesting the contribution of these P450s to neurogenesis. Ketoconazole also inhibit EET\u2010induced relaxation in monkey cerebral artery.TRPV4 is a nonselective cationic channel known to be activated by several endogenous or synthetic chemicals.Our study suggests that arachidonic acid metabolites, 14,15\u2010EETs, produced by cytochrome P450 contribute to neurite outgrowth through activation of TRPV4. Regulation of EET levels using sEH inhibitors, which hydrolyze EETs in the brain, has been explored as therapy for cerebral vascular diseases, such as stroke, because EETs will improve cerebral blood flow as vasodilators.Participated in research design: Oguro A. and Imaoka S.Conducted experiments: Oguro A.Conducted experiments for isolation of rat hippocampal neuronal cells: Kudoh NS.Isolation of rat hippocampal neuronal cells: Inoue T.Performed data analysis: Oguro A.Wrote or contributed to the writing of the manuscript: Oguro A. and Imaoka S.None declared."}
+{"text": "Tardive dyskinesia (TD) is a devastating motor disorder associated with the etiological process of schizophrenia or antipsychotic medication treatments. To examine whether cerebral morphological changes may manifest in TD, we used voxel-based morphometry to analyze high-resolution T1-weighted brain structural magnetic resonance images from 32 schizophrenics with TD (TD group), 31 schizophrenics without TD (non-TD group), and 32 healthy controls (HC group). We also assessed psychopathological symptoms with the Positive and Negative Syndrome Scale (PANSS), and TD severity with the Abnormal Involuntary Movement Scale (AIMS). We compared gray matter volumes (GMVs) among groups, and tested for correlations between GMV changes and psychopathological symptoms or TD severity. The results showed significant differences in GMV in the frontal and temporal cortices, insula and cerebellum among the three groups. Brainstem and inferior frontal and precentral gyri GMVs were significantly larger, whereas cuneus and lingual gyrus GMVs were significantly smaller in the TD group as compared to non-TD group. Further, the cuneus and lingual gyrus GMVs were positively correlated with AIMS scores in the TD group. The current results suggest that TD may be associated with the alterations in GMV that are different from that of schizophrenics without TD. Further studies are needed to confirm and to examine the functional significance of these structural findings. TD reduces quality of life and disrupts a patient\u2019s personal, social, and professional function7. A recent report showed that the annualized incidence of TD was 5.5% in patients treated with first-generation antipsychotics, and 3.5% in those treated with second-generation antipsychotics9. The incidence of TD with atypical antipsychotics is higher than expected, and underscores the fact that TD remains a significant clinical problem10. However, our understanding of the pathogenesis of TD is still lacking11.Tardive dyskinesia (TD) is characterized by repetitive involuntary movements, usually involving the mouth, face, and tongue, and sometimes the limb and trunk musculature, as part of the etiological process of schizophrenia or side effects of antipsychotic medications. TD may occur from days to years after the initiation of antipsychotic treatment and persist even after a switch of medications to atypical antipsychotics13. Recurring dyskinetic movements of facial and limb muscles, indistinguishable in form from tardive dyskinesia, are observed in patients with schizophrenia who have never been exposed to antipsychotics, suggesting that the intrinsic pathophysiology of schizophrenia contributes to tardive dyskinesia4. The leading hypothesis proposes that, as a result of chronic blocking of dopamine receptors, long-term antipsychotic treatments cause compensatory increase in dopamine receptor sensitivity14. On the other hand, TD symptoms may persist even after drug withdrawal, raising the issue that heightened dopamine receptor sensitivity may not account for all of the symptoms of TD.Several hypotheses have been proposed to explain the development of TD, including dopamine receptor hypersensitivity, \u03b3-aminobutyric acid (GABA) deficiency, and neuronal damage due to free radicals15. Voxel-based morphometry (VBM) is a convenient and well-validated quantitative assessment of gray matter volumes (GMVs) by magnetic resonance imaging (MRI), frequently used to document changes in brain volumes in various neurological disorders16. Although not without limitations, VBM allows for an objective and automatic assessment of gray matter (GM) morphology. Few studies have focused on documenting MRI findings specific to TD, and their results were surprisingly inconsistent21. For example, Li et al.19 found that schizophrenics with TD had significantly reduced GMVs in the bilateral inferior and right superior frontal gyri in correlation with symptom severity. Sarro et al.20, however, found that TD-related volume reductions were predominantly subcortical, involving the basal ganglia and thalamus. The inconsistencies do not merely concern the anatomical locations of morphological changes; the putative correlations between morphology and TD symptom severity varied widely in the above-mentioned studies. These inconsistencies may in part be related to small sample sizes, heterogeneity in patient samples, or differences in imaging and analysis methods across the studies.One alternative hypothesis is that TD may be associated with structural brain changesHere, we attempted to address morphological changes in TD with a more homogeneous Chinese patient sample, high-resolution imaging protocol, and standard VBM analytics. We hypothesized that TD-specific changes in GMVs would be identified in patients with schizophrenia and TD, and that the morphological changes would correlate with the clinical symptoms or severity of dyskinesia.p\u2019s > 0.05). However, the TD group had significantly higher PANSS Negative scores (p\u2009=\u20090.002) than the non-TD group  were indistinguishable between the TD and non-TD groups . Further, the TD group demonstrated significantly greater volume reductions in the frontal areas than the non-TD group (p\u2009<\u20090.01)  Fig.\u00a0. The bra01) Fig.\u00a0.Figure 1r\u2009=\u20090.60, p\u2009=\u20090.001) in the TD group (Table\u00a0p\u2009<\u20090.004). .Among the regions that showed significant differences between the TD and non-TD groups, the cuneus and lingual gyrus changes in GMV were positively correlated with AIMS scores  right-handed schizophrenics with TD (TD group), thirty-one schizophrenics  without TD (non-TD group), and thirty-two  healthy controls (HC group) were recruited for this study 32. The TD clinical ratings were performed by two experienced psychiatrists who were clinically trained to consistently perform AIMS testing as part of the general hospital training procedures, although they were not specifically trained for this study. In addition, patients with TD were re-evaluated for AIMS score by the same psychiatrist at least one month later, and were diagnosed with TD only if both evaluations yielded positive results. Psychopathology was assessed using the Positive and Negative Syndrome Scale (PANSS)33. Both scales had intra class correlation coefficients \u22650.75. This study was approved by the Ethics Review Board (IRB) of the Beijing HuiLongGuan hospital. All participants gave written informed consent before participating in the study. All patients were recruited from the inpatient services at Beijing HuiLongGuan Hospital according to a cross-sectional naturalistic design. All study procedures followed this research protocol. We confirmed that all research was performed in accordance with the relevant guidelines.TD was diagnosed using Schooler and Kane\u2019s Research Diagnostic Criteria (1982)Structural MRI data were acquired on a 3.0-Tesla MR scanner  using a T1-weighted (T1W) sagittal 3D-MPRAGE (magnetization prepared rapid acquisition with gradient echo) sequence: echo time (TE)\u2009=\u20092.6\u2009ms; inversion time (TI)\u2009=\u2009800\u2009ms; repetition time (TR)\u2009=\u20091,600\u2009ms; flip angle (FA)\u2009=\u20099\u00b0; field of view (FOV)\u2009=\u2009256\u2009mm\u2009\u00d7\u2009224\u2009mm; matrix size\u2009=\u2009512\u2009\u00d7\u2009448; slice thickness\u2009=\u20091\u2009mm; voxel dimension\u2009=\u20091\u2009mm\u2009\u00d7\u20091\u2009mm\u2009\u00d7\u20091\u2009mm. T2-weighted images (T2WI) were acquired using a turbo-spin-echo (TSE) sequence: 20 axial slices, thickness/gap\u2009=\u20095/1.5\u2009mm, matrix\u2009=\u2009512\u2009\u00d7\u2009416, TR\u2009=\u20094,140\u2009ms, TE\u2009=\u200992\u2009ms, FA\u2009=\u2009150\u00b0, FOV\u2009=\u2009187\u2009mm\u2009\u00d7\u2009230\u2009mm.http://dbm.neuro.uni-jena.de/vbm) technique34, implemented with Statistical Parametric Mapping (SPM8) (http://www.fil.ion.ucl.ac.uk/spm/software/spm8), and executed in MATLAB 2010 (https://www.mathworks.com/products/matlab.html). First, data from each participant were carefully checked three times for any scanner artifacts, motion problems or gross anatomical abnormalities. Then a noise reduction procedure was performed on each participant\u2019s native space T1W structural image using a spatial-adaptive non-local-means denoising filter.Individual high-resolution T1-weighted data were analyzed using the VBM8-Toolbox  was calculated as the sum of GM, WM and CSF volumes. Finally, after image smoothing, the GM images were entered into statistical modeling to obtain cluster sizes for each group.For image preprocessing, T1 images were first segmented into GM, white matter (WM) and cerebrospinal fluid (CSF) and normalized to an Montreal Neurological Institute (MNI) template using the unified standard segmentation option in SPM8p\u2009<\u20090.01), after applying an extent threshold (p\u2009<\u20090.001 uncorrected) with a minimum cluster size of 100 voxels 37. Then we performed correlation analyses to explore the relationship between regions with nominal TD vs. non-TD differences, and that between GMV differences and severity of TD symptoms as assessed by AIMS in the TD group, again using age, sex, education and CPZ as covariates. Volume size, clinical symptoms , and other clinical parameters were also explored. GMV group comparisons were performed in SPM8 and the correlation analyses between volume size of brain regions and AIMS were performed using SPSS 20.0 .ANOVAs were used to compare the age and education among the three groups. Non-parametric tests were used to compare the sex composition among the three groups. T-tests were used to compare the PANSS scores , medication in chlorpromazine (CPZ) equivalents, and duration of illness between the patient groups. Voxel-wise GMV differences among the three groups were investigated using ANCOVA, with age, sex, education and TIV as covariates. To avoid possible edge effects around the margin between different tissue types, all voxels with a GM probability value lower than 0.2 were excluded. Statistical inferences were made with a voxel-level statistical threshold ("}
+{"text": "P\u00a0<\u00a00.001) and OS . The association remained significant in the multivariate analysis that elevated pretreatment LDH level was associated with poor PFS  and OS . A high pretreatment LDH level was significantly correlated with shorter PFS and OS. Pretreatment LDH may serve as a predictive biomarker for advanced NSCLC patients treated with ICIs.The main aim of this study is to investigate whether baseline lactate dehydrogenase (LDH) is associated with the clinical outcome of non small\u2010cell lung cancer (NSCLC) patients treated with immune checkpoint inhibitors (ICIs). We searched Pubmed, the Cochrane Central library and Embase for peripheral blood biomarker of LDH in advanced NSCLC patients treated with ICIs. We extracted the hazard ratio (HR) with 95% confidence interval (CI) for the progression free survival (PFS) and overall survival (OS) and performed meta\u2010analysis of HR. Pooled estimates of treatment outcomes were calculated by stata 15.1. Six studies with 1136 patients were included in this study. The pooled results of univariate analysis suggested that an elevated pretreatment LDH level was correlated with significant shorter PFS (HR\u00a0=\u00a01.53, 95% CI 1.27\u20101.83,  Recent studies have demonstrated that elevated pretreatment level of LDH is associated with poor outcome in several cancer types and baseline LDH level may predict the prognosis of patients treated with ICIs.22.1A search for relevant published and unpublished studies was performed using Embase, PubMed, and Cochrane central Library. The search terms utilized were \u201cimmune check point inhibitor\u201d, \u201ccytotoxic T lymphocyte antigen\u20104\u201d, CTLA\u20104, \u201cprogrammed death\u20101 receptor\u201d, \u201cprogrammed death ligand\u20101\u201d, \u201cPD\u20101 inhibitor\u201d, \u201cPD\u2010L1 inhibitor\u201d, ICI, immunotherapy, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, ipilimumab, \u201clactate dehydrogenase\u201d, LDH, predictor, predict, prognosis, prognostic, lung cancer, \u201cnon small\u2010cell lung cancer\u201d, NSCLC. The last search was updated on June 13, 2018. Both free text and medical sub\u2010headings (MeSH) terms were used in the search strategy.2.2The following articles were included in the analysis: (a) Human studies investigated NSCLC patients receiving ICIs treatment; (b) Determination of the relationship between baseline LDH level and prognosis; (c) Hazard ratio (HR) with 95% CI were presented for OS and/or PFS; (d) If the same population was used by two or more studies, only the one with the largest sample size and latest information was included; (e) the full text was available.2.3The following studies were excluded from the analysis: (a) Case reports, reviews, comments, editorials, letters or articles unrelated with our topics; (b) Publication in a language other than English.2.4For each included study, we extracted the data including first author's name, the year of publication, district of study, type of immune checkpoint inhibitor, the total number of patients, sex, age, cut\u2010off value of LDH, histology, study design, and study outcomes. Two researchers (Zhibo Zhang and Ye Li) independently extracted the data of HRs and the associated 95% CIs for PFS and OS outcomes from both univariate and multivariate analyses. Any discrepancy was resolved by discussion. The present review was prepared according to Preferred Reporting Items for Systematic reviews and Meta\u2010Analyses (PRISMA).2.5As previously reported,2.6I2 statistics to detect any heterogeneity between different studies. A result of P\u00a0>\u00a00.1 and I2\u00a0<\u00a050% indicated that no significant between\u2010study heterogeneity was present. Publication bias was evaluated by examining the funnel plot of the effect size for each study. We set the nominal level of significance 5% and all 95% CIs were 2\u2010sided. All statistical analyses were performed using STATA V.15.1 .We used the method of random\u2010effects inverse\u2010variance\u2010weighted to pool outcomes, which is calculated by HR and its 95% CI to estimate the size of the treatment benefit. We used the 33.1We identified 1199 articles after searching online databases. By verifying related terms in the titles and abstracts, 1110 articles did not meet the inclusion criteria, including 292 duplicate records, 151 irrelevant articles, 585 with no usable data, and 82 without full text. With further reading the whole article, we excluded 83 literatures, all of which were reviews or case reports. Finally, 6 studies were selected for the present meta\u2010analysis.3.2Six studies with 1136 patients were included in our study. Characteristics of the included studies are summarized in Table\u00a03.3P\u00a0<\u00a00.001), and the pooled results of multivariate analysis revealed that elevated baseline LDH level remained significantly associated with poor PFS .Five studies with 1042 cases were included in the final analysis of association between baseline LDH and PFS. As showed in Figure\u00a0P\u00a0<\u00a00.001), the pooled results of multivariate analysis revealed that elevated baseline LDH level was remained significantly associated with poor OS ; For OS, we did not observe significant statistical heterogeneity by either univariate or multivariate analysis .For PFS, no significant statistical heterogeneity was observed in either univariate or multivariate analyses (3.5P\u00a0=\u00a00.165) and PFS (P\u00a0=\u00a00.144).As shown in Figure\u00a04In this study, we demonstrated that the pretreatment LDH was associated with PFS and OS in NSCLC patients treated with ICIs in the univariate and multivariate analysis. These results suggested that pretreatment LDH may serve as a potential predictor for ICIs in patients with non small cell lung cancer.In the last decade, ICIs have brought a shift in the landscape of advanced\u2010stage cancer treatment. Despite of enormous success, not all patients achieve long\u2010lasting responses. Reliable predictive biomarkers remain to be found to identify patients who would benefit from ICIs. Systemic inflammatory status has been found closely correlated with worse prognosis in lung cancer.The aim of this meta\u2010analysis is to investigate whether pretreatment LDH is correlated with clinical outcome of advanced NSCLC patients treated with ICIs. Previous studies have demonstrated that the level of serum LDH was significantly related to the extent of the tumor and poor prognosis in NSCLC patients.Above all, the understanding of LDH is still immature because of the lack of a uniform cut\u2010off value. Although baseline LDH level is correlated with the outcome of patients receiving ICIs, it remains uncertain what value of LDH is best to estimate the survival of patients with NSCLC. Furthermore, since LDH is a dynamic marker, when to measure LDH during a patient's treatment course is also unclear. The last but not the least, whether a single LDH determination or several over a time course is better at predicting survival in patients receiving ICIs has not been established.Our study also has several limitations. First, the results of the meta\u2010analysis may jeopardize by the retrospective nature of included studies because of potential selection bias. Second, the number of studies included in the present meta\u2010analysis is relatively small, but the overall effect size is significant. Last but not least, the cut\u2010off value for LDH varied in these studies. The cut\u2010off values of LDH varied from 217 to 400\u00a0U/L. The level of LDH is influenced by the testing conditions, races, and age, which may be the cause of the difference in the cut\u2010off values of LDH. Even though, the difference in the cut\u2010off values may introduce bias to the results, the difference in the cut\u2010off values was minor.5In conclusion, this study demonstrates that a high pretreatment LDH level is statistically significantly associated with poor outcomes of NSCLC patients treated with ICIs. LDH is a potential useful predictive biomarker to select patients who can benefit from ICIs because of its convenient and non invasive nature. In the future, clinical trials are advocated to determine whether pretreatment LDH level could help stratify NSCLC patients who could benefit from ICIs.The authors declare no conflict of interest.\u00a0Click here for additional data file.\u00a0Click here for additional data file."}
+{"text": "The aim of the present study was to develop an alternative process to spray granulation in order to prepare high loaded spherical nicotinamide (NAM) pellets by wet extrusion and spheronisation. Therefore, a quality by design approach was implemented to model the effect of the process parameters of the extrusion-spheronisation process on the roundness, roughness and useable yield of the obtained pellets. The obtained results were compared to spray granulated NAM particles regarding their characteristics and their release profile in vitro after the application of an ileocolon targeted shellac coating. The wet extrusion-spheronisation process was able to form highly loaded NAM pellets (80%) with a spherical shape and a high useable yield of about 90%. However, the water content range was rather narrow between 24.7% and 21.3%. The design of experiments (DoE), showed that the spheronisation conditions speed, time and load had a greater impact on the quality attributes of the pellets than the extrusion conditions screw design, screw speed and solid feed rate (hopper speed). The best results were obtained using a low load (15 g) combined with a high rotation speed (900 m/min) and a low time (3\u20133.5 min). In comparison to spray granulated NAM pellets, the extruded NAM pellets resulted in a higher roughness and a higher useable yield (63% vs. 92%). Finally, the coating and dissolution test showed that the extruded and spheronised pellets are also suitable for a protective coating with an ileocolonic release profile. Due to its lower specific surface area, the required shellac concentration could be reduced while maintaining the release profile. Laccifer lacca. It is often used as pH-sensitive enteric coating due to its acid character and its high dissolution pH value of about 7.3  is determined as the mass of shellac (shellacm in mg) applied to the pellet surface (A in cm\u00b2): shellacM is the difference between the mass of the sample (samplem in mg) and the mass of the cores (coresm in mg) in the sample. The mass of the cores in the sample was calculated to the following equation:NAM samplem is the NAM mass in the sample (mg) and uncoated pelletsc the NAM content of uncoated pellets (%). The surface area of the sample was calculated from the average radius and the number of pellets (n): coresm to uncoated pelletm. The average mass of pellets was calculated from 500 pellets. The average radius was measured by particle size measurements by laser diffraction using a using the dry feeder of the particle analyzer  with vacuum-driven forced ejection, a vibration power alteration of 120 and a compressed air pressure of 0.3 MPa. A refractive index of 1.47 was used for NAM. The NAM content, after the application of the coating layer, was determined spectrophotometrically at 262 nm after dissolution of about 50 mg pellets in 100 mL Soerensen phosphate buffer at a pH value of 7.4 .The coating level (CL) [mg/cmribed by . MshellaDuring every run, the process yield, which was defined as the extruded mass per minute (g/min), was examined by weighing. The standard deviation of three measurements was calculated to control the process variability.To measure the moisture content during process, freshly produced extrudates were analysed before the spheronisation process. About 2.5 g sample were weighed before and after drying at 105 \u00b0C in drying oven for 24 h. The experiments were done in triplicate. The water content is expressed as the percentage of the total weight of the wet mass.The sieve analysis gives information about the particle size distribution after the extrusion-spheronisation process. The dried pellets of two spheronisation processes were sieved in a sieve shaker AS 200 control  using sieve sizes of 2, 1.4, 1, 0.5 and 0.25 mm to separate the pellets according to their size. The amplitude was set to 0.5 mm and the total sieve time to 3 min. Afterwards, the per cent weight of the fractioned pellets was calculated.The particle size analysis of spray granulated NAM pellets was done by Glatt Ingenieurtechnik GmbH  using a Camsizer XT .The shape of the spheronised pellets (n \u2265 6) was evaluated by optical inspection and by SEM. For SEM, pellets with a diameter between 1 mm and 1.4 mm were prepared on a holder with carbon Leit-tabs . Before examination in a Zeiss EVO 15  at an accelerating voltage of 10 kV, microcapsules were sputter-coated with a layer of 15 nm gold using a Quorum Q150RS  rotary pumped sputter coater. The pellets were categorical evaluated regarding their roundness and their roughness of the surface: roundness (category 1\u20135): 1 = irregular (like agglomerates); 3 = spherical; 5 = dumbbell-shaped or strains; roughness (category 1\u20135): increasing category for increasing roughness.For SEM pictures of the spray granulated NAM pellets were prepared and analysed as described in .The in vitro drug release study was carried out using a standard dissolution paddle apparatus at 100 rpm and 37 \u00b0C  . The expThe target dosage form is a pellet for oral administration. It should have a uniform and spherical shape, a smooth surface and a narrow size distribution for an appropriate application of an additional coating in a further step. The size should be smaller than 2 mm to be able to pass the pylorus independently from gastric emptying. Furthermore, the vitamin load should be high to prevent a high number of capsules, which have to be swallowed to reach the daily dose.The following CQAs were determined based on previous experiments: vitamin load: \u2265 80%; a roundness of category 2.0\u20134.0; a roughness of category \u2264 2 and a maximal useable yield (pellets with a diameter between 1 mm and 2 mm and an acceptable roundness).w/w). The influence of potentially CPPs such as powder mixture feed rate, screw speed and screw configuration (for the extrusion process) and rotation speed, rotation time and mass load (for the spheronisation process) on the CQAs was investigated. The identification of CMAs and CPPs were based on preliminary risk assessment and prior knowledge. Note: CMA is not an ICH  term but it is used here to describe the quality attributes of the materials.The most critical material attributes are the high water solubility of nicotinamide (1000 g/L) and the corresponding vitamin to binder ratio. For these studies, the NAM:MCC ratio was kept constant, 80%:20% . For the extrusion process, two 22 full factorial designs were determined for two different screw designs, which were already shown in w/w NAM and 20% w/w MCC was chosen. In order to get a paste that can be spheronised without blocking the die of the extruder, the range of the solid-to-liquid ratio had to be evaluated before a systematic investigation. Distilled water was added to the extruder with a constant rate of 1.74 g/min. The solid feed rate was increased from 5.3 g/min to 7.9 g/min to evaluate the particle shape changes  can lead to negative effect on the process yield and on the pellet properties [The effect of the solid feed rate on the moisture content of the extrudates is illustrated in operties . As showoperties .r2) varied in a broad range depending on the responses. The r2 of the roughness (0.845) and the useable yield (0.817) indicated a good fitting between the independent variables and responses in the model, in contrast to the moisture content (0.577), the process yield SD (0.544) and the roundness (0.470). However, only the model for the prediction of the roundness gave no significant results (p = 0.1471). The outcomes of all tested combinations are shown in p = 0.0002) and the useable yield (p = 0.0087) of the particles as well as on the process yield variations (p = 0.0154) and the moisture content (p = 0.0041). Furthermore, the combination of hopper speed and screw design influenced the useable yield significantly (p = 0.0221). The interaction profiles between screw design and hopper speed on the responses are shown in To evaluate the effect of the extrusion process parameters on the particle characteristics, the parameters for the spheronisation process  as well as the formulation composition were held constant. During the extrusion process the factors screw design, screw speed and hopper speed  = 0.0382 \u00d7 hopper speed \u2212 3.6991) were varied. The effect of the above factors on the responses such as moisture content and process yield variations (SD of process yield) of the extrudates and roundness, roughness and useable yield of the spheronised particles was examined using a DoE approach. The coefficient of determination  resulted in a higher roughness, although a higher density was documented for a higher water content after spheronisation . Howeverr2 of the roughness (0.976), spheronisation yield (0.958), roundness (0.741) and useable yield (0.614). The spheronisation load had a significant effect on the useable yield (p = 0.0380), the roughness (p = 0.0026) and the spheronisation yield (p = 0.0016), whereby the speed affected significantly the roundness (p = 0.0202), roughness (p = 0.0111) and the spheronisation yield (p = 0.0238). The rotation time showed significant influences on the roundness (p = 0.0426) and roughness (p = 0.0026) of the particles and the spheronisation yield (p = 0.0321). Furthermore, the combination of speed and time significantly affected the particle roughness (p = 0.0026). The interaction profiles of the factors load, speed and time are exhibited in The results of the DoE for the spheronisation process are shown in The corresponding SEM pictures of the ten runs , show irr2) varied according to the responses useable yield (r2 = 0.614), spheronisation yield (r2 = 0.958), roughness (r2 = 0.976) and roundness (r2 = 0.741). The obtained equations for these responses in term of the used factors  are as follows:As already mentioned, the coefficient of determination  of the measured responses: screw design L1, screw speed 200 rpm, hopper speed set 270 (6.6 g/min), rotation speed of 100%, rotation time of 3.5 min and a load of 15 g. Therefore, only the spheronisation model was verified due to similar extrusion conditions as used for the DoE. The run was conducted for about 3 h. Every 15 min, the moisture content of the extrudates was measured. The mean was 23.6% \u00b1 0.58% for 13 measurements in total. The process yield of 44 measurements during the process was 7.95 g/min \u00b1 0.65 g/min, which confirmed a constant process. Furthermore, all runs resulted in well-formed particles with a spherical shape (roundness: 2.5) and a low roughness of 2 . In comp\u00ae) could lead to a higher yield but would also reduce the relative load of NAM. Furthermore, as shown by SEM pictures in As indicated in 2; 7.6 mg/cm2 and 10.0 mg/cm2) of the extruded NAM pellets (w/w) coating weight gain was needed for the application of a coating level of 1 mg/cm2, compared to 12% (w/w) for the spray granulates. Related to 100 g NAM, the required addition of shellac was reduced from 60 g to min. 27.5 g for spray granulated compared to wet extruded NAM pellets, respectively. Due to the resulting thicker coating level for extruded pellets, it is proposed that the required amount could be further decreased (max. to 16.5 g of shellac per 100 g NAM).After the extrusion-spheronisation process, the resulted pellets were evaluated regarding their dissolution profile without and with an enteric coating, according to Fangmann et al.  Figure A,B. In a pellets C, resultAll in all, the extrusion-spheronisation process demonstrates an opportunity to produce highly loaded NAM pellets with a useable yield of about 90%. The pellets were spherical and had a rough surface. In comparison to spray granulated NAM pellets, the extruded NAM pellets resulted in a higher roughness and the useable yield was increased. Finally, the coating and dissolution test showed that the extruded and spheronised pellets are also suitable for a protective coating with an ileocolonic release profile in vitro. The quality by design (QbD) proved to be a suitable method to better understand the effects of the critical process parameters (CPPs) on the critical quality attributes (CQAs) of the desired product in an efficient way. It was shown, that the prediction model was in good agreement with the measured outputs. The design of experiments (DoE), showed the spheronisation conditions speed, time and load had a greater impact on the CQAs of the pellets than the extrusion conditions screw design, screw speed and solid feed rate (hopper speed). The roundness was significantly affected by the rotation speed and time, whereby the roughness varied depending on the spheroniser load, time, speed time and the solid feed rate. Furthermore, the spheroniser load, solid feed rate and the solid feed rate screw design significantly impacted the useable yield. The screw speed showed no significant effect. The moisture content is one of the most critical factors for an optimal spheronisation result. Therefore, the moisture content need to be adjusted to the formulation characteristics to ensure an optimal product. The spheronisation conditions must be adjusted using a multivariate approach."}
+{"text": "Major depressive disorder and associated mood syndromes are amongst the most common psychiatric disorders. To date, electroconvulsive therapy (ECT) is considered the most effective short-term treatment for patients with severe or treatment-resistant depression. In clinical practice, there is considerable variation in the ECT dosing schedule, with the number of sessions typically ranging from 6 to 12, with early antidepressant effects being predictive of increased positive outcomes. We describe here an unusual case of a female patient with severe depression who did not respond to ECT until the 11th session, after which she had shown a drastic improvement in her mental state.A 75-year-old female presented to the old age psychiatry inpatient unit with new onset dysphoric mood, anhedonia, and severe negativity. She scored 23 on the 17-item Hamilton Rating Scale for Depression (HAM-D), and was rated 6 on Clinical Global Impression severity (CGIS) by the responsible clinician. She suffered from post-natal depression fifty years ago and was successfully treated with ECT. She was therefore initiated on a course of ECT treatment. Her condition initially deteriorated, displaying features of catatonia and psychosis, unresponsive to ECT treatment or concurrent psychotropic medications. After 11th ECT session, she started to show signs of clinical improvement and returned close to her baseline mental state after a total of 17 ECT sessions. She remained well 3\u00a0months post-treatment, scoring 4 on HAM-D, Clinical Global Improvement or change (CGI-C) rated as 1 (very much improved). The diagnosis was ICD-10\u2009F32.3 severe depressive episode with psychotic symptoms.we describe here an unusual case of delayed response to electroconvulsive therapy in the treatment of severe depressive disorder. Studies have shown the number of acute ECT treatments to be highly variable, affected by a number of factors including treatment frequency, condition treated and its severity, the ECT technical parameters, as well as concurrent use of pharmacological treatment. This may call for re-consideration of the current ECT treatment guidelines, requiring more research to help stratify and standardize the treatment regime. Major depressive disorder (MDD) and associated mood syndromes are amongst the most common psychiatric disorders. MDD can at times become debilitating, or at worst, life-threatening. In general, antidepressant medications can be effective in treating MDD, but they fail to achieve remission in approximately 1 in 3 patients [Electroconvulsive therapy (ECT) is considered the most effective short-term treatment for patients with severe or treatment-resistant depression, with 70\u201390% of patients showing improvement . ECT invThere is a considerable variation in ECT dosing schedules in clinical practice. During an acute course of the treatment, ECT is given as a twice-weekly regime in the United Kingdom , and twiThe ECT handbook of the Royal College of Psychiatrists stated that clinicians may wish to reassess the need for ECT if there was no response after six sessions. If there is no response within 12 treatment sessions, it is unlikely to have a \u2018sustained response to ECT .\u2019The number of ECT sessions required to elicit improvement tends to vary depending on the intensity of ECT, where a thrice weekly regime yields faster response than twice weekly . Other fOne of the variables predictive of increased positive outcomes is the early antidepressant effects of ECT. A study by the Consortium for Research in ECT has indicated that more than half of patients treated with ECT showed improvement after 3 sessions, and 65% achieved remission after 10 sessions . LikewisIn this article, we outline a case of a female patient, who presented with severe depressive episode. She initially failed to respond to her ECT treatment, until the 11th session, when she had a drastic improvement in her clinical presentation.A 75-year-old female patient presented to the old age psychiatry inpatient unit with a 10-week history of deteriorating depressive symptoms, triggered by a telephone scam. She had repeatedly attempted to slit her wrists with a knife, and developed concerns about having cancer following two brief illnesses with infections. On admission, she presented with dysphoric mood, anhedonia, and severe negativity. She described herself as not having \u2018any thoughts in my head,\u2019 and \u2018frightened.\u2019 She showed evidence of catastrophizing, and appeared hypervigilant. Her food and fluid intake had been poor over the past 10\u2009weeks.In the past, she suffered from post-natal depression fifty years ago and was successfully treated with ECT. In the intervening years, she did not suffer from any episodes of severe mental illness, with no depression or mania. Physically, she suffered from hypertension, type 2 diabetes mellitus, chronic obstructive pulmonary disease, and polymyalgia rheumatica for which she was on a reducing dose regime of prednisolone . She was a non-smoker, and there was no history of alcohol or substance misuse. She lives with her husband and has two supportive children living nearby.On examining her mental state, she was catatonic, displaying excessive motion intermittently including bilateral non-Parkinsonian motion of the upper limbs and lip licking. Her speech was slow and interrupted in flow. She felt low in mood and was negative in her outlook. She was not formally thought disordered, and she was cognitively intact. She had insight into the deterioration of her mental state and agreed to informal admission to the hospital for assessment and treatment.Admission blood tests were normal. She was not anaemic, thyroid function tests were normal.Initially on the ward, she appeared settled and was able to hold conversations with others. She was periodically anxious, requiring encouragement with food and drink intake. Due to her good response previously, the treatment team arranged a course of ECT during the admission, and she was deemed to have capacity to consent to the treatment. Prior to starting ECT, a 17-item Hamilton Rating Scale for Depression (HAM-D) was obtained, for which she scored 23, indicative of severe depression. A Mini-mental state examination (MMSE) was performed, when converted into Addenbrooke\u2019s Cognitive Examination III (ACE-III), gives 69\u201373/100. Clinical Global Impression Severity (CGIS) by the responsible clinician was rated 6 (severely ill).ECT was performed with Thymatron\u00ae System IV, using bitemporal electrode placement with a pulse width of 0.50 millisecond. The details of each ECT session are summarised in Table\u00a0Unfortunately, whilst on the ward, her clinical condition deteriorated. Her catatonia worsened considerably. She displayed virtually no interaction with external world. She showed mutism, fixed, non-reactive gaze. At times she would whisper \u2018locked in,\u2019 \u2018I can\u2019t\u2019. She repeatedly exhibited a rocking motion of her torso with mild rigidity of her limbs. She was unable to be encouraged off her armchair and was unable to walk. She was unable to eat and drink, and was often unable to take oral medications. Prior to the second planned treatment, the team felt that she lacked capacity to consent to further sessions.A Mental Health Act Assessment was arranged, and she was detained under Mental Health Act Section 2. Urgent ECT was prescribed under section 62, whilst a second opinion appointed doctor (SOAD) was sought for to grant her further ECT sessions. A total of 12 ECT sessions were granted.After eight further sessions, the patient\u2019s mental state did not improve. She continued to display minimal engagement. She often refused her medications, and her food and fluid intake was minimal. As a result, she underwent intravenous fluid therapy several times due to dehydration causing a deterioration of renal function. Treatment was given under Mental Health Act 1983 (amended 2007).After 4th and 8th ECT sessions, repeated attempts were made by the ECT doctor to perform a MMSE, but she was deemed too unresponsive to answer the questions.At the same time, several psychotropic medications were trialled. The date of their commencement and dose changes in relation to her ECT treatment are summarised in Table\u00a0As a result of her lack of response to treatment, her diagnosis was reviewed and affirmed by the treatment team. Six further ECT sessions were granted through a Second Opinion Appointed Doctor (SOAD).After 11 ECT sessions, she started to show signs of improvement. She started to walk around the ward and engage in various group activities. She started to accept her medications with less prompting. Her food and drink intake improved significantly, and she could eat independently. Her engagement with staff gradually improved, from making appropriate facial expressions to starting verbal communication. By the end of her ECT course, she could spontaneously engage in conversations and reported to us that \u2018I am back.\u2019 She was able to reflect on how severely unwell she was and felt ready to be discharged home. In view of her clinical improvement, ECT treatment was stopped. Extracts of clinical documentation on her progress have been summarized in Table\u00a0The diagnosis was made during the admission \u2013 ICD-10\u2009F32.3 severe depressive episode with psychotic symptoms . She shoThree months post-treatment, she scored 4 on HAM-D, and scored 89\u201393/100 for ACE-III (converted from MMSE). Clinical Global Improvement or change (CGI-C) was rated 1 (very much improved). Efficacy index was rated at 02 (vast improvement with side effects that do not significantly interfere with patient\u2019s functioning).We describe here a patient with a diagnosis of severe depression. She did not respond to electroconvulsive therapy until the 11th session, after which she had shown a drastic improvement in her mental state. The case is considered unusual. In the literature, the number of sessions during an acute course of ECT typically ranges from 6 to 12, usually given twice a week . Early aCompared to pharmacological treatment, ECT remains the most effective short-term treatment for patients with severe or treatment-resistant depression, with 70\u201390% of patients showing improvement . In thisThere may be some debate surrounding the initiation of the ECT course in this patient. The empirical titration method aims to establish seizure threshold in the first session (titration session), and from session two onwards, therapeutic sessions are given with the stimulus at 1.5 times of the seizure threshold . HoweverOther factors that might have contributed towards the improvement in this patient\u2019s mental state includes a change in her medication regime. By the 10th session, she was taking three adjunctive pharmacological treatments including quetiapine, olanzapine, and lamotrigine. Quetiapine has demonstrated up to 48% response rate in combination with SSRIs and have been approved for adjunctive treatment of MDD by the Food and Drug Administration (FDA) . OlanzapThe anti-epileptic effect of lamotrigine can theoretically inhibit the efficacy of ECT in inducing seizure activity. However, case reports/series have shown minimal or no influence on seizure and/or seizure duration . In thisThe ECT handbook of the Royal College of Psychiatrists stated that clinicians may wish to reassess the need for ECT if there is no response after six sessions. If there is no response within 12 treatment sessions, the patient is unlikely to have a \u2018sustained response to ECT\u2019 . This pa"}
+{"text": "This randomized clinical trial assesses whether emotional memory retrieval in adult patients before receiving electroconvulsive therapy (ECT) weakens underlying cognitive schemas, improves ECT effectiveness, increases ECT response, and reduces relapse rates. Can emotional memory retrieval just prior to electroconvulsive therapy (ECT) sessions improve the outcome of ECT in patients with major depressive disorder?In this randomized clinical trial, 66 patients received emotional memory reactivation or a control condition prior to ECT. The intervention did not influence remission rates, depression scores after the ECT course, total completed ECT sessions, or relapse rates.Personalized reactivation of emotional memories just prior to ECT sessions did not improve ECT effectiveness or speed of response and did not reduce relapse rates. Although electroconvulsive therapy (ECT) is often effective, approximately half of patients with depression undergoing ECT do not benefit sufficiently, and relapse rates are high. ECT sessions have been shown to weaken reactivated memories. The effect of emotional memory retrieval on cognitive schemas remains unknown.To assess whether emotional memory retrieval just before patients receive ECT sessions weakens underlying cognitive schemas, improves ECT effectiveness, increases ECT response, and reduces relapse rates.Diagnostic and Statistical Manual of Mental Disorders  and in whom ECT was indicated. Data analysis was performed from July to November 2019.In this multicenter randomized clinical trial conducted from 2014 to 2018 in the departments of psychiatry in 3 hospitals in the Netherlands, 72 participants were randomized 1:1 to 2 parallel groups to receive either emotional memory reactivation (EMR-ECT) or control memory reactivation (CMR-ECT) interventions before ECT sessions. The Hamilton Depression Rating Scale  was used to measure symptoms of depression during and after ECT, with a 6-month follow-up period. Participants were between ages 18 and 70 years with a primary diagnosis of unipolar major depressive disorder (MDD) according to the EMR-ECT or CMR-ECT interventions prior to ECT sessions.Depression scores and relapse rates within 6 months were assessed with the HDRS and analyzed using logistic and linear multiple regression analyses.P\u2009=\u2009.58), mean (SD) HDRS scores after the ECT course , total mean (SD) number of required ECT sessions for response , and relapse rates  were not significantly altered by the intervention.A total of 66 patients  were randomized to the EMR-ECT group (n\u2009=\u200932) or the CMR-ECT group (n\u2009=\u200934). Regardless of the memory intervention, 42.4% (28 of 66) of patients responded (\u226550% decrease of symptom severity on the HDRS). Of patients who responded, 39.3% (11 of 28) relapsed within 6 months. Remission rates (CMR-ECT group, 29.4% [10 of 34] vs EMR-ECT group, 25.0% [8 of 32]; Study findings suggest that the EMR-ECT intervention just before patient receipt of ECT for depression did not improve effectiveness, increase speed of response, or reduce relapse rates after the ECT course compared with patients receiving CMR-ECT.NL4289Trialregister.nl Identifier:  Electroconvulsive therapy (ECT) has been reported to be beneficial in patients with MDD resistant to pharmacologic treatment,2 although half of the patients undergoing ECT will not achieve full remission.3 Moreover, relapse rates after successful ECT are high, as one-third of patients can be expected to relapse within 6 months.4 More effective targeting of specific underlying psychopathological mechanisms of MDD may help improve ECT effectiveness, be associated with more rapid ECT response times, and decrease relapse rates after successful ECT.Major depressive disorder (MDD) is a common mental disorder associated with substantial reductions in daily functioning. Initial treatment for MDD consists of psychotherapy and/or pharmacotherapy. A 2010 study5 Weakening of negative schemas and change from maladaptive to more adaptive schema processing have been hypothesized to underlie recovery of patients from MDD, but weakening may also lower the relapse rate after cognitive behavioral therapy.6 Cognitive schemas are embedded in strong associative memory structures.7Cognitive schemas are relatively stable thought representations of prior knowledge and experiences. Cognitive theory holds that activated negative schemas play an important etiologic role in MDD, as activated negative schemas may be factors in information processing.8 Studies in the 1960s and 1970s reported that electroconvulsive treatment disrupted reactivated memories in rats9 and that reactivation of obsessive-compulsive symptoms in patients before applying ECT increased effectiveness.10 In a 2014 study,11 a single ECT session selectively impaired memory for a learned emotional story when reactivated just prior to an ECT session. If a single ECT session could weaken memory, multiple emotional memory reactivations (EMRs) in consecutive ECT sessions may improve ECT effectiveness, be associated with more rapid ECT response times, and reduce relapse rates after successful ECT.Research indicates that when memories are reactivated they may become temporarily labile and require restabilization processes to be maintained, a process known as reconsolidation. Pharmacologic interventions that disrupt the restabilization processes may selectively weaken the reactivated memory.In this randomized clinical trial (RCT), patients with MDD were randomized to receive either an autobiographical EMR or a control memory reactivation (CMR) not associated with the patients\u2019 depression just before each ECT session . We hypothesized that reactivation of patients\u2019 own emotional memories related to MDD just before receipt of ECT sessions may weaken their associated negative cognitive schemas, resulting in (1) higher remission rates and lower depression severity scores after the ECT course, (2) fewer required ECT sessions to reach a response, and (3) lower relapse rates within 6 months of the ECT course.Diagnostic and Statistical Manual of Mental Disorders 12 (DSM-IV-TR), for whom ECT was indicated. All patients had a history of insufficient response to previous treatments (pharmacotherapy and psychotherapeutic interventions), which is the primary indication for ECT in the Netherlands.13 Patients were willing and able to understand, to participate, and to comply with the study requirements. Exclusion criteria were the presence of psychotic features (owing to the possibility that psychotic features might worsen because of the cognitive intervention itself); bipolar disorder; schizophrenia or other primarily psychotic disorders; substance abuse; and other cognitive disorders. Psychiatric diagnoses were classified according to DSM-IV-TR criteria using the Mini-International Neuropsychiatric Interview.14 Participants received compensation (\u20ac40) when they completed the study. The study protocol was approved by the Medical Ethical Committee of the Amsterdam University Medical Center and registered in the Dutch Trial Register (NL4289). All patients provided written informed consent, and all procedures were carried out in accordance with the tenets of the Declaration of Helsinki.15 This study followed the Consolidated Standards of Reporting Trials (CONSORT) reporting guideline for RCTs. The trial protocol is available in Patients in this multicenter study were recruited from the department of psychiatry from 3 hospitals in the Netherlands  from 2014 to 2018. Eligible participants were patients aged between 18 and 70 years primarily diagnosed with unipolar MDD, fulfilling all criteria of the Participants were randomly assigned 1:1 to 2 parallel groups, EMR-ECT or CMR-ECT, by means of a predefined randomization list, stratified for each treatment center, using blocks of 4 to ensure equal group sizes. Concealment of randomization was maintained by access to randomization lists only by study investigators who were not directly treating or assessing eligible patients. The ECT teams and clinical outcome assessors were all blinded to randomization.13) with 80% power at 1-tailed \u03b1 of .05. The RCT continued after an interim analysis with 38 patients that was able to detect a statistical trend (P\u2009=\u2009.10) with 80% power. Critical z values with O\u2019Brien-Fleming correction were 3.11 for the interim analysis and 1.97 for the final analysis. The RCT was terminated prematurely after including 72 patients, as inclusion decreased substantially because of a change in the Dutch mental health care policy. The power to detect the same effect size (25% higher remission rate) had decreased to 69%, but the RCT still had 80% power to detect a 29% higher remission rate.Power calculation indicated a total sample size of 98 patients to detect a medium effect size .In patients receiving EMR-ECT treatment, autobiographical memories associated with maladaptive schemas were identified and reactivated according to a standardized protocol. First, an experienced psychologist, in collaboration with the patient, determined which memories to activate. Recurring maladaptive schema thoughts were identified using the Automatic Thoughts Questionnaire-Revised.During the ECT course, the assigned memory reactivation intervention was applied in the waiting room where the patients were prepared for ECT. In the EMR-ECT group at approximately 10 minutes before application of the ECT stimulus, a research assistant reactivated the autobiographical episode by reading 1 of the narratives slowly and carefully, providing the patient time to recall memory in detail and lasting approximately 3 minutes. Only 1 autobiographical memory was reactivated per ECT session, alternating between the 3 selected narratives.17In the CMR-ECT group, an identical procedure was followed. Instead of autobiographical EMR, the research assistant applied a 3-minute control memory intervention that was related to the importance of sleep, physical exercise, and substance use in mental health. After that process, patients received ECT sessions according to Dutch national ECT guidelines.18  was used to measure depressive symptomatology by trained research nurses blinded for treatment randomization.19 The HDRS is a valid observer-rated instrument consisting of 17 items with a maximum score of 52 (mean weighted sum score interrater coefficient: \u03ba\u2009=\u20090.92).20 Remission was defined as an HDRS score less than or equal to 7, response as a 50% or more reduction in HDRS score after the ECT course compared with baseline,21 and relapse as an increase of 10 or more HDRS points on at least 1 assessment in the 1- to 6-month follow-up period.Patients were evaluated before the start of the ECT course; after 6, 12, and 18 ECT sessions; and within 2 weeks after the last ECT session. Follow-up evaluations were done at 1, 2, 4, and 6 months. At each evaluation, the Hamilton Depression Rating Scale22 In addition, the Dutch version of the national adult reading test was used as a proxy for IQ.To quantify treatment resistance at baseline, we sued the Dutch Measure for Quantification of Treatment Resistance in Depression (DM-TRD), which consists of 11 items with a maximum score of 27 and has good psychometric properties and predictive validity.13 ECT courses were discontinued when remission was achieved (HDRS score \u22647) or when no further improvement was observed over a period of 2 weeks. The total number of administered ECT sessions was registered for each patient.After intravenous induction of anesthesia with etomidate (0.2 mg/kg body mass), muscle paralysis with succinylcholine (0.5-1 mg/kg body mass), and application of appropriate oxygenation  until the resumption of spontaneous respiration, ECT was administered using a constant-current (0.9 A), brief-pulse (0.5 milliseconds) device . Lithium was tapered before starting the ECT course; other concomitant medications were kept constant. ECT sessions were performed twice a week. Patients started with 6 right unilateral (RUL) ECT sessions unless clinicians decided to start with bifrontotemporal (BL) electrode placement because of severe clinical conditions or previous effective BL-ECT. During the first session, the seizure threshold was estimated by an internationally accepted, empirical, age-adjusted titration method, and the personalized dose was estimated as 6 or 2.5 times standard treatment for RUL or BL-ECT, respectively.t tests, \u03c72 tests, or Mann-Whitney tests as appropriate. For the whole study group, differences in HDRS scores before and after the ECT course were analyzed using a paired t test, and response and remission rates were calculated as percentages.Baseline characteristics between both groups were analyzed using 2-sample To investigate the relapse rates within 6 months, logistic and linear multiple regression analyses were used, with remission status (logistic) or HDRS score after the ECT course (linear) used as the dependent variable, and the intervention (EMR-ECT or CMR-ECT) as the predictor variable, with sex, age, baseline HDRS score, final electrode placement, and treatment site as covariates. This approach was different from the original analysis plan that consisted of testing only remission rates.P\u2009<\u2009.05 denoted statistical significance. Data analysis was performed from July to November 2019.To explore the secondary outcomes (number of required ECT sessions to reach response and relapse rate), linear (number of ECT sessions) and logistic (relapse rate) multiple regression analyses were conducted in the patients showing response to ECT (n\u2009=\u200928). The number of required ECT sessions was square root transformed to reduce skewness. Because time to relapse was not recorded accurately for the intended survival analysis, we analyzed the relapse rate. In the models, the intervention was entered as the predictor variable, and sex, age, HDRS score before (number of ECT sessions) or after (relapse rate) the ECT course, final electrode placement, and treatment site were entered as covariates. All analyses were conducted using SPSS statistical software, version 25 (IBM Corp), and t65\u2009=\u20098.1; P\u2009<\u2009.01; Cohen zd\u2009=\u20091.00). Twenty-eight of 66 patients (42.4%) showed response and 18 of 66 (27.3%) remission, which was lower than expected13  were included in the final analyses . No diffpected13 .P\u2009=\u2009.58; odds ratio, 1.39). Linear regression analysis showed no significant effect of the memory intervention on post-ECT HDRS scores   .P\u2009=\u2009.39; semipartial r2\u2009=\u20090.02), although the covariate final electrode placement  was significant. As expected, patients treated with BL required more ECT sessions than patients treated only with RUL, as most patients receiving BL initially received RUL-ECT as well.Linear regression analysis showed no significant association between the memory intervention and the required total amount of ECT sessions to reach response .Twenty-eight patients showed response and 11 of those (39.3%) relapsed within 6 months. In ECT responders in the separate intervention groups, 4 of 13 patients (30.8%) receiving EMR-ECT relapsed and 7 of 15 patients (46.7%) receiving CMR-ECT relapsed. Logistic regression analysis showed no significant effect of the intervention on relapse rates within 6 months . Therefore, it was decided to use detailed memories, including negative emotions and cognitions, instead of more abstract underlying negative cognitive schemas. It is possible that limited destabilization of such intended abstract schemas decreased the effect of our memory intervention.26 showed that new learning might be necessary for reconsolidation to occur. As we reactivated only old memories and did not enforce new learning, destabilization of bad memories was not provoked. In future studies, other personalized cues may be invented in which the aspect of new learning for patients is taken into account. At such time, reactivation of underlying negative schemas just before an ECT session may be more beneficial.A 2017 study27 Other studies, however, suggest that this duration may have been too long.29 Conversely, this duration may have been too short, as a reminder duration of 10 to 30 minutes was recently found to be effective for posttraumatic stress disorder.30 In addition, the reactivation procedure took place approximately 10 minutes before the actual ECT session so as to blind the treating physicians and to perform ECT according to regular practice. However, this delay may have been too long, as Kroes et al11 reactivated the memory within a few minutes before induction of anesthesia.The procedure of administering the reminders may have affected our results. In line with a rodent study, our memory reactivation paradigm was 3 to 5 minutes.In this study, the memory intervention was well tolerated by patients, and the overall dropout rate was low, suggesting similar interventions are feasible. Given the possible lessons of our RCT, future studies may consider (1) reactivation of more recent memories; (2) creation of more appropriate reminders ; (3) use of reminders of a different duration; and (4) use of a shorter duration between reactivation of the reminder and the ECT stimulus .This study has limitations. From a methods standpoint, an important problem was the inability of verifying reactivation of negative memories or schemas in patients. However, our research assistants noticed emotional reactions in patients when listening to their personal negative experiences, which may indicate reactivation of negative memory. Furthermore, the control group received potentially useful psychoeducation that was necessary to maintain patient blinding but that might also have contributed to the antidepressant effects in this group and the null findings. Converesely, this psychoeducation might have been forgotten as well because of the intervention, reducing its antidepressant effects. This possible confounder of an antidepressant effect in the control condition usually affects clinical trials with psychological interventions.3 whereas our 27.3% remission rate was consistent with that of a community sample.31 Moreover, our DM-TRD scores appeared to be much higher than others in treatment-resistant MDD groups,22 and we excluded patients with a higher chance of successful ECT , which may have contributed to the low remission rate. Conversely, the low response rate could have maximized the probability to show efficacy of the EMR-ECT.In this RCT, the response rate was lower than expected, limiting the power to detect differences in relapse rates after ECT response; the power to detect differences in relapse rates was reduced at the outset, as this analysis was restricted to treatment responders. Highly selected groups of patients with treatment-resistant MDD may show ECT remission rates of 48%,In this study, personalized reactivation of emotional memories just before ECT sessions for MDD was well tolerated but did not improve ECT efficacy, decrease the time to response, or reduce the relapse rate. This RCT highlights the difficulties of translating insights from laboratory research into clinical practice and may provide direction for future studies to further improve ECT for patients with severe MDD."}
+{"text": "The pathophysiology and temporal dynamics of affected tissues in chronic rhinosinusitis (CRS) remain poorly understood. Here, we present a multiomics\u2010based time\u2010series assessment of nasal polyp biopsies from three patients with CRS, assessing natural variability over time and local response to systemic corticosteroid therapy.Polyp tissue biopsies were collected at three time points over two consecutive weeks. Patients were prescribed prednisone (30\u2009mg daily) for 1 week between Collections 2 and 3. Polyp transcriptome, proteome, and microbiota were assessed via RNAseq, SWATH mass spectrometry, and 16S ribosomal RNA and ITS2 amplicon sequencing. Baseline interpatient variability, natural intrapatient variability over time, and local response to systemic corticosteroids, were investigated.TNF, CCL20, and GSDMA, and upregulation of OVGP1, and PCDHGB1. Members of the bacterial genus Streptococcus positively correlated with immunoglobulin proteins IGKC and IGHG1.Overall, the highly abundant transcripts and proteins were associated with pathways involved in inflammation, FAS, cadherin, integrin, Wnt, apoptosis, and cytoskeletal signaling, as well as coagulation and B\u2010 and T\u2010cell activation. Transcripts and proteins that naturally varied over time included those involved with inflammation\u2010 and epithelial\u2013mesenchymal transition\u2010related pathways, and a number of common candidate target biomarkers of CRS. Ten transcripts responded significantly to corticosteroid therapy, including downregulation of Understanding natural dynamics of CRS\u2010associated tissues is essential to provide baseline context for all studies on putative biomarkers, mechanisms, and subtypes of CRS. These data further our understanding of the natural dynamics within nasal polypoid tissue, as well as local changes in response to systemic corticosteroid therapy. The pathophysiology and temporal dynamics of affected tissues in chronic rhinosinusitis (CRS) remain poorly understood. This study comprised a comprehensive multiomics analysis of polyp biopsies from three patients with chronic rhinosinusitis with nasal polyps, assessing natural variability over time as well as local response to systemic corticosteroids. Overall, these data provide further support for current hypotheses of CRS and polyposis pathogenesis, provide essential temporal context to studies on biomarkers and mechanisms of polyposis, and highlight areas of focus for future targeted therapeutic options. For all metrics, baseline interpatient variability, natural intrapatient variability over time (1 week), local response to systemic corticosteroid therapy , and interactions between the four data sets were investigated.22.1Three patients with CRSwNP listed for bilateral functional endoscopic sinus surgery for CRS were recruited. All patients were male, of New Zealand European ancestry, nonsmokers, aged 46\u201359 years, and with Lund\u2013Mackay clinical severity scores ranging 17 to 23 (Table S1). None of the patients had taken antibiotics or corticosteroids in the 4 weeks before the study. This study was approved by the New Zealand Health and Disability Ethics committee (14/NTA/134), and written informed consent was obtained from all participants.later (Life Technologies). All samples were stored at \u221220\u00b0C until the time of sample processing.Samples were collected at three time points over two consecutive weeks  as outlined in Supporting Information S1, Supplementary Methods, and as per manufacturer's instructions. DNase\u2010treated RNA for transcriptome analysis (RNAseq) was submitted to the sequencing provider (Auckland Genomics Ltd.) for final sample processing, library preparation, and sequencing on two lanes of an Illumina HiSeq machine  were processed and prepared for proteomic analysis as described in Supporting Information S1, Supplementary Methods. Liquid chromatography with tandem mass spectrometry (LC\u2010MS/MS) was conducted for each sample using SWATH acquisition, with fragment ion areas  calculated by PeakView (v. 2.2) with the SWATH MicroApp 2.0  were processed using BBDuk2.3.2Data for each patient (three samples each) were processed independently using Excel, as described in Supporting Information S1, Supplementary Methods. Sums of fragment areas for each peptide were calculated, followed by peptide area sums for each protein. Protein area sums were used for all subsequent analyses.2.3.3Bioinformatics processing of 16S rRNA gene and ITS2 marker amplicon sequences was conducted in USEARCH (v. 10),2.4An initial exploratory analysis of natural transcript and protein variability over time was conducted by calculating ratios of normalized raw read counts between the first and second time points (times i and ii) for each individual patient .2.4.1p values (\u03b1\u2009=\u2009.05). Ensembl IDs were converted to HUGO Gene Nomenclature Committee (HGNC) symbols for ease of interpretation and to standardize reporting between RNAseq and proteome results.The R package DESeq2 (v. 1.14.1)2.4.2p\u2009<\u2009.05 was conducted via two\u2010sample t tests and Tukey's honest significant difference tests . For \u201ctreatment\u201d (\u201ccontrol\u201d vs. \u201ctreatment\u201d), ANOVA results are reported . UniProt IDs were converted to HGNC symbols to standardize reporting between proteome and RNAseq results.Log\u2010transformed  data were tested for the following: (1) baseline interpatient differences; (2) natural variability; and (3) treatment effects (prednisone). Linear models were fitted for each protein, and tested using one\u2010way analysis of variance (ANOVA). For \u201cnatural variability\u201d and \u201ctreatment\u201d comparisons, linear mixed\u2010effects models were fitted with the addition of interpatient differences fitted as random effects. For \u201cinterpatient\u201d (patient 1 vs. 2 vs. 3) and \u201cnatural variability\u201d (times i vs. ii vs. iii), post\u2010hoc testing of variables with ANOVA 2.4.3p\u2009<\u2009.1; proteins with Tukey's p\u2009<\u2009.05) for natural variability and response to prednisone were identified using PANTHER (v. 14.0)p value, \u03b1\u2009=\u2009.05), using GO database release 2018\u201012\u201001.Molecular pathways and Gene Ontology (GO) terms enriched for DEGs and proteins  were identified based on their involvement in a subset of selected PANTHER pathways of interest  together with FDR adjustment (\u03b1\u2009=\u2009.05). Bray\u2013Curtis dissimilarities were calculated for all data sets using the vegan package (v. 2.5\u20101).p\u2009>\u2009.05]). Adonis incorporated patient differences first, followed by treatment.The remaining analyses were conducted in R (v. 3.3.0)\u03b1\u2009=\u2009.05 in both cases).Spearman's correlation analyses were calculated for the following: (1) the 20 most abundant variables from each data set; and (2) variables shared between transcriptome and proteome data sets based on matching HGNC IDs . For the latter, correlation analyses were conducted for each matching data pair (comparing transcription data with its related protein), with a focus on negative correlation patterns. Correlation analyses included pairwise testing of significance via cor_pmat, as well as FDR adjustment calculation  to simplify the presentation of individual transcripts/proteins and associated PANTHER pathways. For visual clarity, figures are color\u2010coded throughout as follows: transcriptome data, red; proteome data, purple; bacterial data, green; fungal data, yellow.2.5Transcriptome and microbiota raw sequence data have been uploaded to the SRA\u2010NCBI repository , and those involved in the electron transport chain . Proteomics data identified 4345 peptides, comprising 921 proteins. Proteins with the highest signal in the data  included the blood protein ALB, globin proteins (HBB and HBA1), cellular and tissue structural proteins , and immunoglobulin proteins . Additional highly abundant variables of interest included transcripts for the genes ALOX15, POSTN, S100A11, and CST1, and the proteins COL3A1, COL6A1, FGA, FGB, FGG, FN1, S100A8, S100A9, and S100A11.In total, 58,734 distinct messenger RNA (mRNA) transcripts were identified . The genes with the highest normalized read counts in the transcriptome data set encoded members of the prostaglandin\u2010endoperoxide synthase family . The most abundant fungal genera included Malassezia, Candida, Rhodotorula, and unclassified members of Malasseziales, Dothideomycetes, Mycosphaerellaceae, and Phaeophaeriaceae . In correlation analyses, a handful of microbial\u2013protein associations were observed . Two ZOTUs of Anaerococcus positively correlated with collagen proteins COL6A3 and COL1A2 , and two ZOTUs of Streptococcus positively correlated with immunoglobulin proteins IGKC and IGHG1 (0.8\u2009<\u2009\u03c1). Of these microbial associations, only that between Streptococcus and IGHG1 remained significant after FDR adjustment.Abundant bacterial genera included d Figure\u00a0. Bacteri\u03c1\u2009<\u2009\u22120.5), including ACADM, ALDH1A1, ATP1B1, CBR1, KRT7, and LCN2 . Notable differences included IGHG3, and the eosinophilia\u2010related proteins EPX, PRG2, and RNASE3 (up to 38\u2013112 times difference between some patients). Patient 1 also had concomitant asthma, and a considerable proportion of the significant interpatient comparisons were in relation to Patient 1 (vs. Patient 2 or 3). Twenty\u2010six pathways_subset proteins . Patient differences may also explain up to 30% of the transcriptome data variability; however, this was outside the significance threshold (p\u2009=\u2009.067).In hierarchical clustering analysis, samples tended to cluster by patients for protein (including the pathways_subset) and bacterial community data , indicating stronger inter\u2010 than intrapatient differences over time. In adonis analyses, interpatient differences significantly explained 69% and 73% of the protein and pathways_subset protein data, respectively, and 43% of the bacterial data , A2M, CCL18, GDF15, TNFAIP3, and CD14 .Testing across all patients, 162 DEGs and 7 proteins differed significantly between time points i and ii, including the genes In PANTHER overrepresentation testing based on DEG and proteins (each tested separately) that varied significantly between times i and ii, significantly enriched GO terms included a number of processes likely involved in inflammation or epithelial\u2013mesenchymal transition (EMT), such as antimicrobial humoral response, extracellular matrix organization, extracellular exosome, cell adhesion, and cornification Table\u00a0.Subtle differences were observed in bacterial and fungal communities within each patient over time . However, in pairwise comparisons testing across all patients, there were no significant temporal differences for the 20 most abundant ZOTUs or diversity indices for bacterial or fungal community data.3.4p\u2009<\u2009.05). The 50 DEGs with the lowest p values are presented in Figure\u00a0p\u2009<\u2009.05; Figure\u00a0Transcripts for 724 genes and 26 proteins differed between pre\u2010 and post\u2010corticosteroid (prednisone) treatment , TNF (9:1), C2CD4C (9:1), and GSDMA (9:1), and upregulation of OVGP1 (2:1) and PCDHGB1 .After FDR adjustment, 10 DEGs (but no proteins) were significant in response to prednisone treatment, including downregulation of p\u2009<\u2009.1, no significantly enriched GO terms were identified after correction for multiple testing. Based on DEG with unadjusted p\u2009<\u2009.05 from DESeq2 analysis (724 transcripts), significantly enriched GO terms included predominantly inflammatory mechanisms. Significantly enriched GO terms associated with proteins that differed in response to corticosteroids included predominantly tissue and cellular structural mechanisms, including laminin\u201011 complex, costamere, and cell\u2013matrix adhesion  treatment. However, the specific corticosteroid responses of individual patients varied considerably, and there was no obvious posttreatment profile \u201ctype\u201d shared by individuals in the experiment . This was further reflected in hierarchical clustering, where treatment samples did not cluster together , and adonis analyses, where treatment did not significantly explain any of the variability in the data.3.5The most abundant transcripts and proteins included those associated with T\u2010cell activation, inflammation mediated by chemokine and cytokine, cadherin, integrin, and cytoskeletal signaling pathways, as well as Wnt, FAS, and apoptosis signaling (transcripts), and B\u2010cell activation and blood coagulation . Notably, DEG and proteins that varied naturally over time, and also those that differed in response to treatment, both included those associated with inflammation mediated by chemokine and cytokine, cadherin, integrin, and Wnt signaling pathways Figures\u00a0 and\u00a04.4Establishing degrees of natural variability over time is essential to provide context for investigations of putative mechanisms and biomarkers of disease, and to inform treatment decisions. This study comprised a comprehensive multiomics analysis of nasal polyp biopsies from three patients with CRSwNP, assessing natural variability over time as well as local response to systemic corticosteroids.4.1Abundant transcripts and proteins identified in this study included several involved in inflammation mediated by chemokine and cytokine, FAS, cadherin, integrin, Wnt, apoptosis, and cytoskeletal signaling, coagulation, and B\u2010 and T\u2010cell activation pathways. Processes associated with inflammation and tissue structural changes have similarly been highlighted previously as central mechanisms underlying CRS and/or nasal polyposis in CRSwNP, supporting the validity of the transcriptome and proteome data presented here.VIM), collagens COL1A1, COL1A2, CO3A1, COL6A1, COL6A2, and COL6A3, fibronectin (FN1), and periostin (POSTN) were all abundant in this study. Proteins involved in the coagulation cascade (including fibrinogen and fibronectin) have also been implicated in CRSwNP, with fibrin deposition (coupled with reduced fibrinolysis) implicated in the pathogenesis of nasal polyposis.EMT is associated with CRS.+/K+ transporting subunit (ATP1B1), carbonyl reductase (CBR1), keratin (KRT7), and lipocalin LCN2 . Posttranscriptional regulation can play important roles in disease, including some variants of cystic fibrosisIt was also of interest to investigate transcription and protein patterns in parallel. However, the resolution of each method differs considerably. In this study, over 58,000 transcripts were identified compared with 921 proteins. Low\u2010abundance proteins, including inflammatory cytokine signaling molecules, are generally omitted from the latter. Nonetheless, patterns were broadly consistent between the overlapping data sets. Exceptions included significant moderate to strong negative correlations between transcripts and their respective protein for ATPase Na4.2Associations have previously been identified between the microbiota and inflammatory signaling and subtypes in CRS.Streptococcus and two immunoglobulin proteins (IGKC and IGHG1). Streptococcus spp. can produce IgG\u2010cleaving proteases,Streptococcus M\u2010proteins to fibrinogen  enables further IgG\u2010related immune evasion.Streptococcus spp. are known to be commonly associated with the sinonasal tract,Streptococcus\u2010mediated IgG inflammatory processes coupled with immune evasion may promote ongoing inflammation, while also providing strong selective pressure on the structure of the associated microbiota. Further study of possible roles of Streptococcus spp. in the pathogenesis of nasal polyposis is warranted.One association of note was a positive correlation between ZOTUs of 4.3In a stratified cohort of patients , numerous interpatient differences were observed, including prominent inflammation\u2010related proteins such as the immunoglobulin protein IGHG3, and the eosinophilia\u2010related proteins eosinophil peroxidase (EPX), proteoglycan 2 , and eosinophilic cationic protein (RNASE3).MUC1) downregulation, elevated MUC4, and increased neutrophilia are markers of reduced response to corticosteroids.While corticosteroid therapy offers relief to a great number of CRSwNP patients, between 20% and 50% of patients are resistant.4.4Identifying biomarkers and better delineating endotypes of CRS is a major focus of current research, and promises improved guidance of treatment decisions.MUC5B and MUC5AC), cystatin SN (CST1), the S100 calcium\u2010binding S100A8, S100A9, and S100A14, alpha defensin (DEFA1), the eosinophilia\u2010related eosinophil peroxidase (EPX), proteoglycan 2 , and Charcot\u2013Leyden crystal galectin (CLC), coagulation\u2010related FGA, FGB, and FGG, claudin 9 (CLDN9), desmoglein (DSG3), periostin (POSTN), immunoglobulin component IGHV1, interleukin (IL)\u20108 (CXCL8), IL19, IL17RB, chemokine ligands 18 and 21 (CCL18 and CCL21), serum amyloids (SAA1 and SAA2), keratin (KRT6A and KRT14), and vimentin (VIM). A number of these markers have been highlighted in previous cross\u2010sectional studies as putative biomarkers of CRS or subtypes of CRS.Between the first two time points (before the treatment phase) there were 162 significant DEGs, suggesting that many processes involved in CRS may vary substantially over relatively short time scales in the natural course of the disease. Numerous targets that may be considered promising candidates for CRS and/or nasal polyposis biomarkers  showed natural variation within patients, including mucins , chemokine ligand 20 (CCL20), and gasdermin A , and upregulation of markers that may reflect a reversal of processes of epithelial dysregulation, including the epithelial\u2010glycoprotein OVGP1, and cell adhesion molecule protocadherin gamma (PCDHGB1) transcription. Multiple changes in both pro\u2010inflammatory and anti\u2010inflammatory transcription have similarly been observed previously.Significant DEG due to treatment effects included marked downregulation of inflammatory mediators tumor necrosis factor , in a comprehensive multilayered approach. Nasal polyp tissue was relatively accessible, enabling biopsy collection in the routine clinic setting over consecutive weeks. Nonetheless, patient recruitment for such a study is challenging, and \u2010omics technologies remain relatively cost\u2010prohibitive for large cohort studies. As a result, this study is limited to three patients and is exploratory in nature. Inferences drawn from comparisons over large numbers of variables in a small patient group should be interpreted with caution. Furthermore, the study design was developed to enable repeated minimally invasive sampling in the clinic setting. Nonetheless, biopsy sampling is inherently invasive, and the local effects of wounding and wound healing over the course of each week may influence the degree of variability in tissue processes observed here. Results should not be interpreted as definitive markers of CRS (or response to treatment), but serve as an important proof of principle, and provide initial insights into markers worthy of more attention.Each data set was generated from a single sample per patient per time point . The focus of this study was temporal intrapatient variability. However, comparably little is known about the natural spatial variability of processes within nasal polyp or mucosal tissue in CRS. The collection of additional biopsy samples at each time point was not feasible in this study, and it remains unclear whether the observed variability of some markers may be accounted for by spatial heterogeneity rather than temporal dynamics. The natural spatial variability of tissue processes represents an additional significant knowledge gap in the understanding of CRS and requires further study.Considerable interpatient variability was observed in all four data sets, and generally patient differences more strongly partitioned the data than changes over time (including response to corticosteroid therapy). Interpatient differences in baseline local inflammatory mechanisms, and an individual response to corticosteroids, may have obscured some genuine associations. The effects of these differences will be especially pronounced due to the small sample size (three patients sampled over three time points). This study provides a template and highlights focal points for subsequent study in larger patient groups. In future, larger cohort temporal studies incorporating finer scale resolution of CRS subtypes (such as inflammatory endotypes) are required to further resolve genuine patterns of change over time.Finally, the focus of this study was mechanisms specific to nasal polyposis within CRSwNP. Findings may not represent nonpolyp sinonasal mucosa processes in CRSwNP or chronic rhinosinusitis without nasal polyps (CRSsNP), and other mucosal markers may better differentiate between different variants of CRS.Despite these limitations, however, this study contributes to several important findings. The observed interpatient variability and intrapatient dynamics both have implications for the interpretation of studies on biomarkers and mechanisms of CRS. Additionally, despite this background variability, several specific local effects of systemic corticosteroids were observed. These included transcripts and proteins related to several pathways previously identified as important in nasal polyp and CRS pathogenesis. Overall, these data provide further support for current hypotheses of CRS and nasal polyposis pathogenesis, provide essential temporal context to studies on biomarkers and mechanisms of nasal polyposis, and highlight areas of focus for future targeted therapeutic options.4.7This study presents a comprehensive multiomic time\u2010series analysis of CRS\u2010associated nasal polyp transcriptome, proteome, and microbiota, in three patients with CRSwNP. To our knowledge, this is the first study to investigate natural transcription and protein dynamics in CRS\u2010affected tissue over time.MUC5B, MUC5AC, S100 calcium\u2010binding proteins, CST1, EPX, CLC, POSTN, and CXCL8 (IL\u20108). Several markers central to inflammation mediated by chemokine and cytokine, cadherin, integrin, Wnt, cytoskeletal, coagulation, and apoptosis signaling pathways were abundant in nasal polyp tissue at baseline. These processes were also often associated with markers that naturally changed over time, and those that responded to corticosteroid treatment.High baseline interpatient variability and differential changes over time were detected in all metrics. Natural temporal variability was observed for a number of transcripts and proteins, including likely agents in the pathophysiology of CRS and nasal polyposis. Several markers that varied naturally over time have been previously identified as putative biomarkers of CRS, including These findings offer promising avenues for future research and candidate targets for the development of novel therapeutics addressing the pathophysiology of nasal polyposis.Michael Hoggard contributed to study design, sample processing, data acquisition, data analyses, and writing of the manuscript. Melissa Zoing and Richard G. Douglas contributed to subject recruitment and sampling. Bincy Jacob and Martin Middleditch contributed to proteomic analysis and data processing. David Wheeler contributed to transcriptomic data processing and analysis. Kevin Chang contributed to statistical analyses. Michael W. Taylor, Richard G. Douglas, and Kristi Biswas contributed to study design and provided laboratory space and materials. All authors contributed to the editing of the manuscript.This study was approved by the New Zealand Health and Disability Ethics Committee (14/NTA/134), and written informed consent was obtained from all participants.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file.Supporting information.Click here for additional data file."}
+{"text": "Segmentation of anatomical structures is valuable in a variety of tasks, including 3D visualization, surgical planning, and quantitative image analysis. Manual segmentation is time-consuming and deals with intra and inter-observer variability. To develop a deep-learning approach for the fully automated segmentation of the inner ear in MRI, a 3D U-net was trained on 944 MRI scans with manually segmented inner ears as reference standard. The model was validated on an independent, multicentric dataset consisting of 177 MRI scans from three different centers. The model was also evaluated on a clinical validation set containing eight MRI scans with severe changes in the morphology of the labyrinth. The 3D U-net model showed precise Dice Similarity Coefficient scores (mean DSC-0.8790) with a high True Positive Rate (91.5%) and low False Discovery Rate and False Negative Rates (14.8% and 8.49% respectively) across images from three different centers. The model proved to perform well with a DSC of 0.8768 on the clinical validation dataset. The proposed auto-segmentation model is equivalent to human readers and is a reliable, consistent, and efficient method for inner ear segmentation, which can be used in a variety of clinical applications such as surgical planning and quantitative image analysis. Technological developments in imaging techniques have allowed (neuro)radiologists to evaluate the human labyrinth, with recent advances increasing the level of detail1. Moreover, applications of artificial intelligence and the quantitative assessment of medical images for the non-invasive exploration of anatomical structures and the classification of diseases have remarkably increased in recent years2.The inner ear, also known as the labyrinth, is a complex structure located in the temporal bone. It roughly consists of the cochlea, the vestibule, and the semi-circular canals. Understanding changes and variations within these structures can help diagnose and predict a number of conditionsradiomics4. A recent study investigated the value of radiomics for diagnosis of Meniere\u2019s disease (MD), an inner ear disorder characterized by episodic vertigo spells, hearing loss, and tinnitus5. Other labyrinthine disorders such as sensorineural hearing loss might benefit from quantitative image analysis as well6.The process of the automated extraction and analysis of large amounts of quantitative information from medical images is known as 9. Over the past years, several automatic and semi-automatic inner ear segmentation methods were proposed for both MRI and CT imaging14, including region-growing, thresholding and edge detection15, model-based14, atlas-based13 and machine-learning techniques11. The inner ear\u2019s small and complex structure makes segmentation challenging, especially in MR imaging due to non-homogenous image intensities11.Image segmentation is a critical step to work towards fully automated diagnostic tools for inner ear disorders. Manual segmentation requires experienced readers, is time-consuming and prone to intra-and inter-observer variability12, yet requires manual intervention. Additionally, segmentation performance of atlas-based methods decreases for complex structures with variable shape and size16.Recent work proposed a statistic shape model (SSM) for inner ear segmentation in MR images (13). However, the\u00a0proposed\u00a0methodology present a high computational burden, both in term of time and cost. Another recently published segmentation model showed very good agreement between an atlas-based segmentation and the manual gold standard17. Among deep learning techniques, the U-Net architecture is a specific type of convolutional neural network (CNN) consisting of multilayer neural networks. These networks have been implemented successfully, especially for auto-segmentation in medical images19. Although U-Net based deep learning approaches do exist for segmentation of the inner ear20, they lack the incorporation of anatomical variations, pathological situations or missing anatomical structures, which are part of daily clinical practice. Hence, there is currently no fully automated, generic segmentation method for the inner ear to meet the growing demand for the developments in 3D visualization and quantitative image assessments.Recent studies have demonstrated the successful application of deep learning techniques for detection, segmentation, and classification tasks in the medical fieldTherefore, this study\u2019s objective was to develop a deep-learning approach for the automatic segmentation of the inner ear in clinical MR images, focusing on the robustness of the method in varying clinical situations, and to evaluate its performance and generalizability with manual segmentation as reference.This study was performed in accordance with the guidelines outlined by Dutch and Belgian legislation. MRI scans were collected and fully anonymized by the local investigators of four centers. Ethics committee of University Hospital Antwerp approved the study  and written informed consent was obtained from the participants.\u00a0The other centers waived the\u00a0ethics approval due to the retrospective nature and full anonymization of the data according to the Medical Research involving Human Subjects act (WMO).The workflow applied in this study consisted of four steps and is illustrated in Fig.\u00a0A total of 1203 images of patients who underwent an MRI scan of the cerebellopontine angle for diverse neuro-otological indications in the period of December 2015 to April 2019 in Maastricht Medical University center (center A) were collected and fully anonymized. All high resolution T2-weighted images were acquired in 1.5 and 3\u00a0T (T) MRI scanners, from different vendors with a variety of high-resolution T2-weigted sequences , with local optimized protocols. MRI scans of the cerebellopontine angle were included if they allowed labyrinth visualization with at least a portion of the labyrinth recognizable and suitable for manual segmentation. MRI scans, which did not allow a clear manual segmentation, were excluded from this study. In total, 259 MRI images were excluded due to unsuitable sequences , poor quality, or skewed MR images. The final training dataset included MRI scans of 944 cases  in the period from 2005 to 2015 , an experienced clinician and researcher in inner ear imaging, to manually segment the labyrinth on both sides in 3D Slicer 4.8.1In order to generate homogeneous MRI volumes as input for the model, the following pre-processing steps were performed. Firstly, all volumes were resampled by B-spline interpolation to an isotropic voxel size of 0.45\u00a0mm. Secondly, the intensities of the MRI volumes were normalized to range [0\u20131] using the minimum and maximum intensity of each volume. Lastly, since the model\u2019s architecture required inputs of the same dimensions, a center crop of 256\u2009\u00d7\u2009256\u2009\u00d7\u200964 pixels was obtained from the pre-processed volumes. This crop size was large enough to contain contextual information of the inner ear. Images smaller than 256\u2009\u00d7\u2009256 pixels in the transversal plane and 64 pixels in slice direction were padded with zeros. More details can be found in Supplementary Information Sect.\u00a021, as illustrated in Fig.\u00a0The model\u2019s architecture is based on a classical 3D U-net22.The model\u2019s architecture was adapted with attention gates, as the relevant features of the inner ear showed large shape variability and were very small compared to the surrounding structuresThe attention gates highlight the regions that correspond to inner ear and suppress the regions that correspond to background. The highlighted features are propagated by the skip connections from the deep stages of contracting paths to the expanding paths. More specifically, attention Gates are used to propagate the important spatial information corresponding to inner ear from the encoding to the decoding part of the model. As shown in the Fig.\u00a0.22.Since different components of inner ear are more easily accessible at different scales, we additionally input the same volume at three different scales along the encoder path, which has been previously described as an input image pyramid by Oktay et al23, ReLu activation24 and Instance Normalization25.Other network parameter changes included an increase in number of convolutional filters from 16 to 128 in the encoder network. Each Maxpooling layer reduced the image spatial resolution by a factor of two. Along the decoder path, transposed convolutions were used for up-sampling which increased the image size by a factor of two at each layer. All the convolutional blocks included 3D convolutions26 and updated by using the Adam optimizer27 at an initial learning rate of 1e\u22124.The model was trained with the pre-processed volumes and their corresponding ground truth labels of the training dataset. Randomly selected input volumes were augmented by vertical flipping or rotation during training. The network weights were initialized by using the He-normal initialization method28 was used as an objective loss function while training the model, which penalized false negatives more than false positives at a false positive penalty score (\u03b2) of 0.3 and a false negative penalty score (\u03b1) of 0.7. This approach emphasizes learning features corresponding to the positive voxels. The loss was calculated in a mini batch of two images per iteration and at the end of each epoch, Tversky loss was calculated on the model\u2019s predictions on the validation dataset to ensure validation loss convergence .Since the number of positive voxels (i.e. part of the inner ear) and the negative voxels were highly imbalanced, Tversky lossThe final model\u2019s performance was evaluated on the multicentric, independent test dataset.The main outcomes of this study were the Dice similarity coefficient (DSC), true positive rate (TPR), false positive rate (FPR), false negative rate (FNR) and false discovery rate (FDR).As a secondary outcome a subjective evaluation of clinical validation was performed by the second author (MvdL) in consensus with an experienced neuroradiologist (A.A.Postma). Towards clinical implementation, it is critical that a deep learning model is able to segment the inner ear under all conditions, including those that might alter the shape of the inner ear . Therefore, eight MR images, with their corresponding masks, were selected by the second author (MvdL) in which the signal intensities of the inner ear were altered either by pathology or post-therapeutic changes. These scans were left out from the training dataset and were used for clinical validation of the performance of the model.An in silico clinical study was performed to make a qualitative comparison between manual and model-generated segmentations for 50 MRI volumes randomly selected from the test cohort. An in-house developed software was used to display pairs of segmentations , at randomized screen positions (left or right) blinded to the participants, overlaid on MRI images, as shown in Fig.\u00a0The final training dataset included MRI scans of 944 cases . The final validation dataset included MRI scans of 99 cases from center A  and 177 cases from centers B, C and D .The segmentation accuracy was evaluated against the ground truth by assessing the DSC. DSC measures the overlap between the reference and the model\u2019s output. The overall average metrics of segmentation accuracy, DSC, TPR, FNR, FDR and FPR are summarized in Table On the held-out clinical validation dataset, the model achieved an average DSC of 0.876, TPR of 87.86%, FDR of 15.2% and FNR of 12.13%. The automated segmentations on this dataset are included in \u221217), indicating that expert users preferred the segmentations generated by the proposed model over the manual segmentations.On average, the participants preferred the automated segmentation in 67% of the cases. A paired one-sided t-test for the hypothesis that this average score is greater than 50% was significant .The in silico based qualitative analysis showed that on an average, the expert users (radiologists and computer scientists) are more likely to prefer model generated segmentations over manual segmentations. The Bland\u2013Altman plot Fig.\u00a0 shows 5 20. The study reported an overall DSC of 0.66 and 0.58 when using 3D-FCN and 2D-FCN, respectively. Another recent study reported a high DSC of 0.95 using SSMs based level set10. However, their model was evaluated on a small dataset (10 cases out of 23 cases were held out for testing) and no independent validation was performed. Directly comparing the present approach with the already published methods in terms of DSC is not possible due to differences in datasets. Nevertheless, it is worth noting that our presented method achieves state of the art performance, which can be ascribed to the robust deep learning approach combined with a wide and varied dataset, both for training and validation, an aspect often neglected in similar studies.A prior study, that used deep learning to facilitate the auto-segmentation of the inner ear, compared the performance of a 3D Fully Connected Network (FCN) to a 2D-FCN29. Next to this, the training dataset was manually segmented by five independent readers. Therefore, the model learned to eliminate noise in the manually segmented labels caused by inter-reader variability. These methodological aspects resulted in a model that is well generalizable, which is reflected in the high-validation performance. Past studies have shown high inter-reader and intra-reader variability on medical image segmentation tasks31. Our method\u2019s consistency  alleviates this issue. Additionally, the interaction time was approximately 10\u00a0min per case for manual segmentation by an experienced reader compared to only 6.5\u00a0s for the automatic segmentation.There are several important strengths of this study. First of all, the model was trained on a diverse set of MR images of the cerebellopontine region. Although all MR images of the training dataset were collected in one center, they were acquired over a wide time span (2015\u20132019) and include different acquisition and reconstruction protocolsOne of the most important strengths of this study is the evaluation on the MR images containing deviant morphological shapes and decreased signal intensities of the labyrinth caused by cerebellopontine pathology. On this held-out clinical validation dataset, the model proved to generalize well with an average DSC and TPR of 0.8768 and 87.86% respectively. So far, previous auto-segmentation studies have trained their models on normal ears or small datasets (13\u201315). To the best of our knowledge, our study is the first to assess generalizability with respect to pathologies.Several limitations of this study should be noted. First of all, the most important limitation is the lack of a gold standard for manual segmentations from highly experienced neuroradiologists. Due to the extent of the segmentation process, manual segmentation of approximately 1500 labyrinths by one or more senior radiologist was not feasible. Therefore, in this study the authors chose to work with independent readers who were trained and supervised by an experienced clinical researcher in inner ear imaging (MvdL) to generate a first proof of concept. This could have induced noise in the manual segmentations. Also, the intra- and inter-observer variability of the segmentation team was not evaluated. Although manual segmentation was performed under strict supervision of the second author and a curating process was performed to detect incorrectly segmented masks, the quality of the manual segmentation could not be fully guaranteed. Since the manually segmented masks were considered as the reference standard for the evaluation of the model, lower DSC scores might have indicated better automated segmentation compared to manual segmentation.32.Nevertheless, efforts have been made to contain this limitation by training a deep learning architecture with large number of parameters and applying Early Stopping to prevent overfitting on the noise in the manual segmentation. Previous studies have proved that overparameterized networks are more robust against noisy labels when Early Stopping is applied33 or shape identification34 prior to automated segmentation especially for abnormal cases.Given the very small are occupied by the inner ear in the whole MRI volume, the performance of our model might be further improved by applying bounding box detection29. In this study, attempts were made to prevent overfitting by training the model on a large dataset from one center and testing its generalizability by holding out 3 independent validation cohorts. Although the overall DSC scores were markedly high, the model performed poorly and failed to generalize in five cases out of 177 (3 cases from center C and 2 cases from center D had DSC\u2009<\u20090.70). This situation could have been mitigated by training the model on all of the centers. This would have made the training dataset more diverse  and the model\u2019s performance could have been evaluated by cross-validation techniques . However, this would degrade the credibility of the generalizability of the model due to concerns regarding overfitting.Secondly, poor generalizability is the most common problems pertaining to deep learning modelsLastly, the model was trained and evaluated on datasets that included only Dutch and Belgian population. The generalizability of the model on MRI images from an international cohort is currently unexplored.35 or learning purposes36. Previous studies have proven the usability of auto-segmentation for pre-operative planning of cochlear implant surgery using CT imaging37 and for the diagnosis of adolescent idiopathic scoliosis using MRI imaging11. Our model proved to be efficient on MRI imaging. However, the proposed methodology can be easily leveraged for similar auto-segmentation applications on different imaging modalities.The future clinical advantages of automated 3D image segmentation of the inner ear are versatile. Image segmentation can be used for 3D visualization, allowing a better understanding of the spatial relations and morphological changes within the inner ear, assisting radiologists in the diagnostic process and providing tools for surgical planning6, volumetric assessment of fluid compartments in the labyrinth38 and the analysis of the morphoanatomy for the vestibular system11 are used to aid diagnosis of vestibular diseases. Radiomics refers to the process of the automated extraction and analysis of large amounts of quantitative features from medical images. These features are sometimes not perceptual for the human eye and might contain information that reflects underlying tissue heterogeneity and pathophysiology39. Quantitative image features involve descriptors of shape, size, volume, intensity distributions and texture heterogeneity patterns39.Nowadays, quantitative analysis of the inner ear is gaining more importance. Techniques like radiomics40. In conventional MRI, the endolymphatic compartment cannot be distinguished from the perilymphatic compartment, and thus, EH is not depicted41. The differences found in radiomic features between MD and controls could hypothetically be explained by the different composition of the fluids in the labyrinth, causing a different distribution of signal intensities5. Possibly, EH is captured in the quantitative image features due to damage to or morphological changes to the endolymphatic space. Since Meniere\u2019s disease is still a clinical diagnosis challenge42, discovering distinctive image features might benefit the diagnostic trajectory of MD. Another study showed that cochlea CT image features can be useful biomarkers for predicting sensorineural hearing loss in patient with head and neck cancers which received chemoradiation therapy6. Different machine learning methods were used for feature selection, classification and prediction. The advantage of using machine learning in combination with radiomics is that the analysis of the labyrinth could be done autonomously in the future5. However, for both studies, setting a Region Of Interest (ROI) by manual segmentation was necessary. The fully automated segmentation of the inner ear contributes to efficient research on quantitative image analysis of the inner ear.A histological feature strongly associated with Meniere\u2019s disease is endolymphatic hydrops (EH), a distention of the endolymphatic compartment in the inner ear38. Contrast-enhanced MR imaging allows the in vivo confirmation and quantification of endolymphatic hydrops43.Next to analyses on conventional MRI and CT imaging, the volumetric assessment of fluid compartments in the labyrinth is also promising for vestibular research45. However, the 3D reconstruction were all rendered semi-automatic. Due to this time-consuming process, the applications for volumetric assessment are yet more scientifically than clinically relevant. A recent study proposed atlas-based segmentation for the volume-based quantification of the fluid spaces of the inner ear12. Which created fast, standardized (auto)segmentation. Further research is necessary to explore the option of the proposed U-net model can be leveraged for contrast-enhanced imaging as well, to facilitate volumetric assessment of the ELS in clinic.Several studies investigated the value of the 3D volumetric assessment of the endolymphatic space (ELS) to better monitor EH in vivo, for example in therapeutic trials in Meniere\u2019s disease, and to better compare the ELS in patients with different otological diseasesAuto-segmentation in its current form, is a step towards fully automated diagnostic tools for inner ear disorders.In this study, a working first proof-of-concept is demonstrated regarding the fully automatic segmentation of the inner ear using deep learning. Overall, the proposed auto-segmentation model is equivalent to manual segmentation and is a reliable, consistent, and efficient method for inner ear segmentation which can be used in a variety of clinical applications, such as 3D visualization, surgical planning and quantitative image analysis. Auto-segmentation of the inner ear in its current form, might open doors towards automated diagnostic tools for inner ear disorders.Supplementary Information."}
+{"text": "Magnolia officinalis, has recently drawn attention due to its anticancer potential. The present study was aimed to explore the effects of Magnolol on restraining the proliferation, migration and invasion of pancreatic cancer in vivo and in vitro. Magnolol showed significant anti-growth effect in an orthotopic xenograft nude mouse model, and immunohistochemical staining of the xenografts revealed that Magnolol suppressed vimentin expression and facilitated E-cadherin expression. The cytoactive detection using CCK-8 assay showed Magnolol inhibited PANC-1 and AsPC-1 concentration-dependently. Scratch healing assay and the Transwell invasion assay proved the inhibiting effects of Magnolol on cellular migration and invasion at a non-cytotoxic concentration. Western blot and rt-PCR showed that Magnolol suppressed epithelial-mesenchymal-transition by increasing the expression level of E-cadherin and decreasing those of N-cadherin and vimentin. Magnolol suppressed the TGF-\u03b2/Smad pathway by negatively regulating phosphorylation of Smad2/3. Moreover, TGF-\u03b21 impaired the antitumor effects of Magnolol in vivo. These results demonstrated that Magnolol can inhibit proliferation, migration and invasion in vivo and in vitro by suppressing the TGF-\u03b2 signal pathway and EMT. Magnolol could be a hopeful therapeutic drug for pancreatic malignancy.Magnolol, a hydroxylated biphenyl extracted from  Pancreatic cancer is a common malignancy and ranks as the 7th leading cancer-associated mortality in developed nations . AlthougEpithelial-mesenchymal-transition (EMT) is an important segment in cancer invasion, metastasis, proliferation and maintenance of stem cell characteristics . When EM18H18O2, CAS Number 528-43-8, PubChem CID 72300) is a natural hydroxylated biphenyl extracted from the root and stem bark of Magnolia officinalis, Magnolia obovata and Magnolia grandiflora  were employed to evaluate the impacts of Magnolol on cell biological behavior. TGF-\u03b21-induced EMT was also investigated as a potential mechanism of action of Magnolol.18H18O2, 5\u2019-Diallyl-2,2\u2019-dihydroxybiphenyl, molecular weight: 266.33, purity \u226598%) was purchased from Aladdin Company . Dimethyl sulfoxide  was used as solvent. A stock solution of magnolol  was stored at \u221215\u00b0C. Magnolol stock was diluted in PBS  and PBS-DMSO (1.2%) was served as a negative control. Recombinant Human Transforming Growth Factor \u03b21 (TGF-\u03b21) was obtained from R&D systems  and dissolved with sterile ddH2O when need.Magnolol  and were cultured with DMEM medium with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 \u03bcg/ml streptomycin in incubators under 5% COApproximately 2,000 cells were inoculated in each well of 96-well plates and incubated with a concentration range of Magnolol. A Cell Counting Kit-8  was applied to test the cellular viability. The experiments were performed five times independently.Approximately 200 cells were seeded in each well of 6-well culture plates, and the plates were incubated for 10\u00a0h. Then, fresh media containing 0, 15, 30 \u03bcM Magnolol were applied to replace the old media, and the samples were then cultured for 14 days. The colonies were fixed in 4% paraformaldehyde  and then stained with crystal violet . Formatted colonies were gently washed and air seasoned before photos of them were taken. The experiments were performed three times independently.Transwell chambers with 24 wells and 8.0-\u03bcm pore membranes (Corning USA) were applied following the manufacturer\u2019s protocol. Approximately 100,000 cells were seeded in each well in the upper chamber. The upper chamber was filled with serum-free medium which was covered by thin layers of matrigel basement membrane matrix while the lower chamber was filled with complete medium. The invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution after being incubated for 24\u00a0h at 37\u00b0C. Photos were taken using light microscopy and the invaded cells in five random fields were counted.Cells were seeded and confluently cultured in 6-well plates. The scratches were made by scraping with a sterile 1-ml pipette tip in confluently cultured cells. The medium was changed to medium containing 0, 15, or 30 \u03bcM Magnolol, and the samples were incubated for 48\u00a0h. Photos were captured using a phase-contrast microscope at 0, 24, and 48\u00a0h. The rate of cell migration equals the ratio of the coalesced area of the scratch in 48\u00a0h to total the area of the scratch at 0\u00a0h. The experiments were performed three times independently.2), for which a high value indicates a high degree of roundness in cell shape  was applied to obtain total protein from cells. Same amounts of protein were isolated by SDS-PAGE and then passed on to a polyvinylidene difluoride membrane (Millipore). The membrane was incubated with primary antibodies against E-cadherin , vimentin , pSMAD2/3 , SMAD2/3 , and \u03b2-actin  and were then incubated with HPR-conjugated secondary antibody. Chemiluminescence reagents (WanleiBio) were applied to visualize Protein bands. The experiments were performed three times independently.6/50 \u03bcl) were injected into the pancreas of immunocompromised mice. Pinholes were sutured with Prolene 7-0. Once orthotopic xenograft became palpable (approximately 7 days after injection), the mice were divided into two groups randomly (four mice per group). The MAG group received i.p. injection of Magnolol , and the Control group received vehicle  only. Tumor growth was assessed per week by bioluminescence imaging following intravenous d-luciferin injection (150 mg/kg). Final imaging was obtained at 28 days, and then mice were sacrificed. The primary orthotopic xenografts were harvested, weighed, and fixed in Bouin\u2019s solution.Five to six-week-old BALB/C nude mice were purchased from Shanghai SLAC Animal Center . An orthotopic xenograft nude mouse model was established by Orthotopic Injection Technique as described previously . BrieflyParaffin specimens of harvested xenografts were cut into 4-\u03bcm-thick sections and mounted on silanized slides. The immunohistochemical staining was performed as published . The antAll data are expressed as the mean \u00b1 SD and were analyzed using Student\u2019s t test or one-way ANOVA where necessary. GraphPad Prism V6 was applied to analyze and visualize the\u00a0data. All experiments were performed independently at least 3 times. A P value more than 0.05 was defined as statistically significant.We assessed the antitumor effect of MAG with a pancreatic orthotopic xenograft in a mouse model. We chose AsPC-1 for its high tumorigenicity in mice. The experimental design is shown in EMT plays a critical role in cancer proliferation and maintenance of stem cell characteristics, which is reported to be significant from experimental and clinical points of view . We furtThe impact of Magnolol on the viability of PANC-1 and AsPC-1 cells was determined using an MTT assay. PANC-1 and AsPC-1 cells were incubated with a concentration range of Magnolol for 24, 48, and 72\u00a0h. The data revealed that Magnolol suppressed the viability of these cells both time- and concentration-dependently ; Natural Science Foundation of Shaanxi Province (No: 2019JQ-969); and the Xi\u2019an Jiaotong University Education Foundation, XJTUEF (No: xjj2018141).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "The present study aimed to specify diagnostics for peritonsillar abscesses (PTAs) and to clarify the role of minor salivary glands. This prospective cohort study included 112 patients with acute tonsillitis (AT) and PTA recruited at a tertiary hospital emergency department between February and October 2017. All patients completed a questionnaire concerning their current disease. Serum amylase (S-Amyl) and C-reactive protein (S-CRP) levels, tonsillar findings, and pus aspirate samples and throat cultures were analyzed. Eight of 58 PTA patients (13.8%) had no signs of tonsillar infection. The absence of tonsillar erythema and exudate was associated with low S-CRP (p<0.001) and older age (p<0.001). We also observed an inverse correlation between S-Amyl and S-CRP levels . Therefore, we observed a group of PTA patients without signs of tonsillar infection who had significantly lower S-CRP levels than other PTA patients. These findings support that PTA may be caused by an etiology other than AT. Variations in the S-Amyl levels and a negative correlation between S-Amyl and S-CRP levels may indicate that minor salivary glands are involved in PTA development. Acute tonsillitis (AT) is a highly prevalent infection that is responsible for a large number of consultations. Peritonsillar abscess (PTA) is the most common deep head and neck infection, with an incidence of 10\u201341/100,000 , and traShared symptoms of AT and PTA are sore throat and fever. PTA patients also suffer from trismus, and the pain is typically asymmetrical , 11. In Salivary amylase levels can serve as a marker of salivary function, and recent studies have shown that both serum and pus amylase levels are highly elevated in PTA patients compared to patients with other neck abscesses and dental abscesses. In PTA patients, the mean serum amylase level is 50 U/l and mean pus amylase level 3045 U/l  \u201317.Fusobacterium necrophorum [FN]) as major pathogens in AT Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: YesReviewer #3: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified.The Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: YesReviewer #3: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Although a well written paper with a coherent statistical analysis of the data, it is not a new suggestion in the literature regarding the relationship of PTA and the minor salivary glands of Weber (this eponym should have been quoted ...). Other than that, for validation of microbial etiology swabs of the tonsilar surface in the PTA patients should also have been done as for individual comparison of microbial etiology and for comparison with the AT patients. As regarding the s-Amyl levels, the findings could possibly have other causes; in this way, the s-Amyl levels should also have been measured after disease resolution as to exclude false-positive results during PTA crisis.Reviewer #2: This prospective cohort study of 112 patients nicely compares serum amylase and CRP levels in patients with tonsillitis and peritonsillar abscess, including 8 patients who did not have acute tonsillitis on physical exam. Those who did not have acute tonsillitis but had a PTA had lower CRP and were older. The paper is well-written.Suggestions:Would add to keywords; peritonsillar abscess, tonsillitisAbstract line 11: suggest changing to: \"These findings support that some cases of PTA may be caused by anetiology other than AT. \"Page 11 line 25: Suggest not using the abbreviation TE (eg write out the words)Page 12 line 4: It is a stretch to say that there is a group of patients who share features with parotitis. Suggest limiting the conclusion to what is known- i.e. the inverse relationship between CRP and S-amyl.Reviewer #3: interesting topic with good study drawn, intereting number of patines and well designed studysome limitations recognized by the authors and interestingly discussedgood statistical analyses with consistency on results**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  8 Mar 2020Reviewer 2: Abstract line 11: suggest changing to: \"These findings support that some cases of PTA may be caused by an etiology other than AT. \"Response: Abstract lines 11-12 changed.Reviewer 2: Would add to keywords; peritonsillar abscess, tonsillitisResponse: peritonsillar abscess, tonsillitis added to keywordsReviewer 1: Weber\u00b4s glands should be quoted Response: Weber\u00b4s glands are added to introduction, page 4, line 9.Reviewer 2: Page 11 line 25: Suggest not using the abbreviation TE (eg write out the words)Response: Page 11 line 4 as suggestedReviewer 2: It is a stretch to say that there is a group of patients who share features with parotitis. Suggest limiting the conclusion to what is known- i.e. the inverse relationship between CRP and S-amyl.Response: Page 11, line 15 changed as suggested.Reviewer 1: Although a well written paper with a coherent statistical analysis of the data, it is not a new suggestion in the literature regarding the relationship of PTA and the minor salivary glands of Weber (this eponym should have been quoted ...). Other than that, for validation of microbial etiology swabs of the tonsillar surface in the PTA patients should also have been done as for individual comparison of microbial etiology and for comparison with the AT patients. As regarding the s-Amyl levels, the findings could possibly have other causes; in this way, the s-Amyl levels should also have been measured after disease resolution as to exclude false-positive results during PTA crisis.Response: Thank you for your comments. We are not suggesting that the relationship of PTA and minor salivary glands is our original idea. See the introduction Page 4, Lines 6-9: \u201cOver the last three decades, PTA has been speculated to not necessarily arise from AT, but as a consequence of poor dental health, smoking, and salivary dysfunction. Minor salivary glands have been suggested to play a significant role in PTA. \u201cThe idea about validation microbial etiology by comparing the superficial throat swabs between AT and PTA patients is good, and we are definitely considering that in our next study. BUnfortunately, because we did not take the superficial throat swabs from PTA patients in the present study, the comparison at this stage is not possible .Other causes that could elevate the S-Amyl levels are listed in discussion: page 9, lines 23-26. The follow up of S-Amyl levels after recovery would increase the reliability of our findings, but because of the nature of prospective study we have no possibility to get this information afterwards. However, by excluding the known causes of elevated S-Amyl levels we can quite reliably assume that the elevated S-Amyl levels are caused by PTA . 10 Mar 2020Peritonsillar abscess may not always be a complication of acute tonsillitis: A prospective cohort studyPONE-D-20-00508R1Dear Dr. Sanmark,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication.https://www.editorialmanager.com/pone/, click the \"Update My Information\" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Jorge Spratley, MD, PhDAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 20 Mar 2020PONE-D-20-00508R1 Peritonsillar abscess may not always be a complication of acute tonsillitis: A prospective cohort study Dear Dr. Sanmark:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. For any other questions or concerns, please email Thank you for submitting your work to PLOS ONE.With kind regards,PLOS ONE Editorial Office Staffon behalf ofProfessor Jorge Spratley Academic EditorPLOS ONE"}
+{"text": "Salix babylonica and divided into three classes based on the conserved domain analysis, phylogenetic tree and gene structure: tau, phi and DHAR. The tissue-specific expression patterns were substantially different among the tau and phi GSTs. The Salix GST proteins showed functional divergences in the substrate specificities, substrate activities and kinetic characteristics. The site-directed mutagenesis studies revealed that a single amino acid mutation  resulted in the lowest activity of SbGSTU7 among the Salix GSTs. These results suggest that non-synonymous substitution of an amino acid at the putative glutathione-binding site may play an important role in the divergence of enzymatic functions of Salix GST family.Glutathione S-transferases (GSTs) are ubiquitous enzymes that are encoded by a large gene family, and they contribute to the detoxification of endogenous or xenobiotic compounds and oxidative stress metabolism in plants. Although the GSTs gene family has been reported in many land plants, our knowledge of the evolution and function of the willow GSTs is still limited. In this study, 22 full-length GST genes were cloned from  Glutathione S-transferases  are encoded by a large gene family and widely distributed in both prokaryotes and eukaryotes. They are multifunction proteins whose functions include detoxification, since they mainly catalyze the detoxification of a series of xenobiotics by conjugating the reduced glutathione (GSH) to various hydrophobic and electrophilic compounds . GSTs arSalix, a member of the Salicaceae family, is widespread throughout China, and represents an essential part of the urban and rural ecosystems , and could be considered as excellent species for the phytoremediation of heavy metals pollution , we studied its structural and functional characterizations in this study. S. babylonica is considered to be a promising species for bioenergy production due to the high biomass yields through the short-rotation coppice systems  of S. babylonica was searched using the TBLASTN program with the default algorithm parameters and 81 full-length GST protein sequences of Populus trichocarpa. The GST candidates of S. babylonica were then looked up in the National Center for Biotechnology Information (NCBI) Conserved Domains Database1 to confirm the presence of typical GST N- and C-terminal domains in the protein structures. Next, primers were designed, based on the identified GST gene sequences, to amplify the genomic and coding sequences of each S. babylonica GST , and sequenced in both directions. Finally, the amplified coding sequences of the S. babylonica GST genes were mapped to their corresponding genomic sequences to verify the intron/exon structures.In order to identify the GST genes from nica GST , such thS. babylonica GSTs were named according to the system described by Sb to represent Salix babylonica, then the subfamily name was denoted by GST plus the logogram of each class. For example, GSTU, GSTF, DHAR, correspond to the tau, phi, DHAR classes, respectively, and the full phi GST genes names are SbGSTF1, SbGSTF2, etc.The 4CHS) as a template by the SWISS-MODEL software2. The simulated structure of the SbGSTU7 gene was then manually processed using the Discovery Studio 4.0 Client software.The GST protein sequences were separated into two distinct parts according to the N-terminal and C-terminal domains. The protein sequences of the full-length, N-terminal domain and the C-terminal domain were, respectively, aligned using the MUSCLE online service. Next, the alignments were manually further adjusted using the BioEdit v7.0.0 software , then thS. babylonica trees. The primary leaves with a length of 2\u20133 cm that were newly-expanded and the mature leaves with a length of 10\u201312 cm were collected from the shoot top and the middle of the shoot, respectively. After the inverse transcription of the RNA using the RNA PCR Kit (AMV) , the real-time PCR (qRT-PCR) was performed using the SYBR Green PCR master mix (Tiangen) and Bio-Rad iQ5 Real-Time PCR system . Three biological replicates and three technical ones were performed for each qRT-PCR procedure. The S. babylonica actin gene  cells. The BL21 cells, containing the recombinant vectors, were then cultured to an optical density (A600) of 0.5, and isopropyl-\u03b2-D-thiogalactopyranoside (IPTG) was added to the culture to induce the expression of the GST proteins. The final concentration of IPTG was 0.1 mM. After the induction (12 h at 20\u00b0C), the cells were harvested by centrifugation  and resuspended in binding buffer . The cells were disrupted by sonication in ice-cold binding buffer. Next, the homogenate was centrifuged . In order to check the solubility of the recombinant proteins, the resulting particulate material and a small portion of the supernatant were analyzed using SDS-PAGE. Regarding the soluble recombinant proteins, the rest of the supernatant was loaded onto a Nickel-Sepharose High Performance column , and the GST proteins were then eluted with elution buffer .In this study, 22 GSTs  were selected for the enzymatic activity assay. Each of these 22 GST proteins was subcloned into a pET30a expression vector to obtain a 6 \u00d7 His-tag. The primers that were used to construct the GST expression vectors are listed in In order to obtain the mutated proteins, the site-directed mutagenesis of the protein sequences was performed by the methods that were previously reported . The priS. babylonica GST proteins were performed using a 752 UV visible single beam spectrophotometer . According to the methods described by The enzymatic activity assay of the purified Salix tau GSTs, the apparent Km values for GSH and CDNB were separately determined. The concentration of GSH ranged from 0.1 mM to 1 mM, and the concentration of CDNB was fixed at 1.0 mM to determine the KKhttp://hyper32.software.informer.com/. All the activity and kinetics assays were determined at 25 \u00b0C and performed at least three times. The statistical analysis for the enzyme activities between the wild type and mutant proteins were analyzed using the SPSS software ver. 16.0 .In order to examine the steady-state kinetic parameters of the Salix GST proteins were identified to belong to the GST classes of tau, phi or DHAR, and their coding sequences were successfully cloned from Salix babylonica , 22 putative bylonica . The physupports . In orde support . The resSalix GSTs , since they were more variable among the Salix tau GSTs compared with the phi GSTs. The Salix tau GSTs were divided into two clades  were expressed as soluble proteins in Escherichia coli (E. coli), while 3 phi GSTs  and the SbDHAR1 gene were expressed as inclusion bodies in E. coli. In order to assay the enzymatic activities of Salix GSTs, seven GST substrates were used: CDNB, NBD-Cl, DCNB, NBC, fluorodifen, Cum-OOH, and DHA CDNB value, and SbGSTU7 had the lowest kkcat/Km)CDNB values.The termined . Except Salix tau GSTs, SbGSTU7 showed the lowest enzymatic activities to the CDNB, NBD-Cl and fluorodifen substrates, to which SbGSTU6 had the highest enzymatic activities  No.: 4CHS). The GSH-binding sites of GmGSTU10 were Ser13, Lys40, Ile54, Glu66, and Ser67  under the accession numbers listed in H-LY and X-LZ conceived the project. X-LZ and Z-JX performed research. All authors contributed to data analysis, writing of the manuscript, and reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "However, the data show, we do expect considerable epistemic responsibility on the speaker\u2019s behalf: In order to appropriately assert a claim, the speaker must have good reasons to believe it.The recent controversy about misinformation has moved a question into the focus of the public eye that has occupied philosophers for decades: Under what conditions is it appropriate to assert a certain claim? When asserting a claim that  Human action and interaction are heavily governed by conventions, norms, and laws. Over the last few decades, philosophers have explored whether assertion\u2014the backbone of linguistic communication, and thus all language-dependent human practices\u2014is regulated by norms. The topic could not be more pertinent to the current misinformation controversy that shapes public discourse in the United States and many other countries , which hx? The field is roughly divided into two main camps  have a true, justified belief that x [the \u201cknowledge account\u201d  used a classic vignette from epistemology in which Bob has good reason to believe that his colleague Jill drives an American car. In one condition, Jill still drives an American car (true justified belief); in the other, she has changed to a German car . Participants were randomly assigned to one of the two conditions. They were asked whether Bob, when prompted, 1) should say that Jill drives an American car (assertibility) and, as a manipulation check, 2) whether it is true that Jill drives an American car (truth). The questions used a forced-choice Yes/No response mechanism.Study 1 (Left): Although truth did have some impact on assertibility  = 43.05, P < 0.001, \u03d5 = 0.31), at least three out of four participants considered it appropriate to assert a false justified belief . Country was nonsignificant  = 0.56, P = 0.755, \u03d5 = 0.035). In short, the question as to whether the assertibility of a claim requires truth was answered with a resounding \u201cno\u201d across all three cultures and languages tested. Since knowledge entails truth, there is no need to investigate the factive accounts further. Study 2 thus explored the nonfactive accounts  employed the \u201cairport scenario\u201d from ref. Study 2 (P < 0.001); see Right. Assertibility was low when the speaker did not have good reasons . A regression analysis  = 568.09, P < 0.001, Nagelkerke R2 = 0.84) revealed justification to be a significant and powerful predictor of assertibility . Country was nonsignificant (P = 0.437), and the same held for the interactions . Inconsistent with some previous findings for the United States . As preregistered, inattentive subjects, those spending less than 10 s on the main task (reading the short scenario and answering the assertibility question), and nonnative speakers of English, German, or Japanese, respectively, were excluded. The final datasets comprised 143 subjects for the United States , 158 subjects for Germany , and 160 subjects for Japan .For study 1, 495 participants were recruited in the United States, Germany, and Japan via crowdworking platforms (details in P < 0.001). As concerns the main analysis, due to a cell count of zero for \u201cunassertible\u201d in the German true belief condition , a logistic regression analysis could not be performed. However, a logistic regression with the full sample  was possible and is reported in SI Appendix, Table S3. Consistent with the Pearson\u2019s Chi Square results, country had no impact on assertibility (P = 0.423). There was some impact of truth value , although the model explained only about 20% of the variance (Nagelkerke R2 = 0.196). All interactions were nonsignificant .As a further manipulation check, people were asked whether the protagonist\u2019s belief was justified. As intended, the vast majority of participants attributed justification in both conditions in all three countries , 171 from Germany , and 177 from Japan .2 = 0.52, P = 0.469, \u03d5 = 0.030). Besides the manipulation check on justification, a second check was run to ensure that participants understood that the protagonist did indeed believe the proposition at issue and thus was not interpreted as lying. As intended, at least about four in five participants ascribed belief in all conditions in all three countries .To increase external validity, the formulation of the assertibility question was also manipulated: It either asked whether Carlos \u201cshould have said\u201d or whether it \u201cwas appropriate for Carlos to say\u201d that the flight leaves from gate 24. Due to a cell count of zero in the justified belief condition in certain countries, formulation could not be entered as a predictor into the regression analysis, but a Pearson Chi Square test revealed formulation to be nonsignificant  page: https://doi.org/10.17605/OSF.IO/H6M49.For both studies, the materials  and detailed analyses are reported in Supplementary File"}
+{"text": "Ruta graveolens, essential oil (REO) against clinical strains of Candida albicans, Candida parapsilopsis, Candida glabrata, and Candida tropicalis. Data obtained showed that C. tropicalis and C. albicans were the most sensitive strains showing minimum inhibitory concentrations (MIC) of 4.1 and 8.2 \u00b5g/mL of REO. Time\u2013kill kinetics assay demonstrated that REO showed a fungicidal effect against C. tropicalis and a fungistatic effect against C. albicans. In addition, an amount of 40% of the biofilm formed by C. albicans was eradicated using 8.2 \u00b5g/mL of REO after 1 h of exposure. The synergistic effect of REO together with some antifungal compounds was also investigated. Fractional inhibitory concentration index (FICI) showed synergic effects of REO combined with amphotericin B. REO Lead a disruption in the cellular membrane integrity, consequently resulting in increased intracellular leakage of the macromolecules, thus confirming that the plasma membrane is a target of the mode of action of REO against C. albicans and C. tropicalis.Drug resistance in antifungal therapy, a problem unknown until a few years ago, is increasingly assuming importance especially in immunosuppressed patients and patients receiving chemotherapy and radiotherapy. In the past years, the use of essential oils as an approach to improve the effectiveness of antifungal agents and to reduce antifungal resistance levels has been proposed. Our research aimed to evaluate the antifungal activity of Colombian rue,  Candida spp. with the appearance of white lesions generally affecting the oral or oropharyngeal mucosa [Candida species are the most common pathogen isolated in patients in the critical care setting. It is commonly found in elderly subjects, diabetic patients, and solid organ transplant recipients, and it is also an etiological agent of urinary and vaginal tract infections [The mucosal surfaces primarily affected by candidiasis are the oral cavity, esophagus, angles of the mouth, and genitals . Oral cal mucosa . Despitel mucosa . Reportsl mucosa . Furtherfections .C. albicans is the predominant pathogenic fungus responsible for the OC [albicans Candida (NAC) species are starting to be frequently isolated in Candida infections. The incidence of species such as C. glabrata, C. parapsilosis, C. tropicalis has been widely reported within the past 10-year period [C. glabrata and C. parapsilosis are frequently isolated in North and Central Europe and North America, and C. tropicalis in South America and Asia [Although r the OC , non-albr period . In partand Asia . Moreoveand Asia .Satureja montana; Thymus capitatus, and Melaleuca alternifolia EO in Candida albicans inhibition [In order to find new classes of antifungals, the use of essential oils (EOs) has been proposed, and many studies have focused on studying EOs properties and application in fungal control ,10. Sevehibition .Ruta graveolens is a plant used in traditional and herbal medicine. It was used in some medieval rites to protect the house against negativity. In folk medicine, rue has been used to treat cough, diphtheria laryngitis, colic, headache, and as an antidote in case of mushroom poisoning, snake bites, and insect bites; in addition, it has been used for its stimulating, stomachic, emmenagogue effects consumed as an infusion and to treat headache, muscular and joint pain, as well as an anti-inflammatory using the oil or extract [R. graveolens essential oil (REO) in vitro against phytopathogens as Colletotrichum gloeosporioides [Cladosporium herbarum, Aspergillus fumigatus, Fusarium oxysporum, Aspergillus flavus, and Alternaria alternata [ extract . In the  extract ,15. In torioides ,17, Cladlternata . Moreovelternata , goosebelternata , tomato lternata , and pealternata  showed aR. graveolens antifungal activity against multi-resistant Candida spp. of clinical origin at the same time evaluating the time\u2013kill kinetics and the ability to reduce biofilm formation in order to find new alternatives to help overcome drug resistance in Candida spp.The present study aimed to clarify Candida spp. was selected for this study: C. albicans (6), C. parapsilosis (6), C. tropicalis (6), C. glabrata (6). The isolates were cultured from specimens isolated from the oral cavity of patients with head and neck cancer at the Otolaryngology Clinic, Department of Medical, Surgical and Experimental Sciences, University of Sassari, Italy. All microorganisms were identified by standard methods: germ tube test (GTT) and YBC Vitek Card  [A collection of 24 clinical isolates belonging to 4 different  France)  and storC. tropicalis ORL20 and ORL21 was successively confirmed using the amplification of the ITS region  with universal fungal primers  [C. tropicalis ORL20: KX664640.1 and C. tropicalis ORL21 KX664611.1.The identification of 1, ITS4) . The GenFluconazole (FLC) was obtained from Sigma-Aldrich. Stock solutions of FLC were prepared in dimethyl sulfoxide. The final concentration of DMSO was not higher than 0.14%. In addition, RPMI 1640 (Thermo Fisher Scientific) was used in this study. Rue essential oil (REO) was obtained from Kr\u00e4uter SAS  lot n \u00b0 SSTE01.Candida spp. strains were determined according to the broth microdilution assay in 96-well microtitration plates, as described by the M27-A3 method from the Clinical and Laboratory Standards Institute  [6 CFU/mL. Each strain was tested in duplicate and positive growth control (the strain under test without REO) and a negative one (medium only) were included in each test. The plate was incubated at 37 \u00b0C, and the minimal fungicide concentration (MFC) was determined by taking 10 \u00b5L from each well and spreading them on Sabouraud dextrose agar. The plates were incubated at 37 \u00b0C for 24/48 h and checked to detect microbial growth. MFC is considered the lowest concentration capable of inhibiting 99% fungal growth. Three independent experiments were performed.The minimum inhibitory concentrations (MICs) of antifungal agents (REO and FLC) against the y NCCLS) . TwofoldCandida species, the broth dilution method was used, as recommended by the Clinical and Laboratory Standard Institute (CLSI. 2008). Yeasts were cultivated at 37 \u00b0C on Sabouraud dextrose agar plates  for 24 h. The inoculum was prepared by a dilution of the colonies in a salt solution, at a concentration of 0.5 McFarland and confirming the concentration by spectrophotometric reading at a wavelength of 530 nm. The sensitivity test was carried out in RPMI 1640, using 96-well plates. Oil concentrations were prepared by serial one to two dilutions from 131 to 1.0 \u00b5g/mL. After shaking, 100 \u00b5L of each oil dilution and 100 \u00b5L of yeast suspension at a concentration of 106 CFU/mL were added to each well and then incubated at 37 \u00b0C for 48 h.In order to establish the MFC of In order to determine the MFC value, 10 \u00b5L were seeded on Sabouraud dextrose medium, the plates were incubated for 24\u201348 h at the temperature of 37 \u00b0C. Minimal fungicidal concentration (MFC) was considered as the lowest concentration inhibiting fungal growth. Moreover, each yeast strain included in the study was tested for its sensitivity to fluconazole, voriconazole, and amphotericin B. Each experiment was performed in duplicate and repeated three times.C. albicans (ORL3 and ORL8) and two C. tropicalis (ORL20 and ORL21) strains that were selected for their sensibility to REO and amphotericin B, were then chosen for further studies.Two Ruta graveolens essential oil, the checkerboard method was performed to obtain the Fractional inhibitory concentration indices (FICIs) of REO in combination with amphotericin B and fluconazole, following the methodology proposed by [6 CFU/mL) in each well and mixed well. The plates were incubated at 37 \u00b0C for 48 h. The inhibition of the growth of fungal cells was indicated by the absence of the red color.In order to determine the synergy between antifungal antibiotics and posed by . The micThe FICIs were calculated using the following formulas:The interpretation of FIC indices (FICIs) was made following the approach used by Fratini et al. . This meC. albicans and two C. tropicalis strains was performed following the method proposed by Chaves-L\u00f3pez et al. [In order to further evaluate the REO effect, the time\u2013kill assay in two z et al. , with soIn order to determine REO fungicidal or fungistatic features, a reduction of <3 log CFU/mL after the treatment in the growth of the starting inoculum was defined as fungistatic activity of REO, and a reduction \u22653 log CFU/mL as fungicidal activity according to the method proposed by Scorneaux et al. . SubsequCandida strains were evaluated to quantitate the reduction of biofilm in the presence of REO in 96-well polystyrene microplates according to the methodology reported by Rossi et al. and Chaves-L\u00f3pez et al. [Standardized samples from four z et al. ,31. To b590) for each strain were measured using a Biolog MicroStation system , and the biofilm productions were grouped into: OD590 < 0.1 = nonproducers (NP), OD590 0.1\u20131.0 = weak producers (WP), OD590 1.1\u20133.0 = moderate producers (MP), and OD590 > 3.0 = strong producers (SP). The biofilm reduction was calculated using the following equation:CO = absorbance sample control and AbsREO = absorbance sample treated with REO.The absorbances values at 590 nm . Photographs were taken with Samsung COLOR CAMERA SAC-410 PA interfaced with a PC.Cell membrane damage produced by the REO was evidenced using Evans blue staining according to Chaves-Lopez et al. . BrieflyExperimental results were expressed as means \u00b1 standard deviations: data were evaluated by analysis of variance (ANOVA) and compared by 95% Tukey\u2019s HSD test, using Statistica 13.5 software .Data regarding the REO characterization by mass spectrometry\u2013gas chromatography (MS\u2013GC) were reported in our previous work  Supplem. REO shoCandida species tested, 8 isolates were resistant to FLC with MIC values ranging from 8.2 to 256 \u00b5g/mL, and 8 isolates were sensitive to FLC with MICs ranging from 1 to 4 \u00b5g/mL. The MICs of REO were in a range of 8.2\u2013131 \u00b5g/mL against all the Candida spp. isolates ; in addition, C. glabrata was also resistant to voriconazole (MFC: 2 \u00b5g/mL after 24 h and 4 \u00b5g/mL after 48 h). The antifungal effect of REO was therefore highlighted against C. albicans and C. tropicalis also with respect to synthetic drugs such as amphotericin B and fluconazole.The MFC data for clinical It should be pointed out that, generally, the efficiency of REO for the yeast species here studied is quite different not only for different species but even among the same species.Ruta graveolens in combination with fluconazole and amphotericin B using the checkerboard method was evaluated. As shown in C. albicans ORL3 and ORL8 and C. tropicalis ORL21 with FICI values of 0.38, 0.5, and 0.8, respectively. REO did not present interaction with the amphotericin B in C. tropicalis ORL20. Regarding the combination of REO\u2013fluconazole, no interactions were evidenced with C. tropicalis or C. albicans ORL8. However, antagonistic activity was revealed in C. albicans ORL3. As is observed in To overcome the mechanisms of bacterial resistance against antibiotics, in the last years, some studies have proposed the use of association of plant extracts with antibiotics. In our study, the synergistic potential of essential oil of C. albicans and C. tropicalis that showed high sensibility to REO were taken into consideration.For the TKK assay, two representative strains of Candida strains tested are depicted in C. tropicalis strains were more sensitive to the treatment than C. albicans. In addition, differences between the strains of the same species were also observed. Then, 15 min after the treatment, indeed, the cell count was reduced by about 1.4 log CFU/mL. and 1.0 for C. tropicalis ORL21 and C. tropicalis ORL20, respectively. After 2 h of treatment, there was a further decrease of about 1.7\u20131.5 log CFU/mL for both strains. With exposure time increase, both strains were reduced, further reaching values of 1.5 log CFU/mL and 1.15 log CFU/mL, respectively, thus evidencing a fungicidal activity (a kill of \u22653 log CFU/mL).REO and fluconazole mean time\u2013kill graphs and standard deviations against the four C. albicans, showing only 0.98 log CFU/mL after 2 h of exposure in C. albicans ORL08 and 0.25 log CFU/mL in C. albicans ORL03. Additionally, in this case, there was a further reduction of the yeast population achieving counts of 3.58 and 2.92 log CFU/mL for Candida albicans ORL08 and ORL03, respectively, reflecting a fungistatic activity. Additionally, it was found that concurrent time\u2013kill experiments on isolates with fluconazole failed to show reductions in starting inoculum.The effect of REO was more reduced in C. tropicalis, the time needed to reach 50% of the reduction was less than an hour but it presented differences between ORL20 and ORL21 strains, being the latter the most sensitive to REO with 0.39 h. After about 1.5 h. a 90% reduction of growth was evidenced by both strains, reaching 99.9% at 1.8 and 2.9 h for C. tropicalis ORL21 and ORL20, respectively. Concerning C. albicans, the reduction of 50% of growth with respect to the starting inoculum was reached in a range of 3.6 and 4.5 h. No was achieved a reduction in the CFU of 90% and 99.9%.The time required by REO to achieve a reduction of growth of the starting inoculum was determined for each strain . For C. C. tropicalis and C. albicans ORL3 strains formed strong biofilm biomass, while C. albicans ORL08 showed a weak production. When 8.2 \u00b5g/mL of REO was applied to the formed biofilm, we observed a significant detachment of the biofilm biomass after 1 h of REO exposure, overall, in C. albicans . On the contrary, we observed a negligible biofilm detachment in samples with C. tropicalis during the first hour of treatment.All the strains tested produced significant biofilm biomass on polystyrene microplates at 37 \u00b0C, as shown in p > 0.05) differences between the two treatments. It is worth mentioning that the strongest biofilm formed by C. albicans ORL03 was more difficult to eradicate with both REO and amphotericin B.Comparing the REO efficacy with that of amphotericin B, on the biofilm eradication, we observed slight but not significant  increase in the cellular release, which was intensified with the exposure time. Indeed, an early release of the intracellular compounds was observed already after 30 min of exposure to REO, in comparison with the untreated sample. A minimal and constant cellular release in the Candida yeasts was observed with the fluconazole.As observed in Candida cells exposed to REO, fluconazole, and untreated are presented in C. Tropicalis, yeasts treated with REO showed a constant behavior in the initial 30 min after treatment, followed by a significant increase (p < 0.05) of 15.2 and 14.2% after 120 min treatment for ORL21 and ORL20, respectively, concerning the control. C. albicans yeasts showed a minor but significant increase (p < 0.05) about control with 3.7 for ORL08, and 7.6% for ORL03. No significant differences were evidenced in the yeasts with the fluconazole after 120 min post treatment.The extracellular pH of C. albicans and C. tropicalis.To investigate if there was a disruption of cell membrane integrity to the exposure to the REO, the cells were stained with Evans blue staining. As indicated in In addition, we observed a cell shrinkage after the treatment with REO probably due to the release of the intracellular components.C. albicans, C. neoformans, and Malassezia sp. [C. albicans seems to be due precisely to a reduced susceptibility of the target enzyme. The other mechanism of resistance to azoles may occur due to the inability of antifungal agents to accumulate in the cell due to a high outflow of the drug in turn due to an alteration of the functionality of transporters located on the membrane of the fungus. Two types of transporters mediate this mechanism: the \u201cABC transporters\u201d, encoded by the CDR genes, and the major facilitators, encoded by the MDR genes. These mechanisms have been described in isolates of C. albicans, C. glabrata, C. parapsilosis, and C. tropicalis [Resistance may be due to an altered intracellular accumulation of the drug, an altered composition of membrane sterols, an alteration of ERG11 , or an alteration of the functionality of the efflux pumps . These lezia sp. ,36. The opicalis ,38.Ruta graveolens essential oil had a satisfactory antifungal activity against C. tropicalis and C. albicans associated with oral candidiasis. Preliminary studies demonstrated that the antifungal activity of this oil is due overall to the main components 2-nonanol and 2-undecanone, which exhibited the most potent antifungal effect [C. albicans, with 86% of growth reduction in comparison to the positive control (amphotericin B); these authors showed that the antifungal activity is related to the abundance of ketones and alcohols in the REO. In addition, Attia et al. [C. albicans clinical strains, a C. glabrata with MIC of 1.14\u20132.5 \u00b5g/mL; moreover, morphological changes were observed including cell surface deformation, disruption, and prevention of germ tube production; additionally, it was demonstrated a direct correlation between the percentage of ketones and the antimicrobial activity.In this study, we demonstrated that l effect . On the l effect  tested Ra et al.  also fouAntibiotic resistance is a big concern around the world and many strategies have been adopted to reduce this problem. In the last years, a rational approach to deal with antibiotic resistance problems using a combination therapy combining conventional antibiotics and essential oils has been proposed. In this work, we evaluated the effect of these combinations using the checkboard method and the determination of the FICs was made using the novel approach proposed by Frattini et al. . This apCandida species has been reported. For example, Saad et al. [Candida species [Citrus aurantium essential oil showed synergistic potential with fluconazole and amphotericin B against C. albicans and enhanced the antifungal efficacy of the clinical drugs by 8.3 to 34.4 folds [C. albicans and C. tropicalis. Combination of clinical antibiotics with essential oils and phytocompounds targeting resistant fungi may have a different mechanism of action including (i) sequential inhibition of common biochemical pathways, (ii) amplified diffusion of one antifungal agent subsequent from the action of another antifungal agent on the fungal cell membrane, (iii) inhibition of different targets, and (iv) the inhibition of carrier proteins [In this context, the interaction between plant extracts and antibiotics with synergistic activity against d et al.  reported species . In the .4 folds . In thisproteins .C. albicans and C. tropicalis tested. REO evidenced the highest levels of killing against C. tropicalis strains showing antifungal activity. The time to achieve a 50% reduction of starting inoculum growth was less than an hour with a decrease in growth concerning control of 76.1% for C. tropicalis ORL21 and 82% for C. tropicalis ORL20 after 48 h of treatment. However, the REO effect was not so fast with C. albicans strains with just a 50% reduction after 3.5 h, a final reduction of 48% for C. albicans ORL08, and 50% for C. albicans ORL03, indicating fungistatic activity of the REO. These results suggest that REO effects depend on the Candida species. Similar results have been reported with other essential oils showing different fungicidal or fungistatic activity according to Candida species. In one study, Ocimum gratissimum L. essential oil was fungicide against C. tropicalis but showed fungistatic activity against C. albicans [C. glabrata and C. albicans takes a time of over 1.5 h to reach a 50% of reduction of growth [In our study, the time\u2013kill kinetics demonstrated that REO had an effect against the four strains of albicans . Some auf growth .Candida biofilm is a well-organized formed by planktonic and mycelial yeast form, surrounded by extracellular polymeric substances; this structure is an effective microbial protection and can generate the well-known drug resistance [C. albicans strains evaluated; this activity was detected within just one hour of treatment. The percentage of biofilm eradication here obtained were similar to those obtained with amphotericin B, a drug with known efficacy against Candida biofilm [Candida activity of different EOs such as peppermint, eucalyptus, ginger grass, clove, and thyme essential oils ranging between 28 and 85% [Candida albicans; furthermore, the hydrophobic character of EOs may increase the absorption through charged extracellular polymers, producing that oil has greater contact and permeation in the membrane to the cells [C. gloesporioides, where a compromised membrane was observed after one hour of exposure [C. tropicalis strains. Al-Fattani et al. [C. tropicalis are mainly constituted by hexosamine matrix in comparison with a glucose-rich matrix of the C. albicans biofilm; such a structure in the latter allows faster drug penetration. Moreover, one study involving several candida species aiming at measuring drug diffusion rates reported that the slowest rates of penetrations were presented by C. tropicalis [C. tropicalis biofilm.sistance . REO dem biofilm . Studies and 85% ,44. Somehe cells ,47. REO exposure . No signi et al.  reportedopicalis . ConsideCandida. Some authors have suggested that the antimicrobial activity of essential oils involves phenomena such as changes of cell membrane integrity, leading to an alteration of permeability and consequent leakage of cell contents [Anethum graveolens essential oil induced a lesion of the cell membrane in C. albicans. On other hand, Ahmad et al. [Coriaria nepalensis essential oils disrupt membrane integrity in different Candida isolates. A similar result was reported by Rajkowska et al. [Candida.During recent years, the antifungal activity of the REO has been reported ,19,20,21contents . In our contents . Some aucontents  reportedd et al.  reporteda et al.  using diRuta graveolens essential oils as a natural fungicidal agent and as a biofilm eradication agent and gave insights into its mode of action on Candida albicans and Candida tropicalis. Our study showed strong antifungal and biofilm eradication activity using 8.2 \u00b5g/mL of REO. In addition, we observed irreversible cellular membrane damage inducing leakage of the intracellular compounds very few times after the treatment. Fractional inhibitory concentration index (FICI) showed synergic effects of REO combined with amphotericin B. These results suggest the possible effective use of REO alone or in combination with amphotericin B, against multidrug resistant overall on C. tropicalis strains. In addition, the present study demonstrated that REO essential oil is a promising alternative for the treatment of Candida biofilms eradication.In this work, we explored the use of"}
+{"text": "Through emergency medicine\u2019s evolution as a specialty, residency leadership embraced advanced training in educational theory and practice. It is not clear that leaders in medical toxicology training programs have followed this paradigm. Medical toxicologists have long been recognized as master educators with a range of experience educating fellows, residents, members of the public, and allied health care providers. However, enthusiasm and historic prowess in bedside and small group teaching may not translate to designing and running an outstanding medical toxicology training program.While toxicology fellowship directors possess content expertise, there is a paucity of information on their training as educational leaders. We distributed a survey to fellowship directors via the American College of Medical Toxicology (ACMT) program director mailing listserv to assess participation and interest in formal activities focused on education and presented our findings at the 2020 American College of Medical Toxicology Annual Scientific Meeting. Only half of the responding program directors reported participation in faculty development activities focused on educational methodology, and less than a third reported development in educational theory specifically. Most reported an interest in more opportunities for educational training on theory and administration.There is an opportunity and need to strengthen a field which constantly adapts to new circumstances and historically embraced advancements in medical education. Medical toxicology subspecialty training has long been the most common fellowship training for emergency medicine program directors; however, there has been a recent increase in the number of emergency medicine program directors who have completed advanced training or a fellowship in medical education . SimilarLike the person in the burning building who\u2019s more interested in putting out the fire than understanding the laws of thermodynamics, fellowship directors in our survey seem more focused on concrete educational issues. While the majority of current program directors support the need to understand educational milestones and curricular design, less than half agreed educational theory was vital to understand. This may suggest a misunderstanding of how educational theory informs development of effective curriculum and evaluation of milestone achievement. Much of the role of fellowship director relies on these areas of development. The Accreditation Council for Graduate Medical Education\u00a0(ACGME)\u00a0requires core faculty in toxicology must \u201cdevote a significant portion of their entire effort to fellow education and/or administration, and must, as a component of their activities, teach, evaluate, and provide formative feedback to fellows .\u201dIronically, the same impulse which encourages someone to pursue advanced training in medical toxicology makes it less likely that they will also do a formal education fellowship or obtain an advanced degree in educational theory. New fellowship directors are frequently spending time running the fellowship or establishing their credentials in the field; many do not have the time to pursue formal education professional development until later in their career. More so, because they frequently attend meetings and conferences focused on toxicology, they are less likely to have the access or resources to independently implement professional development in formal education training. While residencies have assistant program directors who spend years preparing to lead the program by learning the nuances of the position, medical toxicology fellowships rarely have such a position. One program director commented, \u201cHaving been a program director for a long time, I think the administrative burdens have expanded tremendously. I\u2019m not aware of any preparation for that aspect besides mentorship and the right disposition to get that work done.\" In our survey, we found most of those with more than five years of experience had participated in formal educational activities, unlike those with fewer years of experience. Enthusiasm for formal training is not lacking, as our survey suggests the more junior fellowship directors reported interest in such activities. The very time spent running a fellowship can improve educational skills, but a structured approach grounded in theory might provide greater impact and rigor. Examples where formal educational training may serve better than mere enthusiasm include remediating a poorly performing fellow or faculty member, curriculum assessment, and research on educational methodology , 5.It is likely many emergency medicine fellowships, other than medical education, also\u00a0need to embrace this key transition point and focus on program director development as lead educators within their field. Medical toxicology should shepherd this charge. We need to lead in this process by forming alliances with other national organizations (such as Council of Residency Directors (CORD)\u00a0in Emergency Medicine, American College of Emergency Physicians\u00a0(ACEP)\u00a0Teaching Fellowship, Academic Life in Emergency Medicine, Harvard Macy Institute for Educators in Health Professions). Partnerships with our education expert colleagues  may benefit further needs assessments to identify scope of the problem and approach to implementation of change.Our national organizations should be a forum for further dissemination of educational training. The addition of educational sections at toxicologic national conferences could benefit medical toxicology faculty who are more likely to attend medical toxicology specific events. Implementation of online training sessions may allow a broader reach to faculty members of other emergency medicine subspecialties. Finally, the creation of a formal certificate program for early career medical toxicologists interested in education would allow for synchronous and asynchronous longitudinal sessions focused on education theory, curricular design, learner assessment, and remediation. Formal mentorship from senior faculty would be a source of support and pressure to complete such an optional curriculum, but over time such coursework could serve as a model for other fellowships.There is an unmet need among our medical toxicology educational leaders. Establishing and supporting high standards for our educators is necessary to advance our specialty. Now is the time to take the next step, embrace early adaptation, and lead the way forward. We owe it to our patients, our learners, and our predecessors who inspired us to join this rarefied field."}
+{"text": "Some of community mitigation efforts on COVID-19 created challenges to ongoing public health programs, including HIV care and prevention services among men who have sex with men (MSM). The goal of the current study was to explore sociodemographic factors and the impact of COVID-19 on HIV testing among Chinese MSM during state-enforced quarantine.We conducted a community based survey between May 1st to June 30th, 2020 on COVID-19 related impacts on HIV testing among 436 China MSM during the COVID-19 state-enforced quarantine.One-third (33.7%) of MSM received HIV testing during the quarantine period. Few participants reported difficulty accessing facility-based testing  or obtaining HIV self-test kit online . However, 12.1% of participants reported being afraid of getting facility-based HIV test due to concerns about the risk of COVID-19. In the multivariate logistic regression model, participants who were married , reported increased quality of sleep , and increased difficulty in accessing health care  were more likely to get an HIV test during the state-enforced quarantine.The mitigation measures of COVID-19 have created various barriers to access HIV related prevention services in China, including HIV testing. To mitigate these impacts on HIV prevention and care services, future programs need to address barriers to HIV-related services, such as providing high-quality HIV self-testing. Meanwhile, psychological services or other social services are needed to those experiencing mental distress. Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1:\u00a0YesReviewer #2:\u00a0No********** <font color=\"black\"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1:\u00a0YesReviewer #2:\u00a0No********** <font color=\"black\"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data\u2014e.g. participant privacy or use of data from a third party\u2014those must be specified. </font> The Reviewer #1:\u00a0NoReviewer #2:\u00a0No********** <font color=\"black\"> 4. Is the manuscript presented in an intelligible fashion and written in standard English? </font> PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1:\u00a0YesReviewer #2:\u00a0No********** <font color=\"black\"> 5. Review Comments to the Author </font> Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1:\u00a0The authors evaluated the impact of COVID-19 on HIV testing among MSM in China. Overall, it was a well-written paper and the analyses will sound. However, there are some typos throughout the paper that should be addressed before publication. I have a few other comments as well, noted below.Methods: Is the survey nationwide or in the Jiangsu province. Line 139 says the province, but line 141 says nationwide.For eligibility criteria, did the men also have to be HIV negative?The survey was face-to-face. That could impact response rates during a pandemic. Could you address this in the discussion? What was the response rate? Was there an incentive?Results: Remember to discuss results in past tense.Discussion: I think a big limitation in comparing testing rates between your survey and others is the different lengths of time that are assessed. This is addressed briefly, but should be discussed further. When you summarize the results for other studies you should include the time frame . This is important information when comparing to your 3-month testing rate, since you would expect the proportion to be lower than the proportion testing in past 12-months. You should try to include results from other studies that also ask about a 3-month window for a more comparable comparison.Tables: In Table 2 you don't need to include both the did test and didn't test columns since you one is the inverse of the other. I would only include the \"did test\". You can also add a total column and a col percent column, so then most of the info in Table 1 can be shown in Table 2. Table 1 then can just show location of test among those that tested, and the the results of the three questions asked of non-testers. You should also make it clear that those three questions are only asked of the non-testers, so it is clear why it's a smaller sample size.The description of the location in the title of the table should also be consistent between the two tables.Reviewer #2:\u00a0Apart from all other issues in this paper, I do not think it is possible to to analyze the impact of state enforced Covid-19 quarantine on HIV testing if one of the enrollment criteria was \"have not taken any HIV test during the past 12 months\" (line 145-146). An association between enrollment criteria and the outcome of the study is a serious fallacy. Moreover, the past 12 months also cover the period of covid state enforced quarantine. Hence the impact thereof cannot be evaluated in this study.********** <font color=\"black\"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose \u201cno\u201d, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. </font> Reviewer #1:\u00a0NoReviewer #2:\u00a0Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at\u00a0figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,\u00a0 12 Dec 2021Thank you for the opportunity to revise and resubmit our manuscript, \u201cPsychosocial and behavioral correlates with HIV testing among men who have sex with men during the COVID\u201019 pandemic in China\u201d. The recommendations of the reviewer were constructive, and we have made appropriate changes.AttachmentResponse to reviewers.docxSubmitted filename: Click here for additional data file. 26 Dec 2021Psychosocial and behavioral correlates with HIV testing among men who have sex with men during the COVID\u201019 pandemic in ChinaPONE-D-21-17838R1Dear Dr.Ling-en-Shi,We\u2019re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you\u2019ll receive an e-mail detailing the required amendments. When these have been addressed, you\u2019ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they\u2019ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Professor Kwasi Torpey, MD PhD MPHAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 30 Dec 2021PONE-D-21-17838R1 Psychosocial and behavioral correlates with HIV testing among men who have sex with men during the COVID\u201019 pandemic in China Dear Dr. Shi:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofProfessor Kwasi Torpey Academic EditorPLOS ONE"}
+{"text": "The development ofporous materials with hierarchical porous structuresis currently of great interest. These materials exhibit propertiesrepresentative of different pore scales and thus open up the possibilityof being used in new applications. In this paper, a method for thepreparation of silver foams with hierarchical porous structures isdiscussed. Here, the replication method, which is typically used toproduce coarse-pore foams, is merged with dealloying, which is commonlyused to manufacture small-pore foams. For this purpose, packed NaClparticles (hard template) were infiltrated with 75%Al\u201325%Agalloy . Both thehard and soft templates were removed by water dissolution and dealloyingwith HCl or NaOH solutions, respectively. Extensive characterizationof the resulting materials revealed pores ranging from a few nanometersto hundreds of micrometers. The materials were characterized by theirantibacterial performance against Gram-positive and Gram-negativebacteria and showed significantly higher activity than both silverfoams prepared by sintering pure Ag particles and silver nanofoamsproduced by chemical dealloying. The combinations of pores of differentsizes and the resulting high internal specific surface area have adecisive influence on the antibacterial capacity of these new materials. However, in the last decades, intensiveefforts have been made to produce porous materials with pores in thenanometer range, characterized by a high specific surface area.6 These materials have been considered for variousapplications including electrodes, catalysts, sensors, actuators,and filtration systems.3 Activated carbon,7 activated alumina,8 or zeolites6 have been shown to be effectivein the chemical catalysis of certain reactions, either by directlyparticipating in the reaction mechanism or by supporting other catalystmaterials.7 More recently, foams of various types  with interconnectednanometer pores have been prepared by spinodal decomposition followedby selective dissolution of one of the phases present.9 In addition to the inherent advantages of high specificsurface area, small-pore materials have distinct disadvantages dueto restricted diffusion of species into and out of the pores, especiallyat large sample dimensions.10 This effectis particularly important for the innermost pores of a material, whichoften become nonfunctional due to access difficulties of diffusingspecies.10Inrecent years, there has been growing interest in the developmentof porous materials with specific pore sizes for applications as diverseas impact absorbents, filtration systems, thermal management systems,prosthetic implants, and catalysts.11 This has led to materialswith hierarchical porous structures at the nanometer scale, formingnanoarchitectures that are often used to control diffusing species.12 Other materials developed a hierarchical combinationof pores at the macro/micro- and nanometer scales with specific applicationsin catalytic processes.13 An exciting applicationof certain porous metallic materials is their ability to eliminatebacteria.14 Metal ions such as copper,zinc, or silver have been shown to have potent antibacterial activity.15 Silver ions have a broad antibacterial spectrumas they negatively affect the growth of Gram-positive and Gram-negativebacteria. This is due to their ability to form ligand complexes withproteins or enzymes in bacterial cells.17 In recentwork,14 the effect of micro/macroporoussilver foams on the growth of certain bacteria was investigated. Thefoam to be used as an antibacterial material is expected to have abroad antibacterial spectrum, excellent mechanical resistance, andlong-term biocidal activity. In addition, essential conditions mustbe considered, such as that it is not harmful to the environment andhuman health and does not cause toxic effects.18Materials with hierarchicalporous structures have had a significantimpact on the scientific literature because they offer a combinationof properties characteristic of different pore scales. In this scenario,the hierarchical pore size scales largely determine the intended applicationsfor each material.The primary objective of this study is to developeffective antibacterialfoams with hierarchically arranged pores ranging from millimetersto nanometers. The underlying principle is to merge pores of differentsizes into a material with a large surface area , which in turn allows efficient molecular transport (whichrequires larger pores). The process used to produce these materialsis a combination of the replication method, typically used to producelarge-pore foams, and the selective dissolution method, generallyused to manufacture small-pore foams. Production began by alloyingaluminum and silver metals in a 75:25 ratio, and the resulting alloywas then used for gas pressure infiltration of packed NaCl particlepreforms. NaCl was chosen as the hard template, while the aluminumin the alloy served as the soft template. After detailed structuralcharacterization, experiments were performed to evaluate the foamsfor their antibacterial properties against Gram-positive and Gram-negativebacteria. These evaluations served to demonstrate the different rolesof pores at different size scales in the elimination of bacteria andto explain the reason for the excellent antibacterial behavior ofthe foams with hierarchical porous structures prepared here.22.1The materials used forthe preparation of the alloy Al\u2013Ag were high-purity 99.999%aluminum and 99.99% pure silver cast grain purchased from Alfa Aesar. NaCl particles with 99% puritywere purchased from Panreac . These initial particleswere sieved, and the fraction with an average diameter of 350\u2013600\u03bcm was collected. Analytically pure HCl and NaOH, purchasedfrom Alfa Aesar , were usedas selective dissolution reagents.2.2\u20131 so that the samplecould acquire a spinodal structure. The prepared metal with cylindricaldimensions of 15 mm diameter and 15 cm length was cut into smallerpieces of 5 cm height for use in infiltration experiments.The Ag\u2013Al alloy with atomiccomposition of 75%Al\u201325%Ag(hereafter referred to as Al\u201325Ag) was prepared in an inductionfurnace with up to 15 kW maximum power. For this purpose, appropriateamounts of the two metals were placed in a graphite crucible coatedwith boron nitride (BN). The crucible was heated to 1100 \u00b0C,and the liquid alloy was poured into a large graphite-coated steelmold after a waiting time of 3 min. The waiting time and the liquidmetal movement generated by induction ensure the homogeneity of thesample composition. Casting into the steel mold allowed the metalto cool at about 300 K min2.3Ag foams with hierarchical porous structureswere prepared by the following three-step method: (i) packing NaClparticles to conform packed particulate preforms and infiltratingsuch preforms with liquid Al\u201325Ag alloy, (ii) removing theNaCl templating agent by water dissolution to form Al\u201325Agfoams (hereafter referred to as AlAg), and (iii) dissolving the Al-richphase by a chemical attack with aqueous solutions of HCl or NaOH toform the final Ag foam. Each step is explained in detail below.\u20131. After another5 min, necessary to ensure temperature homogenization, the vacuumwas stopped and a pressure of 0.1 MPa was applied to the chamber.This pressure was sufficient to infiltrate the liquid Al\u201325Agalloy into the porous preform. Then, the crucible and sample set weredirectionally cooled by moving them to the lower part of the chamber,where they fit into a water-cooled copper cooler. The resulting solidificationrate (up to 5 K s\u20131) is nearly equivalent to whatcan be achieved by pouring molten metal into a metal mold. Once themetal had solidified, the sample was extracted and the excess metalwas removed.The NaCl particles were packed into cylindrical BN-coated graphitecrucibles with a height of 30 mm and a diameter of 18 mm. A methoddeveloped by the Alicante University group was used, which involvedalternating vibrations and impacts applied with a metal plunger. Theattained volume fraction was 0.58 \u00b1 0.01, which is slightly lowerthan the value encountered in the literature for random packing ofspherical particles . A piece of Al\u201325Ag alloy was placed overthe packed preform, and the assembly was placed in an infiltrationchamber. The infiltration chamber consists of a metal chamber thatcan be pressurized to a maximum pressure of 0.45 MPa and is equippedwith an electric resistance furnace that enables metal melting. Thevacuum was set to 0.1 mbar. At the same time, the temperature wasincreased to 1033 K at 5 K min20 for details).The result is an AlAg foam with pores that replicate the characteristicsof the NaCl particles. Subsequently, the Al-rich phase of Al\u201325Ag,which is considered a soft templating agent, is removed. For thispurpose, the samples are treated with chemicals such as HCl or NaOH. Acidic treatment was performedby immersing the Al\u201325Ag foam sample in a 5% HCl solution at300 K for 8 h. The alkaline medium treatment consisted of immersingthe sample in a 20% NaOH solution at 300 K for 8 h. The diagram in The resulting material is a composite of Al\u201325Agmetal matrixand NaCl particles. Both the NaCl particles and the Al-rich phasesin the matrix can be considered as templates that allow differenttypes of porosities to be obtained after dissolution. NaCl particlesare a rigid templating agent and can be removed by a two-step dissolutionprocess (see refs2.4\u20131.Sintered foams were produced by a simple sintering process consistingof two steps: (i) packing spherical pure silver particles (99.99%)with an average diameter of 0.7\u20131.3 mm into a quartz tube witha diameter of 16 mm and (ii) treatment at 873 K for 300 min in anelectric furnace with an inert nitrogen flow atmosphere at 5 mL min2.52.5.1The foam morphology was studied by scanning electron microscopy(SEM) using a Hitachi S3000N equipment and by field-emission scanningelectron microscopy (FESEM) using a Zeiss Merlin VP equipment. Forthis purpose, SEM and FESEM images were acquired at different magnificationsand analyzed using image analysis software Buehler-Omnimet Enterprise(Illinois). An X-ray Bruker XFlash 3001 EDX probe connected to theformer electron microscope was used to study the chemical composition.A Bruker D8 Advance X-ray diffractometer was used to determine thecrystalline phases in the metal during the various processing steps.2.5.2To obtain information aboutthe pores in a size interval below 50 nm, an indirect study was performedby analyzing the nitrogen adsorption curves of the foam samples. Thesecurves were recorded at 77 K using an Autosorb 6-b Quantachrome Instrumentsequipment (Florida). The isotherms were analyzed using two complementarytheories. The first one is the well-known standard BET theory, whichallows obtaining the specific sample surface area. In addition, thetheoretical framework developed in ref  was used2.5.3Escherichia coli and Staphylococcus aureus were performed according toASTM E2149-13a.22 Bacterial populationgrowth curves of both bacteria were generated in Luria broth (LB)media before testing antibacterial activity. A colony from a singlecolony plantation was seeded in a nutrient broth and incubated at37 \u00b0C for 24 h. An aliquot of this culture was transferred toa 250 mL Erlenmeyer flask containing 50 mL of liquid medium. Incubationwas carried out in an orbital shaker incubator at 310 K and 200 rpm,and the optical density was measured. The reason for choosing 310K as the incubation condition for bacteria is that at this temperatureboth bacteria can divide and multiply optimally. Based on the growthcurves of the bacterial population, the maximum growth time of thebacterial population was determined. These times were set at 5.5 hfor E. coli and 6 h for S. aureus using a known concentration of the bacterialinoculum. The inoculum of E. coli and S. aureus was prepared using a total volume of 9mL of liquid culture medium containing 1.70 \u00d7 105 and1.81 \u00d7 105 CFU mL\u20131, respectively.Samples were placed in the prepared liquid medium and incubated at310 K with a rotation of 200 rpm. The bacterial concentrations weredetermined at different time intervals , andthe values of percentage reduction and logarithmic reduction werecalculated.Antibacterialtests for 33.1\u20133, respectively)than that of the AlAg sample (1.87 g cm\u20133). TheAg-sint sample has a distinct appearance as it consists of sinteredspherical particles with a size of 0.7\u20131.3 mm, leaving poresof similar size due to their low sintering degree. Its density (6.55g cm\u20133) is significantly higher than that of theother materials, as Ag has high density and is also present in greaterquantity . The metal volume fraction in the Ag\u2013HCl andAg\u2013NaOH samples is much lower because some of the metal isdissolved by the chemical treatment with HCl and NaOH .3.22Al phases can be identified.These peaks correspond respectively to the dark and light phases previouslycharacterized by microscopy and EDX. Therefore, the concentrationof Ag in the dark phase measured by EDX  thus correspondsto silver dissolved in a primary aluminum crystal matrix. The equilibriumphase diagram of the system Al\u2013Ag indicates that the \u03b1-Al(Ag)and Ag2Al phases can coexist at room temperature and thatthe maximum silver solubility in the \u03b1-Al(Ag) phase at the eutectictemperature is 23.5 at. %. A wide range of percentages of silver dissolvedin the \u03b1-phase has been reported in the literature for the Al\u2013Agsystem based mainly on solidification conditions: from 2.2 at. %23 for relatively slow solidification conditionsto 23.5 at. % for a supersaturated \u03b1-phase obtained by ultrafastcooling.24 The lower value of dissolvedsilver in the primary \u03b1-Al(Ag) phase (dark) of the Al\u201325Agalloy after infiltration (7.86 at. % compared to 9.34 at. % recordedfor the as-cast condition) can then be explained by milder solidificationconditions. The EDX results allow calculating that the percentageof phases in the Al\u201325Ag alloy after infiltration is about71% for \u03b1-Al(Ag) and 29% for the \u03b2-Ag2Al phase,which is in perfect agreement with the results from image analysisby optical microscopy.26 Analysis of the XRD pattern for the Ag\u2013HCl sample shows notonly that the \u03b1-Al(Ag) phase was completely dissolved but alsothat the Al contained in the intermetallic \u03b2-Ag2Alwas dissolved concurrently. It was shown that dissolution of the primary\u03b1-Al(Ag) crystals in HCl acid creates penetration pathways thatexhibit a catalytic effect for the dissolution of \u03b2-Ag2Al in biphasic Al\u2013Ag alloys. Thereby, the metal matrix afteracid treatment consists of a single Ag phase with the FCC structure.EDX analysis of the Ag\u2013HCl sample confirms a pure Ag compositionwith impurities of C, Al, and O  and alkaline (NaOH)media. 30 and can be affected by the properties of the solutions such as temperature,ionic composition, and concentration. The less noble components aredissolved out of the precursor alloy, while the remaining noble componentdiffuses and agglomerates to form a nanoporous structure. Both thesurface diffusion of Ag atoms in Al\u2013Ag alloys and the reactionbetween Al and chemical etchant determine the nanoporous structure.The silver surface diffusion coefficient can be estimated by the followingequation29k is the Boltzmannconstant (1.3806 \u00d7 10\u201323 J K\u20131), \u03b3is the surface energy of Ag (1.24 J m\u20132), d(t) is the ligament size of the as-dealloyedAl\u201325Ag alloy at the dealloying time t, a is the lattice parameter of Ag (4.086 \u00d7 10\u201310 m), and T is the dealloying temperature. The ligamentsdeveloped in 25Al75 alloy and obtainedligaments of different sizes by treating it with 5 wt % HCl at differenttemperatures and times. They concluded that the surface diffusionof Ag is an activated process according to the Arrhenius law withan activation energy of 75.66 kJ mol\u20131 and a pre-exponentialfactor Do = 4.432 \u00d7 10\u20134 m2 s\u20131. Considering these values inthe Arrhenius equation, we can deduce that the surface diffusion coefficientof Ag for the temperature used in this study (300 K) is Ds = 2.973 \u00d7 10\u201317 m2 s\u20131. Using Porous structures formed duringthe dealloying of alloys containingnoble elements such as Ag are influenced by the surface diffusivityof the noble atoms31 In ref (\u2013) in the HCl solutioncan accelerate the surface diffusion of undissolved noble elementsand thus induce ligament coarsening in the final nanoporous structures.The authors of ref , followed by Ag\u2013HCl (2.10m2 g\u20131). These materials show comparabletotal pore volume but differ significantly in the distribution offiner pores. As previously reported in the literature, the surfacediffusivity of Ag during alkaline solution dealloying of Al-basedalloys is significantly lower than in acidic medium due to the formationof MN-hydroxy  compounds. Therefore, the coarseningability of silver atoms is much lower and larger amounts of smallerpores were obtained compared to the acidic medium.31 The AlAg and Ag-sint samples show low specific surface areas  due tothe absence of microporous or mesoporous structures. The specificsurface area of NaCl particles is 0.740 m2 g\u20131, which corresponds to 1.60 m2 cm\u20133 (thedensity of NaCl was assumed to be 2.16 g cm\u20133).Provided that these particles are packed to a volume fraction of 0.58,the specific surface area of a packed preform made of these NaCl particlesis approximately 0.928 m2 cm\u20133, whichcorresponds to 0.496 m2 g\u20131 for AlAgfoam , respectively. In terms of log(reduction), E. coli and S. aureus, respectively). Itshould be recalled that substances and materials are deemed antimicrobialif they can achieve a 2-log or greater reduction in colony-formingorganisms.38 Nevertheless, there are nointernationally accepted methods for antimicrobial testing exceptISO 22196:2011, ASTM 2149-13a, and ASTM E2180-18.38 In recent years, numerous articles have been publishedon silver-based products and materials with bactericidal activity,including silver nanoparticles42 and silver ion-releasing materials.43 Studies with both materials indicate greater bactericidal activitythan the materials prepared here. By adding 0.2 ppm Ag+ ions to the culture media, log-reduction values of 6 can be achievedfor S. aureus after 3 h and for E. coli after 1 h.38 Theuse of silver nanoparticles or silver derivatives generally leadsto a significant decrease in activity compared to the direct use ofAg+ ions. In fact, metallic silver has no antibacterial effect.+ ionsdiffuse from the substrate and exert a strong inhibitory effect (remarkableat concentrations as low as 0.001 ppm) on a wide range of microorganismssuch as bacteria, molds, and viruses.38 In combination with polyurethane matrices and polyurethane foams,silver nanoparticles showed high bactericidal activities.51 Nevertheless, the treatment of human consumer goods such as wateror food with Ag+ ions or silver nanoparticles poses risksthat need to be addressed. On the one hand, Ag+ ions increasetheir activity with increasing concentration, requiring relativelyhigh concentrations of 0.2 ppm or more. This is the permissible valuein the water consumption limit (0.2 mg L\u20131).52 In addition, some experiments with silver nanoparticleshave shown that they can detach from parent materials and be releasedinto the environment; aggregates of these particles are more hazardousthan asbestos.53 For this reason, materialswith an appropriate balance between their bactericidal capacity andtheir ability to release Ag+ ions or particles are preferred.The activity of silver relies on the fact that Ag2 g\u20131 for the HCl-treated alloy and 4.17 m2 g\u20131 for the NaOH-treated alloy. The graph contains information thatdeserves special attention. The AlAg, Ag\u2013HCl, Ag\u2013NaOH,and Ag-sint samples show bactericidal activity that scales with theirspecific surface area, such that the most active materials are Ag\u2013NaOHand Ag\u2013HCl, both of which exhibit a pore hierarchy. This scalingis not fulfilled in the new samples, which have developed only nanoscaleporosity. Despite their specific surface area, which is comparableto those of Ag\u2013HCl and Ag\u2013NaOH samples, their comparativelylow activity is probably due to the fact that the nanopores presentin these samples do not form a network of channels with suitable dimensionsto allow sufficient diffusion of Ag+ ions into the medium.These results are very significant as they suggest that the combinationof pore sizes in hierarchical foams potentiates the diffusion of Ag+ ions into the medium, thereby increasing their bactericidalactivity. In this context, we refer to a pore hierarchy in these foamsnot only in terms of size but also in terms of functionality. Thesmaller pores are responsible for the large specific surface areathat promotes the dissolution of silver into Ag+ ions,and the larger pores promote the transport of these ions into themedium.4Silver foams with hierarchical porousstructures canbe prepared by infiltrating martyr porous preforms consisting of packedNaCl particles with Al\u201325Ag alloy. The infiltrated materialsare immersed in water to dissolve the NaCl particles, which are consideredas hard templates and lead to the formation of coarse pores. Smallerpores can be created in these foams by dealloying with acidic (HCl)or alkaline (NaOH) solutions.\u20131, is sufficientto obtain suitable fine phase distributions to produce foams withlarge specific surface areas. Selective dissolution by chemical treatmentwith aqueous HCl or NaOH solutions gives different results. Treatmentwith HCl leads to complete elimination of the Al-rich \u03b1-Al(Ag)phase and aluminum in the Ag-rich \u03b2-Ag2Al phase,leaving Ag as the product. However, treatment with NaOH does not leadto the complete dissolution of Al, which coexists with the Ag phaseafter treatment as \u03b2-Ag2Al.The solidificationrate of the Al\u201325Ag alloyupon infiltration, about 5 K s2Al phase remains. However, due to the surface texture, which stronglydepends on the conditions of the treatment medium, the specific surfacearea generated by NaOH dealloying is higher than that obtained byHCl dealloying.Microstructural characterization of the materials showsthat NaOH treatment leads to materials with higher densities and lowertotal porosity volumes than analogous materials obtained by HCl dissolution.This is mainly due to the fact that NaOH treatment cannot completelyeliminate the Al content and the less dense \u03b2-AgS. aureus) and Gram-negative (E. coli) bacteria,they prove to be very active materials against bacterial growth dueto their 24 h bacterial logarithmic reductions of 4.5 and 4.2, respectively.Materials with pore hierarchy show much stronger antibacterial behaviorthan those with pores only in the nanometer range. The pore hierarchyimplies a functional hierarchy in which the small pores provide alarge specific surface area that facilitates the dissolution of silverinto silver ions, and the coarse pores form a channel network thatfacilitates transport into the environment.The materials obtainedby NaOH dealloying exhibit higherantibacterial activity than their HCl analogues due to their largerspecific surface area. For Gram-positive (From the results and the discussionon the preparation and characterizationof the presented materials, the following conclusions can be drawn."}
+{"text": "With success and effective long-term antiretroviral treatment (ART), HIV-infected patients live longer and frequently developed non-communicable diseases (NCDs). Few studies have been conducted in low-income countries, particularly in West Africa.We carried out a cross-sectional study in the referral HIV centre of the Service des Maladies Infectieuses et Tropicales (SMIT) in Abidjan. From April to September 2015, we consecutively included HIV-1 infected patients aged 18 years and older, and on ART for a minimum of 12 months. Data were collected using a structured questionnaire, and entered into the centre\u2019s computerised HIV database. Clinical assessment, laboratory tests, electrocardiogram, transthoracic echocardiography and vascular Doppler ultrasound were performed. The main outcome was the prevalence of patients with severe cardiovascular abnormalities (SCA). Univariate and multivariate logistic regressions were used to identify factors associated with SCA.3 (IQR: 347\u2013529). Basically, cardiovascular abnormalities were mainly non-obstructive carotid plaques (19.1%) followed with left ventricular diastolic dysfunction (16.5%). The overall prevalence of SCA in the study population was 7.6% . The prevalence of SCA 7.6% . In multivariate analysis, age > 50 years and nadir CD4 count > 200 cells/mm3 were significant predictors of SCA.Out of 278 patients , 74.5% were female. Overall, the median duration of ART was 84 months (IQR: 54\u2013126). One hundred and ninety-nine (71.6%) patients were on first-line ART regimen and 229 (82.4%) were virologically suppressed with a median CD4 count of 511 cells/mmThe prevalence of SCA is high in West African HIV-treated patients. Given the high mortality associated with cardiovascular diseases in the general population, refining disease preventive strategies in HIV-positive subjects is essential to continue prolonging their life. These are significant causes of severe morbidity and mortality observed in HIV-positive individuals as compared to the general population.6 Apart from the traditional risk factors including advanced age, male sex, family history of CVD, higher smoking rates and dyslipidemia, individuals may also develop CVD because of non-traditional factors such as inflammation, the direct effects of the virus on the vasculature and the toxicity of specific antiretroviral drugs, causing metabolic syndrome and insulin resistance.8Antiretroviral treatment (ART) has dramatically reduced AIDS-related morbidity and mortality.10 With the changes in lifestyle and the increasing number of people living in urban cities, CVD are becoming an increasing public health issue in low-income countries heavily affected by HIV and AIDS where unfortunately, few data are available of patients on ART. Most previous studies were carried out in Eastern and Southern Africa (Sub-Saharan Africa Survey of Heart Failure and Soweto study cohort), and reported a significantly lower rate of CVD in HIV-infected individuals compared to industrialised countries.12 Eholie et al. estimated the 10-year cardiovascular risk at 3% using Framingham score, during follow-up of HIV-infected patients on ART with no difference amongst sub-Saharan patients living in C\u00f4te d\u2019Ivoire or France.13 However, the patterns of CVD in HIV-treated patients have not yet been documented in C\u00f4te d\u2019Ivoire. Our study aimed to estimate the prevalence of severe cardiovascular abnormalities (SCA) as measured by electrocardiogram, echocardiography, vascular Doppler in patients on ART and to assess associated factors which could result in public health interventions so as to reduce these non-AIDS events in people living with HIV (PLHIV).In developed countries 9% \u2013 20% of HIV-positive patients have a moderate to high risk of myocardial infarction over a 10-year period, identified as the main CVD in these countries with smoking as the main factor.14 At the time of our study, the ART-start CD4 threshold was 350 cells/mm3 in asymptomatic HIV-infected adults.14 Second-line ART for adults consisted of two nucleoside reverse-transcriptase inhibitors (NRTIs) and a ritonavir-boosted protease inhibitor (PI). Atazanvir/ritonavir (ATV/r) and Lopinavir/ritonavir (LPV/r) heat-stable fixed-dose combinations are the preferred boosted PI options for second-line ART. Antiretroviral treatment and biological examinations such as CD4 cell count, haematology and biochemistry were provided free of charge by the national HIV/AIDS control programmes according to their individual care package. Lipid and cardiovascular assessment were not subsidised. We included in this analysis HIV-1 infected patients aged 18 years or older, treated for at least 12 months.A cross-sectional study was conducted in the Service des Maladies Infectieuses et Tropicales (SMIT) of CHU Treichville Hospital in Abidjan from April to September 2015. The SMIT is a referral HIV center for the management of HIV-positive individuals, working closely with several others institutions in Africa and Europe. On the whole, 16 906 HIV-infected patients were in care in the SMIT and 9881 received ART. Moreover, 3575 patients had been on ART regimen for at least 12 months. All ART-naive patients started first-line ART with a WHO-recommended regimen containing at least two nucleoside reverse transcriptase inhibitors (NRTIs) and one non-nucleoside reverse transcriptase inhibitor (NNRTI).Exclusion criteria were: HIV-2 infected or HIV-1/2 dually reactive patients, ART-na\u00efve patients, and patients with any acute infectious episode.15 Smoking was categorised into never smokers, current smokers or former smokers using the Natural Language Processing (NLP) tools.16 The standardised examination consisted of a targeted assessment of medical history and a physical examination including two separate measures of blood pressure and anthropometrics measurements . We performed laboratory analyses , HIV-1 RNA viral load testing .Demographic data, self-reported walking time, cigarette smoking status, alcohol intake and the history of ART was collected at inclusion. The self-reported walking time was categorised into two modalities: greater than 30 min per day or less than 30 min per day. Alcohol consumption was assessed using the Alcohol Use Disorders Identification Test (AUDIT).The cardiac examination was performed by the same cardiologist. A resting 12-lead electrocardiogram (Schiller A-T 110 machine) was used to diagnose conduction disorders and rhythm abnormalities.17Electrocardiography abnormalities were classified using the Minnesota ECG Code classification system.7 to obtain LVM index (LVMI).18 Left ventricular ejection fraction was assessed using Simpson\u2019s biplane rule using conventional apical 4- and 2-chamber views.19A 2-dimensional echocardiography (Vivid S6 machine) was used to assess the left ventricular mass and ejection fraction and evaluate the cavity size and valves of the heart. The m-mode measurements of left ventricular (LV) and left atrial dimensions were obtained in the parasternal long-axis view. Left ventricular mass was calculated according to Devereux et al.\u2019s formula and normalised to body surface area and height20Vascular Doppler (Vivid S6 ultrasound machine) equipped with a 7 MHz linear probe with high-axial resolution was used to measure carotid intima media thickness (cIMT) and determine the plaque presence on the left and right carotid bifurcations and internal and common carotid arteries. Carotid artery plaque was defined as a focal structure that encroaches into the arterial lumen of at least 0.5 mm or 50% of the close IMT value or demonstrates a thickness > 1.5 mm, as measured from the media-adventitia interface to the intima-lumen interface.21All cardiovascular abnormalities observed after CVD examinations in patients were reported. Based on the 10th review of the International Statistical Classification and Related Health Problems (ICD-10), the cardiovascular abnormality meet the definitions of \u2018severe\u2019 if it requires a cardiology consultation, with cardiovascular medication and requires hospitalisation or endangers the life of the patient .2, and left atrial (LA) enlargement as LA diameter > 2.6 mm/m2. Left ventricular hypertrophy (LVH) was defined as an indexed LV mass (LVMI) > 131 g/m2 in men and LVMI > 108 g/m2 in women. We used Appleton\u2019s criteria to diagnose and classify LV diastolic dysfunction.22In the electrocardiogram LV Hypertrophy was defined using Cornell index (RaVL + SV3) > 28 mm in men and 20 mm in women. Repolarisation disorders were found corresponding to ST segment elevation and ST-T wave changes. Left ventricular (LV) dilation was defined as indexed LV diameter in diastole > 34 mm/m23Dilated cardiomyopathy (DCM) is defined as left ventricular (LV) dilation and systolic dysfunction in the absence of coronary artery disease or abnormal loading conditions proportionate to the degree of LV impairment.24The diagnostic of pulmonary hypertension (PH) is based on a mean pulmonary artery pressure of more than 25 mm Hg at rest, or more than 30 mm Hg with exercise, measured by Doppler echocardiography.25Carotid artery stenosis is a narrowing or constriction of any part of the carotid arteries. Any stenosis greater than 50% was considered significant according to the San Francisco consensus conference.Subclinical atherosclerosis was defined as cIMT \u2265 0.9 mm and/or the presence of \u2265 1 carotid plaque.Thromboses were the formation of a blood clot inside a blood vessel, obstructing the flow of blood through the circulatory system. Thrombosis may occur in veins (venous thrombosis) or in arteries .\u03b1 risk of 0.05 and 1-\u03b2 power of 80%). To account for patients who could refuse the survey (10%), a total of 270 HIV-infected patients on ART were required in the present study. Categorical variables were described with numbers and percentages, and continuous variables with median and interquartile range (IQR). The prevalence of SCA was calculated as the number of patients meeting the definition out of the total population with their corresponding 95% confidence interval (95% CI). We used a univariable analysis and then a multivariable logistic regression analysis to identify factors associated with SCA. Statistical analysis was performed using Stata statistical software for professionals .Calculation of the sample size was based on an expected 20% prevalence of CVD in the study population. Therefore, the inclusion of 245 patients would have led to at least 49 cases of CVD , respectively. The majority of patients were asymptomatic. Only 10 (3.6%) patients reported exertional dyspnea. One hundred and seventy-two (61.9%) participants were on two nucleoside reverse transcriptase inhibitors and one non-nucleoside reverse transcriptase inhibitors regimens (2NRTI + 1NNRTI). Seventy-nine (28.4%) patients were on 2NRTI and one protease inhibitor regimens (2NRTI + 1 PI). The median duration of ART was 84 months  at the time of evaluation. Additional characteristics are shown in The median age was 46 years (IQR: 41\u201352) and the majority  of them were women. Most patients  were not sedentary and reported walking more than 30 min per day. Frequent alcohol consumption was reported by 110 (40%) patients and few of them 20 (7.2%) were regular smokers. Eleven (4%) patients had a history of diabetes mellitus. Eighty-six and 36 patients had hypertension and were obese  patients, 32% of whom were elderly (over 50 years) patients. Electrocardiography abnormalities were observed in 70 (25.2 %) patients included. Repolarisation disorders and left ventricular hypertrophy were the most common reported . Echocardiography abnormalities were diagnosed in 95 (34.2%) patients, half of whom were elderly patients. Left ventricular diastolic dysfunction was the most frequent abnormality observed on echocardiography in 46 (16.5 %) participants. Subclinical atherosclerosis was found in 59 (21%) patients with predominance of non-obstructive carotid plaques. Severe cardiovascular abnormalities was prevalent in 21 (7.6% [95% CI: 4.7\u201311.3]) patients distributed as follows: dilated cardiomyopathy, \u02c2 30 min, hypertension (\u2265 140/90 mmHg), ALT levels. > 40 UI, blood glucose levels > 1.1 g/l, and nadir CD4 cell count > 200 cells/mm3. Nevertheless, there was no significant association between gender, smoking status, BMI, PI-boosted ART, total cholesterol/high density lipoprotein ratio and viral load.3 were  less likely to present SCE than those with nadir CD4 cell count \u2264 200 cells/mm3 . Indeed, patients aged > 50 years were more likely to present SCA than those aged \u2264 50 years . Furthermore, patients with nadir CD4 cell count > 200 cells/mmells/mm3 .28 The prevalence of PH in our study is consistent with a recent narrative review indicating a PH prevalence between 5% and 13% in the same population.29 Besides this review, Bigna et al. in a systematic review found a pooled prevalence of 14% (95% CI 6%\u201323%) of PH amongst African HIV-infected adults between 2006 and 2014 from the three African WHO regions.30 This is significantly higher than the estimated 0.5% prevalence of HIV-associated pulmonary hypertension in developed countries.31 With regard to dilated cardiomyopathy (DCM), we found low rates (1%) of these events which are confirmed by a recent study on HIV-infected children and adolescents with high intake of ART in Uganda.32 Indeed, Patel et al. showed a significant impact of ART marked by a 50% reduction of DCM in developing countries. This may explain the low rate observed in our study because more than two-thirds of our patients had full viral suppression.33Our main findings showed an estimated 7.6% prevalence of SCA. They were dominated by pulmonary hypertension (PH), dilated cardiomyopathy and obstructive carotid plaques. These exposed patients to life-threatening events such as myocardial infarction, cerebral stroke and decompensated heart failure, if they are not diagnosed and treated on time.N = 348) showed significantly increased rates of CVD. These results are similar to ours except that CVDs are mainly represented by coronary artery diseases and myocardial infarction.34Advanced age and nadir CD4 cell count were the main factors associated with SCA. In a prospective, multicentre cohort study, Esser et al. reported an estimated 10.1% prevalence of a broad range of CVD in HIV-positives subjects estimated at 10.1%. In this report, aging HIV-positive patients  certainly resulted in a loss of power and precision in the analysis. Finally, the absence of control groups (e.g. HIV-naive subjects) limits the validity of the results. Carefully collected epidemiologic data comparing HIV-positive to HIV-negative patients in low- and middle-income countries are critical to improve understanding. Ongoing longitudinal studies are necessary to determine whether our findings have any significant impact on future heart function and the real contribution that treatment-related factors have on progression, incidence and prevention of cardiac disease.Our study has several limitations. The cross-sectional design restricts any causal inference. The relatively small sample size of the study (The prevalence of SCA is high in West African HIV-treated patients. Therefore, a standardised screening and risk reduction intervention should be routinely undertaken amongst elderly HIV-infected patients receiving ART. In view of the importance of this issue, it appears essential to conduct longitudinal studies to further evaluate the impact of HAART on CVD in the sub-Saharan region and beyond."}
+{"text": "Giant parathyroid adenoma is a type of parathyroid adenoma weighing >\u20093.5\u00a0g and having a size of more than 2\u00a0cm.This report describes giant primary parathyroid adenoma with reference to the literature. We report the case of a 48-year-old Persian man referred to the clinic with knee and lower back pain. He had a history of mitral valve replacement and several episodes of bilateral nephrolithiasis. After a thorough assessment, a neck mass with a possible thyroid origin was detected, but further assessment showed it was of parathyroid origin. The resected mass was 9\u2009\u00d7\u20096\u00d7\u20094\u00a0cm and weighed 122\u00a0g, and histopathology showed a giant parathyroid adenoma.Giant parathyroid adenomas that weigh more than 110\u00a0g and are larger than 8\u00a0cm can lead to significant hypercalcemia. Despite giant parathyroid adenomas and high parathyroid hormone levels, a calcium crisis may not always occur in these patients, and the masses may be initially misdiagnosed as a thyroid mass. Primary hyperparathyroidism (PHPT) is the most common disease of parathyroid glands and the third most common endocrine disease , 2. The Clinical manifestations of PHPT include nephrolithiasis, osteoporosis\u2013osteopenia, pancreatitis, depression, cognitive disorders, and others. The severity of symptoms correlates with the adenoma\u2019s weight and PTH level , 3. PatiPreviously, the largest reported GPA was 8\u2009\u00d7\u20095\u2009\u00d7\u20093.5\u00a0cm with a weight of 110\u00a0g  and was A 48-year-old Persian man from Urmia, Iran, presented to Imam Hospital with knee pain, lower back pain, fatigue, and dizziness for the last 2\u00a0months. The patient had a history of mitral valve replacement 30\u00a0years earlier, several bilateral nephrolithotripsies, and diabetes mellitus under control with oral medication. His vital signs were typical. There was a palpable large and soft nodule in the left lobe of his thyroid, which moved with swallowing. A scar of previous cardiac surgery was visible on the chest. The rest of the examination was normal.99mTc-MIBI scintigraphy. His complete blood count (CBC) was normal, and the results of the biochemical tests are presented in Table Color Doppler ultrasonography of the thyroid and parathyroid indicated a single isoechoic nodule in the right lobe (12\u2009\u00d7\u20099.5\u00a0mm) and two cystic nodules in the left lobe (40\u2009\u00d7\u200923\u00a0mm and 30\u2009\u00d7\u200916\u00a0mm) of his thyroid. A cystic mass in the region of the left lobe of the thyroid was visible on computed tomography (CT), in contrast to the patient\u2019s biochemistry, indicative of parathyroid carcinoma  two-finger width above the suprasternal notch, subplatysmal flaps were created as a routine thyroidectomy procedure. The raphe between strap muscles was opened, and the muscles were incised on the left near the upper pole of the thyroid.On initial evaluation after the thyroid was exposed, it appeared as a thyroid mass displacing the carotid sheath laterally and extending into the mediastinum as a retrosternal goiter. However, further evaluation showed a central posterior neck mass with extension to mediastinum that displaced carotid sheath laterally and the left lobe of the thyroid superior and medially. The inferior thyroidal artery crossed the mass toward the carotid sheath. These findings favored a parathyroid origin, most likely from the superior parathyroid glands. The inferior thyroid artery was ligated as laterally as possible, and then the lateral side of the tumor was dissected out with sharp and blunt dissection, and the tumor was pulled out of the mediastinum to the neck Fig. . The recOwing to the PTH value, size of the tumor, and adhesion to adjacent tissues, the patient was diagnosed with parathyroid carcinoma, and the tumor, right lobe of the thyroid, and adjacent lymph nodes were resected. After inserting a Hemovac drain, the neck wound was closed. The resected mass had a size of 9\u2009\u00d7\u20096\u2009\u00d7\u20094\u00a0cm and weighed 122\u00a0g. Since the mass was descended to the retro-esophageal space and was located posterior to the recurrent laryngeal nerve, we believe it originated from the superior parathyroid gland.3 and calcitriol pearls gradually tapered until discontinued after 2\u00a0weeks.The postoperative period was eventless. In the first 24\u00a0hours, an intravenous infusion (drip) of calcium gluconate was given to prevent bone hunger syndrome. Liquid diet was started in the second 24\u00a0hours, and the patient was discharged on the third postoperative day with oral CaCOHistopathological assessment of the resected mass revealed a parathyroid adenoma and multinodular goiter of the resected thyroid lobe Fig. . No lymp99mTc-MIBI scintigraphy facilitate the diagnosis of giant parathyroid adenoma [Primary hyperparathyroidism (PHP) is the third most common endocrine disorder and is usually due to parathyroid adenoma. Parathyroid carcinoma is the least common cause of PHP but should be considered in the differential diagnosis of PHP when the serum calcium (mg/dL) level is above 14 or PHP is associated with a palpable neck mass \u20139. The sThe patient had been suffering from recurrent bilateral nephrolithiasis. Considering his back and bone pain, the adenoma might have been chronically functional, so the patient had not experienced calcium crisis symptoms. Diagnosis of a parathyroid mass was made on the basis of lab findings, including high calcium and PTH levels. While the CBC was as expected, the level of PTH was almost 50 times its expected value. Previous studies have shown that parathyroid carcinoma occurs with equal frequency in both sexes and usually increases serum PTH to five- to tenfold of the normal upper limit , 9, 11. However, the calcium level was not as high as the PTH level, which could be due to chronic elevation of PTH or genetic resistance to PTH .At that point, we had no evidence to prove either option. After tumor resection and putting the patient on calcium and calcitriol for several months, then tapering them, the patient no longer had symptoms of hypo- or hyperparathyroidism, which ruled out any genetic disorder of Ca metabolism.In summary, hyperparathyroidism should be considered in the differential diagnosis of any neck and cervical mass, even though it is a sporadic tumor.This case report describes the largest giant parathyroid adenoma that has ever been diagnosed, although the size of the mass, location, and imaging might be misleading. It should be noted that very high levels of parathormone may not always lead to calcium crisis signs and symptoms."}
+{"text": "MobiDB aggregates disorder annotations derived from the literature and from experimental evidence along with predictions for all known protein sequences. MobiDB generates new knowledge and captures the functional significance of disordered regions by processing and combining complementary sources of information. Since its first release 10 years ago, the MobiDB database has evolved in order to improve the quality and coverage of\u00a0protein\u00a0disorder annotations and its accessibility. MobiDB has now reached its maturity in terms of data standardization and visualization. Here, we present a new release which focuses on the optimization of user experience and database content. The major advances compared to the previous version are the integration of AlphaFoldDB predictions and the re-implementation of the homology transfer pipeline, which expands manually curated annotations by two orders of magnitude. Finally, the entry page has been restyled in order to provide an overview of the available annotations along with two separate views that highlight structural disorder evidence and functions associated with different binding modes.The MobiDB database (URL:  Intrinsically disordered proteins (IDPs) and regions (IDRs) are characterized by the lack of a fixed three dimensional structure, they are generally more extended and exhibit an extreme dynamic behavior. Many functions of IDRs, such as entropic springs, flexible linkers or spacers are directly associated with their structural attributes ,2. Main Biological databases play a central role in accelerating biological discovery, making experimental information accessible in a standardized and structured way . HoweverMobiDB celebrates 10 years of active development since it was first published in 2012 . The MobIn this article, we present the latest innovations introduced in MobiDB. First, we introduced AlphaFold predictions  which haMobiDB has been developed to serve both experimental scientists, interested in comprehensive information of single protein systems, as well as bioinformaticians, who seek large homogeneous collections of proteins sharing the same features to build statistical classifiers. In order to make its content more accessible to both scientific communities, MobiDB adopted the concept of \u2018annotation pyramid\u2019. The height of the pyramid represents the annotation quality while the horizontal axis is the coverage of known proteomes. Also, the MobiDB pyramid is staired to indicate different levels of evidence. In Table Curated annotations are pulled from the corresponding databases processing the data and checking their consistency. Curated entries are also used as input to infer homology and project their annotation to the rest of UniProtKB sequences . DerivedIn the current version of MobiDB, manually curated annotations are transferred to other proteins based on homology inference\u00a0, gaps must not exceed the 20% of the length of the alignment and the subject (homologous region) must be 80% identical to the query region. In the case of multiple regions being identified on the same target protein, in order to remove overlaps, a greedy algorithm which prioritizes longer regions, is applied. Despite an expansion of two orders of magnitude, the homology transfer for low complexity regions is limited as they are masked by BLAST by default.https://github.com/BioComputingUP/AlphaFold-disorder. In MobiDB, AlphaFold structures are downloaded from AlphaFoldDB , referring to their extended conformation. Similarly to disorder evidence, in MobiDB there are different levels of evidence (annotation confidence) and different features, the binding modes, that can be associated with a LIP. In Table As for the binding modes MobiDB is committed to enrich the functional knowledge and the biological role of IDPs. The formation of dynamic liquid droplets and the phenomenon of protein phase separation are thought to be driven by disordered regions forming transient interactions . The newThe MobiDB database schema has now reached its maturity and has proved to be effective and fast in serving data, allowing complex queries. The last MobiDB release has focused on the simplification and acceleration of content updates. Now the entries are splitted into two different collections depending on their annotation level (see MobiDB annotation pyramid). The subset of entries with \u2018curated\u2019, \u2018derived\u2019 or \u2018homologous\u2019 annotations, and those that are extracted from AlphaFoldDB entries are stored in a so-called \u2018gold\u2019 collection. The rest of the proteins are annotated only with MobiDB-lite predictions. The \u2018gold\u2019 collection is relatively small (1.5 millions proteins) and it is regenerated from scratch at each release update. The data is processed automatically through a workflow that takes a couple of weeks of calculation. All annotations integrated from member databases undergo stringent sanity checks that verify sequences and identifiers and \u2018out-of-index\u2019 regions. MobiDB uses UniProtKB as the reference for protein identifiers and sequences. When the member database sequence does not match the sequence provided by UniProtKB for that identifier, the annotation is discarded, meaning the member database has not been updated.The separation of entries into different collections is completely transparent to the user since database queries are always issued on both collections and results are combined at the server level.Given the amount of different annotation sources and processing procedures employed, the new MobiDB release provides a controlled vocabulary (CV) that is used as a reference to describe precisely the different annotation features. Most of the current terms in the CV were already used in the previous release, now they are fully described and clearly exposed in the website. CV terms are split into three main categories, or name spaces: (i) evidence, (ii) feature and (iii) source of information. The evidence namespace represents the different levels of the MobiDB pyramid. Feature terms represent the type (or flavor) of the annotation, while the source can be a database, a piece of software or in general provide information about the method used to generate that annotation. All annotations in MobiDB are fully identified by triplets of CV terms, one for each category. Currently 4, 39 and 37 terms populate the three groups, respectively.The MobiDB website has been renewed, in particular the entry page, to help the user appreciate and explore more easily the amount of different annotations that are provided. Other notable changes regard the integration of the AlphaFold predicted structures and a page that shows the complete controlled vocabulary used to identify and describe all types of annotations available in MobiDB. Moreover, we have improved the API documentation by implementing a Swagger UI where the user can build and try a custom query directly on the MobiDB website. The page includes documentation of all output fields and their values.The entry page has been extensively refactored preserving the functionality and the style already available in the previous release. Beside the general protein information pulled from UniProtKB , MobiDB now provides its annotations grouped into three different tabs: i) overview, ii) disorder and iii) binding. This separation allows the user to focus on specific aspects and delve into the hierarchical structure of the provided information more easily.As in the previous version, the central component of the entry page is the feature viewer , which sWhen the entry page is open for the first time, the structure viewer loads the AlphaFold prediction which is available for about all MobiDB entries. For those annotations derived from experimental PDB structures, e.g. LIPs in protein complexes or mobile residues in NMR ensembles, the structure viewer can load the corresponding PDB entry through a button placed aside the corresponding track in the feature viewer.Other improvements regard an inset card that pops-up at the bottom of the page when a region is selected on the feature viewer. The new card provides detailed information about the region position and the origin of the carried information.The great accuracy recently reached by AlphaFold  in the pThe new version of MobiDB improves the focus on the increase of functional and structural knowledge about conformational ensembles, also exploiting the great added value provided by the visualization of AlphaFold structures. A new homology transfer pipeline increased the number of entries with high quality annotations by two orders of magnitude. The new design of the entry page provides a better visualization of disorder functions. The formalization of a controlled vocabulary clarifies the source, type and classification of all annotated features and improves their accessibility.https://bioschemas.org)   which alhttps://mobidb.org.All the data and link\u00a0to used software are available at"}
+{"text": "The consequent distinct bioreactivity and toxicity towards macrophages are largely attributed to the changes of species of surface O-functional groups. Importantly, we unveiled the specific interactions between aged PMs and macrophages due to the variant contents of the surface carboxyl group, resulting in the divergent inflammatory activations and immune balance in the lung. Collectively, this study unearths the significance of ageing in altering particle cytotoxicity, and also provides additional understandings for consecutive investigations on the adverse effects of air pollution on the respiratory system.There are still significant concerns about the detrimental effects and health risks of particulate matters (PMs) on the respiratory system. Notably, a largely overlooked knowledge gap is whether the environmental ageing process would change the physicochemical properties of PMs as well as the toxic influences of PMs on macrophages. Here, we applied ambient treatment of model PMs to mimic the real O The long-term process of the aerosol heterogeneous reaction of PMs in the atmospheric environment is often referred to as ageing [2SO4 changes the oxidative potential of PMs [Recently, air pollution has raised mounting public concern about health risks, both in academic research and for strategic agendas worldwide ,2,3. Spes ageing . Normalls ageing ,10,11,12l of PMs . Althoug3 aging involved a change in the oxygen-containing functional groups of CB particles, and the content of epoxy groups would induce higher ROS generation and then higher oxidative stress to erythroid cells [Once inhaled into the respiratory tract, UFPs  could enter and further be cleared by reticuloendothelial cells in the lungs ,15,16. Aid cells . Wu et aid cells . Notablyid cells . The M1 id cells ,33,34. Tid cells ,37,38,393, and to interrogate how the ageing process changes the interaction and toxicity profiles of UFPs towards macrophages. In addition, the influences on the immune states of lungs by UFPs with different surface properties were also scrutinized. Together, our combined results unveiled that the alteration in the toxicity effects of UFPs largely resides in the changes in surface physicochemical properties owing to the ageing process.Therefore, we aimed to investigate the physicochemical alterations of the model UFPs during the ambient ageing process with O3 corresponded to 83 ppb, according to the China National Environmental Monitoring Centre (http://www.cnemc.cn/sssj/ accessed on 18 July 2020) in June 2018, in Beijing, China, and the equivalent O3 exposure corresponded to the true ageing periods of 1 and 7 days according to Equation (1):Degussa Inc. Corp.  provided commercial CB particles (Printex U). The ozonation of CB particles was accomplished using a well-established process, as reported in previous work ,13. BrieThe CB particles were named as CB-0 (pristine CB), CB-1 (ageing for 1 day), and CB-7 (ageing for 7 days), respectively, depending on how long they were exposed to ozone.Dh) and zeta potential of the CB suspensions (at 10 \u00b5g/mL) in deionized (DI) water at 25 \u00b0C.A scanning electron microscope  was used to examine the physical dimensions and morphologies of the CB particles. X-ray photoelectron spectroscopy  was used to determine element components and energy bands, and the results were then evaluated using the program XPSPEAK41. A ZetaSizer Nano ZS  was used to assess the hydrodynamic diameter (2 and in Dulbecco\u2019s modified Eagle medium (DMEM) supplemented with 100 U/mL of penicillin\u2013streptomycin  and 10% fetal bovine serum .The murine macrophage cell lines J774A.1 and RAW264.7 were obtained from the Shanghai Cell Bank of Type Culture Collection . These cells were cultured at 37 \u00b0C in a humidified incubator with 5% CO2. After incubation for 3 days, the medium was changed every day until day 7. The induced BMDMs were then used for further experiments.Primary bone marrow-derived macrophages (BMDMs) were obtained from mouse marrows, as described previously . Briefly4 J774A.1 cells/well, Corning, NY, USA) plate overnight, J774A.1 cells were treated with CB particles at various concentrations  for 24 h. Cell viability was then assayed through the cell counting kit 8 (CCK-8) method following the standard manufacturer\u2019s instructions . Briefly, 10 \u00b5L of CCK-8 solution was added to the cell culture medium in each well. The absorbance was then monitored at a wavelength of 490 nm on a plate reader Varioskan Flash  after incubation for 4 h in the dark at 37 \u00b0C.For cell viability assessment, after seeding in a 96-well  overnight, J774A.1 cells were treated with CB particles at 10 \u00b5g/mL for 12 h. Then, the cell membrane was stained with tetramethylrhodamine TRITC Phalloidin  and the nuclei were stained with 4\u2032,6-diamidino-2-phenylindole , following the instructions from the manufacturer. The fluorescence of the cell membrane (red) and nucleus (blue) were subjected to examination using a TCS SP5 laser scanning confocal microscope . ImageJ software  was applied to measure the length and width of the cells.After seeding in glass-bottom dishes (3.0 \u00d7 105 J774A.1 cells/well) overnight, J774A.1 cells were exposed to CB particles at 10 \u03bcg/mL for 12 h and then harvested with phosphate-buffered saline (PBS). Cells were then washed 3 times with PBS and carefully collected after treatment. The collected cells were fixed in 2.5% glutaraldehyde, and thereafter sliced into sections according to the standard protocols. The ultrastructure of the cell was imaged using a Tecnai Spirit TEM at 120 kV .After seeding in a 6-well plate (3.0 \u00d7 104 J774A.1 cells/well) plate overnight, J774A.1 cells were pre-incubated with 10 mM of dichloro-fluorescein diacetate  probes for 30 min. Thereafter, the cells were washed with PBS at least 3 times prior to exposure to CB particles at 10 and 50 \u03bcg/mL for up to 6 h. The DCF-DA fluorescence was then read on a microplate reader  with an excitation wavelength of 488 nm. Untreated cells were used as the negative control and 0.2% H2O2 was otherwise used as the positive control.After seeding in a 96-well (1.0 \u00d7 105 J774A.1 cells/well) overnight, J774A.1 cells were exposed to CB particles at 10 \u03bcg/mL for 24 h. Thereafter, cells were collected and lysed in PBS with 5% Triton X-100 lysis buffer  for 30 min. To evaluate the number of CB particles that were endocytosed by macrophages, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed with an equal amount of each lysate . After electrophoresis, the gel was collected and stained according to the manufacturer\u2019s instructions . Briefly, the gel was fixed with fixing solution  for 1\u2009h, and then stained with staining solution  for 45\u2009min. After that, the gel was de-stained with the de-staining solution  until there was no visible background staining. The protein bands were then recorded, and the ImageJ software  was applied to differentiate the CB signals in images. CB signals\u2019 intensity was analyzed relative to the CB-0 group.After seeding in a 6-well plate (3.0 \u00d7 105 J774A.1 cells/well) plate overnight, J774A.1 cells were exposed to CB particles at 10 \u03bcg/mL for 24 h. Cell culture medium was then collected from wells and measured using a kit made by the manufacturer .After seeding in a 6-well . The RNA concentration in each sample was measured using a Nanodrop ND2000 instrument . Then, using M-MLV reverse transcriptase , 2 \u00b5g of RNAs were reversely transcribed into cDNAs. Using a CFX96TM Real-Time Technique, the expression levels of target genes were measured using a standard SYBR Green qRT-PCR system . The protein cyclophilin (CyC) was utilized to normalize the relative expression of the target genes in this research. The information on the primers is listed in 5 J774A.1 cells/well) overnight and then pretreated for 6 h with the inhibitor Resatorvid  to suppress TLR4 signaling, followed by CB particles\u2019 exposure at 10 \u03bcg/mL for another 24 h. After that, the cells were washed 3 times with PBS before collection for qRT-PCR analysis.For pretreatment of the inhibitor against Toll-like receptor 4 (TLR4) signaling, J774A.1 cells were seeded in a 6-well plate (3.0 \u00d7 105 J774A.1 cells/well) overnight, J774A.1 cells were exposed to CB particles at 10 \u03bcg/mL for 24 h and then harvested with PBS. Post-treatment, the harvested cells were stained with PE-cy7-conjugated anti-mouse CD86 antibody  for 30 min to recognize the polarization states of the macrophages. The Attune NxT Flow Cytometer  was used to perform the flow cytometry analysis. J774A.1 cells without antibody staining were used as the template control.After seeding in a 6-well plate  and p < 0.001 (#).All data were presented as mean \u00b1 standard deviation (SD). The statistical analysis of the experimental data was implemented with an independent 3. The O3 concentrations applied in these experiments were determined according to our previous study, in parallel to the real O3 levels in June 2018, in Beijing, China [Dh was measured to be 1707.2 nm for CB-0 in DI water solution, and sharply decreased to 515.0 nm for CB-1 and 205.2 nm for CB-7, respectively. Consistently, the changes in Dh were confirmed by the aggregation states of CB particles in DI water, as the inserted panels showed  were elevated to 10.87% in CB-7, compared to the original CB-0 at 8.21% and 8.01% in CB-1. Of note, the carboxyl groups (-COOH) increased from 3.91% for CB-0 to 12.19% for CB-1 and 17.40% for CB-7, respectively. These data thus unraveled the significant alterations in the physicochemical properties of the CB particles, i.e., the oxidative reaction between O3 and the carbon backbones [Afterwards, the surface functional groups, as characterized by XPS, of the parental and aged CB particles showed considerable changes. Overall, the total surface O-content increased after Oreatment , in analackbones . Togethep < 0.001). Mild and significant cytotoxicity was demonstrated in CB-1-treated J774A.1 cells, with a 19% reduction in cell viability at 30 \u00b5g/mL compared to the untreated control (p < 0.001). Otherwise, negligeable cytotoxicity was found in CB-7-treated J774A.1 cells even at 30 \u00b5g/mL, respectively  was used to avoid dramatic cell death. As shown in membrane c. In themembrane . The tremembrane . It shoumembrane d. Howevep < 0.05). As documented, upon stimuli, macrophages would tend to be in an activation state, and the increase in length/width ratio is a typical marker [\u03b2) and interleukin-6 (IL-6), was investigated. As shown in \u03b2 during 24 h exposure . In addition, similar changes in IL-6 were demonstrated, supporting the findings on the robust inflammation reactions upon CB-7 treatment. For comparison, the expressions of IL-1\u03b2 and IL-6 induced by CB particles in other model macrophages, i.e., RAW 264.7 cells and BMDMs (namely the murine primary culture cell), were also investigated, and the results were in parallel to those of J774A.1 cells, as the CB-7 induced the most severe inflammatory reactions , suggesting that TLR4 signaling was closely implicated in the CB-7-induced inflammatory response. Collectively, CB-7 particles were able to induce the M1 polarization of macrophages, and may drive the immune micro-environment of the lung into an inflammatory state.Supported by our previous studies, carbon-based nanomaterials can trigger pro-inflammatory responses of macrophages through the activation of TLR4 on the cell membrane . Accordi0.1), are thought to be the most concerning in terms of their detrimental health effects on the lung, since they can escape from the mucous barrier and enter the lungs [3 oxidation process and figured out the surface O-functionalities as one physicochemical determinant for changing the toxicity profiles. In line with previous studies, the ageing of CB particles would greatly change the stability and species of surface O-containing groups [3-induced ageing. Based on our results, a trend of gradient cytotoxicity in macrophages, namely CB-0 > CB-1 > CB-7, could be concluded. Although the CB-7 particles exhibited the least cytotoxicity, CB-7 could induce the most serious inflammatory reactions in macrophages. Intriguingly, our results indicated no significant variation in ROS generation upon these different types of CBs under a sublethal concentration. Nonetheless, CB particles still induced other detrimental outcomes in macrophages, e.g., inflammation, especially CB-7. The remarkable biological effects of CB-7 might be due to the significant increase in carboxyl groups on the surface after ageing, increasing colloidal stability [Of various air pollutants, ambient fine particles, especially UFPs , light, and humidity, giving rise to remarkable alternations in surface O-functionalities. Under this premise, despite numerous toxicity studies on fine air particles, rather limited knowledge has been obtained to understand ageing-determined toxicity profiles. Airborne particles after inhalation can enter the lung deeply and then be endocytosed by macrophages. Our data unraveled that the species of surface O-containing groups essentially dictated the bioreactivity and cyto-compatibility of aged CB particles towards macrophages, and even the inflammatory reactions in macrophages. These findings, therefore, offer a new point of view in proving the risks of particulate pollution and the effects associated with the ageing process.We reported the impact of the ageing process on the physicochemical changes of UFPs, and also the cytotoxicity profiles and the inflammatory reactions in macrophages. Airborne UFPs are subjected to various atmospheric conditions, such as air pollutants (e.g., SO"}
+{"text": "Salvia officinalis L. (common sage). The populations with the highest genetic diversity were located in the central parts of the Balkan distribution range. A large group of closely related haplotypes was distributed throughout the Balkans and the central Apennines, while the private lineage occupied the southern Apennines. In addition, two highly differentiated lineages were scattered only over the Balkans. The results suggest that a single refugium of the studied species from the last glacial period was located in the central part of the range in the Balkans. Numerous microrefugia, probably spanning several glaciation cycles, were scattered across the Balkans, while colonisation of the Apennines from the Balkans occurred at least on two occasions.Studying the population-genetic and phylogeographic structures of a representative species of a particular geographical region can not only provide us with information regarding its evolutionary history, but also improve our understanding of the evolutionary processes underlying the patterns of species diversity in that area. By analysing eight highly polymorphic microsatellite loci and two chloroplast DNA regions, we have investigated the influence of Pleistocene climate fluctuations on the evolutionary history of  During climatic oscillations, the distribution ranges of many species exhibited substantial fluctuations due to changes in various climatic factors7 as well as sea level changes linked to global temperature10. Sea level oscillations were especially important in areas surrounding shallow seas, where they were responsible for the opening and later closing of land bridges between geographically isolated landmasses11.During the Quaternary period, the global climate was characterized by pronounced global temperature fluctuations. Periodical glaciations across the Northern Hemisphere became regular occurrences7, the refugium is \u201can area where distinct genetic lineages have persisted through a series of Tertiary or Quaternary climate fluctuations owing to special, buffering environmental characteristics\u201d. Comparative phylogeographic analyses of numerous plant and animal species recognized three northern Mediterranean peninsulas as major European refugia14. Although Hewitt14 suggested that each of the three peninsulas should be treated as a single refugium, the \u201crefugia-within-refugia\u201d model15 seemed more appropriate on numerous occasions22. The model suggests that after climate conditions became favourable, geographically scattered lineages that survived in isolated locations would reclaim the previous distribution range of the species, while the genetic fingerprints of glacial isolation would still be visible. Refugial populations are expected to be characterized by a specific molecular signature, such as the presence of unique yet highly divergent haplotypes that emerge as a consequence of long-term isolation through multiple glacial cycles23. The level of geographic structuring of the lineages can provide information regarding the expansion following the occurrence of favourable environmental conditions. If lineages are spatially structured, recolonization from localized refugia likely occurred, enabling the geographical expansion of the divergent haplotype. However, highly localized haplotypes of divergent lineages scattered throughout the distribution area of studied taxa indicate their pulsating range distribution, with repeating cycles of spatially localized expansions and contractions that did not increase their distribution area22.According to Medail and DiademaFagus grandifolia in North America based on the known dispersal mechanisms of the species , the concept of microrefugia was borne25. The term \u201cmicrorefugia\u201d was first introduced by Rull et al.26, and it is now widely accepted and used in the context of postglacial colonization. It suggests that recolonization did not occur through a single large-scale expansion event from an area  where species survived the period of unfavourable climatic conditions. Instead, recolonization occurred simultaneously from multiple areas \u201cwith local favourable environmental features, in which small populations can survive\u2026protected from the unfavourable regional environmental conditions\u201d27.From the inability to explain the postglacial expansion of 28. Consequently, during the last glacial maximum (LGM), most of today\u2019s northern and central Adriatics were land connecting two peninsulas, while the sea remained only in the southern parts30. Such geographic rearrangements have strongly influenced the ranges and phylogeographic structures of numerous amphi-adriatic species or groups of closely related species, of which there is ample empirical evidence35. The majority of these studies suggested that the expansion occurred from the Balkans to the Apennines, and only on rare occasions did the results suggest expansion in the opposite direction36.The Adriatic Sea is located in the northern Mediterranean between the Apennines and the Balkans. Its northern and central sections are rather shallow, while the southern section is characterized by a depression more than 1200\u00a0m deepSalvia officinalis L., 1753) is an endemic species of the northern Mediterranean and one of the most common species within its distribution area. It grows on shallow limestone soils37, which make up most of the western coastal region of the Balkans39. However, it covers substantially smaller portions of the Apennines40, which reflects the species distribution. In the Balkans, it grows abundantly from the Gulf of Trieste in the north to central Greece in the south41, while fewer and localized populations are present in the central and southern parts of the Apennine Peninsula45. Until now, comprehensive population genetic and phylogeographic analyses that would cover the entire distribution area of the species have not been performed. Re\u0161etnik et al.46 performed an SSR-based population genetic analysis of a limited number of populations from the Balkan Peninsula, with emphasis on the detection of naturalized populations throughout the area. Recently, Jug-Dujakovi\u0107 et al.47 analysed populations from Croatia, thus covering only a fraction of the species distribution area.Investigation of population-genetic and phylogeographic structures of a representative species in a specific habitat or geographic area can not only provide us with information regarding the species\u2019 evolutionary history, but it also improve our understanding of evolutionary processes that underlie biodiversity patterns in the studied area. Common sage , suggesting the presence of two well-differentiated genetic groups, each located on one side of the Adriatic Sea Fig.\u00a0, Fig.\u00a02bFor the DIYABC analysis, the first scenario had the highest posterior probability  even after repeated attempts. Sixteen haplotypes were identified by the analysis Fig.\u00a0, and theHd) and nucleotide diversity (\u03c0) were calculated for the genetic clusters recognized by STRUCTURE.\u00a0The highest values of both parameters were found in the CBalk group of populations , followed by the SBalk group . In the remaining groups , moderate values of Hd were found , while the lowest \u03c0 values were detected in the NBalk (0.002001) and Apennines groups (0.00048 in SAp and 0.00058 in the CAp).Haplotype diversity levels quantified as unbiased haplotype diversity  was significantly higher (P\u2009<\u20090.01) than the GST value (0.733), thus reflecting the fact that distinct haplotypes mixed in the same population were, on average, more closely related than distinct haplotypes from different populations.The presence of phylogenetic structure was tested by comparing two estimates of genetic variation: D and Fu's FS were nonsignificant (\u2212\u20090.142 (P\u2009=\u20090.527) and 3.119 (P\u2009=\u20090.831), respectively).No signs of severe recent population expansion or bottleneck were detected, as estimates of both Tajima's Based on the results of the multicollinearity test, 13 bioclimatic variables were excluded from further analysis, while the remaining six variables  (\u00d7\u2009100), BIO4\u2014temperature seasonality (standard deviation\u2009\u00d7\u2009100), BIO8\u2014mean temperature of wettest quarter, BIO9\u2014mean temperature of driest quarter, BIO14\u2014precipitation of driest month) were used. Their relative contributions to the niche model were 7, 0.1, 19.4, 20.1, 23.4 and 30.1%, respectively. The model characterized by the smallest AICc value  was selected. The model obtained for the present time was consistent with the distribution area of the species in the Balkans, while in the Apennines, this was not the case, as the suitable area suggested by the model was substantially larger  occupying neighbouring peninsulas. At a higher resolution level (K\u2009=\u20095), spatial structuring remains clear, with clustering of the populations according to their geographical positions. In contrast to other studies of species occurring throughout the western Balkans53 Table .54, based on the obtained number of ca. 580 generations that have passed since then, we can assume that the common sage generation time ranges from 17 to 26\u00a0years. Based on this value, we can further estimate the earlier divergence time (t2) ca. 180,000\u2013119,000 YBP, which corresponds with the time of the penultimate glacial maximum that lasted from 194,000 to 135,000 YBP55 and the following last interglacial, which peaked approximately 120,000 YBP56. Such conclusions are in agreement with both SSR and cpDNA results, which suggested that the split of the southern Apennines populations occurred sometime before the central Apennines cluster split from the Balkan populations. Although the results undoubtedly support the colonization of the Apennines on two separate occasions, we should be cautious when assessing the time scale of these events since only medians of the generation numbers were taken into account here. Confidence intervals of the estimated values were wide, making the conclusions based on these results speculative.In the Apennines, some populations from the central parts were characterized by admixed structures, with various proportions of the Balkan clusters\u2019 genetic material included. Thus, the recent gene flow with the Balkan populations seems obvious. In contrast, populations from the southern Apennines retained genetic purity, suggesting their prolonged isolation Fig.\u00a0b. These 29 was likely characterized by suitable climatic conditions for species survival. It seems that during the LGM, the proximity of seawater masses and the resulting temperature-buffering effect could be of great importance for sustaining the species along the paleo-sea coastline57. For the northern Adriatic and southern Balkans regions, modelling suggests that, in contrast to the central parts of the Balkans, during the LGM, they were unsuitable for species survival, at least on large geographic scales. Consequently, it seems likely that the common sage experienced a severe contraction in its distribution range during the last glaciation, followed by range expansion when the conditions became more favourable. At this point, it is worth noting that ENM for the present time gave very accurate results for the Balkans but not the Apennines, likely because the patchiness of the limestone soil throughout the Apennines40 is a limiting factor for the expansion of this species. Such a result serves as a reminder that the actual distribution of the species is influenced by numerous biotic and abiotic elements, and bioclimatic variables represents just a fraction of them.The obtained ENM results confirmed that the land bridge that stretched across the middle parts of today's Adriatic Sea59 or were focused solely on the phylogeography of the studied species60. Nonetheless, there are a few available studies comparable to ours in which taxa with similar distribution ranges and ecological preferences were analysed. Ku\u010dera et al.51 addressed the population-genetic structure of Cardamine maritima (Brassicaceae), and the results suggested that the more ancient populations were detected in specific habitats (open mountain slopes and gorges oriented towards the sea) and not any specific geographic region. Grdi\u0161a et al.61 presented results from the population-genetic analysis of Tanacetum cinerariifolium (Asteraceae), which also grows throughout western parts of the Balkan Peninsula. The results were in contrast to ours, as the northern Adriatic region was suggested as the likely LGM refugium for the species. Finally, the results from Surina et al.20 were more similar to ours, as higher gene diversity and rarity index values were detected in southern populations of Edraianthus tenuifolius (Campanulaceae) in the same region where highly diversified populations of common sage were recognized.Numerous studies have already investigated various Balkan and amphi-Adriatic taxa in search of genetic fingerprints of past demographic oscillations. However, the majority of these studies were either focused on species occupying mountain or inland areas62, these lineages are not geographically structured, which blurs their role during recolonization. Nonetheless, their locations, which likely sheltered them throughout multiple glaciation cycles, represent the species microrefugia. Meanwhile, the absence of these haplotypes from the Apennines implies that the species was much earlier present in the Balkans than in the Apennines.Barely any similarities in patterns between cpDNA and SSR results were detected. The most prominent feature of the haplotype network was the existence of two highly differentiated lineages distributed along the eastern coast of the Adriatic Sea without any representatives in the Apennines. Without a doubt, populations that harbour these haplotypes are of ancient origin (in further text: ancient populations). In contrast to similar studies65 and that Pleistocene recolonization patterns can hardly be explained without them27. However, such assumptions were not supported by our results. If these populations had such a role during the postglacial colonization, their haplotypes would be, if not more abundant than they are, geographically structured, which they are not. Did ancient populations play a stimulating role during the expansion of other lineages through genetic support by sharing genes of importance for local adaptation? Is it possible that these diverged haplotypes represent once-dominant ecotypes well adapted to the colder climate that was dominant throughout much of the Pleistocene27? Or are they the remnants of extinct and closely related species assimilated by a more abundant species, the common sage? From the obtained results, it is impossible to answer any of these questions. On some occasions, similar phylogeographic results were explained as evidence of cryptic species' existence67. However, at least some support for such a conclusion from the nuclear-genome results is expected71. In contrast to plastid genomes of ancient populations, their nuclear genomes do not stand out in any aspect compared to those of surrounding populations. Since plastids are inherited maternally72 and SSRs biparentally, it is not a surprise to see such divergence between plastid and nuclear genomes. The nuclear-level genetic signature that would possibly distinguish ancient populations from the others was likely washed away by intense and persistent pollen-mediated gene flow. At the same time, the nonrecombinant plastid genome inherited via species-specific barochory-type seeds73 was retained in numerous microlocations across today's distribution range.Since diverged haplotypes were distributed across large geographic areas and were found in many populations, it is obvious that they did not evolve numerous times in situ. Sometime during the species evolutionary history, they were substantially more abundant than they are today, occupying larger parts of the paleo-distribution area. Over time, unfavourable conditions and perhaps other lineages suppressed them, and they became quarantined in scattered microrefugia. In general, it is assumed that microrefugia played an important role during postglacial expansions74. Consequently, the inability of a lineage to accumulate new mutations will result in the constant presence of poorly differentiated haplotypes closely related to assumed ancestral ones. The absence of any substantial differentiation of these haplotypes across large areas can be additionally explained by rapid migrations and colonization that possibly occurred over long distances, which resulted in the absence of any significant divergence among remote sites75.During a prolonged period that possibly spanned over multiple glaciation cycles, the majority of the populations harbouring haplotypes closely related to the ancestral ones (H01\u2013H10) have repeatedly vanished and re-emerged through recolonization. This group of populations, with H01 as the prevailing haplotype, dominated the scene for a reason beyond our comprehension. Being closely related to hypothetical ancestral haplotypes, it seems plausible that this group retained low levels of differentiation because of strong oscillations in its distribution range and abundance throughout evolutionary history. If a population periodically experiences severe genetic drift events, it is expected that emerging low-frequency mutations will constantly be purgedA comparison of haplotypes from the central and southern Apennines revealed that they were not as closely related as perhaps expected. For the most part, the central Apennines group harbours haplotypes found throughout the Balkans, with H01 being the predominant haplotype. At the same time, the group from the southern Apennines consisted of unique, more differentiated haplotypes not found anywhere else. It seems that Apennines groups of populations originated in two \"out of Balkans\" expansion events. From the first, more ancient colonization, the southern group emerged, while the second colonization event resulted in the formation of the central Apennines group of populations. Since the southern group haplotypes form a lineage that is exclusively found only in that region, it can be assumed that the colonization occurred just once and was followed by persistent isolation. The second colonization event likely occurred during the LGM. Since most of the haplotypes from the central Apennines are commonly found in the Balkans as well, there is no reason to think this group experienced a prolonged period of isolation. As already discussed, during the LGM, the land bridge between the Balkans and the Apennines was present and characterized by suitable environmental conditions that could easily support species range expansion.27 stated that \u201cmicrorefugia are no more than a theoretical necessity for explaining Pleistocene recolonization patterns\u201d. However, it seems that there is also the possibility of microrefugia acting as a quarantine and not a pole position for recolonization. Regardless, although it was shown on more than several occasions that the \u201crefugia within refugia\u201d concept was applicable for explaining the present-day genetic structure of both plant and animal species in major refugium areas, this was not the case for the common sage.Microsatellites and plastid DNA molecular markers allowed us to elucidate the evolutionary history of the common sage, not only during the recent periods but also more ancient ones. We proved again that the Apennines were colonized by the Balkans species, and in the case of the common sage, this occurred on two separate occasions. Although the results provided answers to numerous questions, they have also raised new ones. RullSixty-two common sage populations distributed throughout the species distribution range were sampled Fig.\u00a0. The col\u00ae). After the extraction, the concentrations were measured on a spectrophotometer and the samples were diluted to 1\u00a0ng/\u03bcL for SSR and cpDNA analyses.For population genetic analysis, leaf material from 20 to 25 individuals from each population was collected and stored in silica gel for rapid desiccation. For the phylogeographic analysis, five samples were randomly chosen from each population sample set . Genomic DNA was extracted from dried leaf material using the GenElute\u2122 Plant Genomic DNA Miniprep Kit (Sigma\u2013Aldrich77. Details of the polymerase chain reaction (PCR) conditions and the scoring of the amplified PCR products can be found in Radosavljevi\u0107 et al.78.To assess the levels of genetic variability and structure of the sampled populations, eight previously characterized microsatellite markers were used HO) and the expected heterozygosity (HE) were calculated by Genepop v. 4.779, while the allelic richness (Nar) and the private allelic richness (Npar) of each population were estimated by HP-Rare v. 1.080.The average number of alleles per locus, the observed heterozygosity  were calculated for the established genetic clusters.Genetic structure was inferred by a model-based clustering method implemented in STRUCTURE v. 2.3.482), was performed using DIYABC v.2.0 software83. In accordance with the results obtained from the STRUCTURE analysis at K\u2009=\u20095, five populations were defined. In total, 41 sampled populations were selected for the ABC analysis, all characterized by a high proportion of membership of a specific ancestral population (Q\u2009>\u20090.7). Based on the obtained results, five competing scenarios were constructed  Therps16-trnK and rpl32-trnL) of the plastid genome were analysed. PCRs were performed in a total volume of 15 \u03bcL containing 1.5 \u03bcL 10\u2009\u00d7\u2009PCR buffer (TaKaRa Taq\u2122 Hot Start Version), 1.2 \u03bcL dNTP solution , 0.6 \u03bcL 5\u00a0\u03bcM of each forward and reverse primer, 0.1 \u03bcL TaqHS polymerase  and 3 \u03bcL of 1\u00a0ng/\u03bcL DNA. The PCR conditions were 94\u00a0\u00b0C for 5\u00a0min, followed by 30 cycles of 95\u00a0\u00b0C for 1\u00a0min, 50\u00a0\u00b0C for 1\u00a0min and 65\u00a0\u00b0C for 4\u00a0min, and a final elongation step of 5\u00a0min at 65\u00a0\u00b0C. PCR products were cleaned using Exonuclease I  and FastAP Thermosensitive Alkaline Phosphatase  following the manufacturer\u2019s instructions. Amplicons were sequenced in both directions using an ABI 3730XL analyser (Applied Biosystems).To assess the phylogeographic structure of the studied species throughout its distribution area, two regions  in the cpDNA were treated as substitutions (single events)86.DNA sequences were aligned using CLUSTAL X v. 2.1h), the number of haplotypes per number of individuals (h/n), the number of private haplotypes (hpr), unbiased haplotype diversity , and nucleotide diversity  using Arlequin v. 3.5.2.289. Arlequin was also used to calculate the haplotypes distance matrix.Haplotype diversity within populations or groups of populations was quantified by calculating the number of haplotypes  for better presentation.The median-joining haplotype network was constructed by PopART v. 1.792 was used to estimate genetic differentiation , considering only haplotype frequencies (unordered haplotypes), and the corresponding measure NST92, considering genetic similarities between haplotypes (ordered haplotypes). A comparison of differentiation for ordered (NST) vs. unordered (GST) haplotypes was performed according to Pons and Petit92. Significance was tested based on 1000 random permutations. Phylogeographic structure is indicated when NST is higher than GST because closely related haplotypes are found more frequently in the same population than would be expected by chance.The program Permut cpSSR v. 2.0D93 and Fu's FS94 statistics were calculated using Arlequin to test for evidence of range expansion95. These parameters are very sensitive to deviations from population equilibrium. A significant value for D may be due to factors such as population expansion or bottlenecks97 and a significantly large negative value for Fs may be due to population expansion94. The significance of both test statistics was tested with 1,000 bootstrap replicates.Tajima's 98. The analysis was based on 62 occurrence records equally distributed throughout the species distribution range  following the procedure explained in Radosavljevi\u0107 et al.78 with all 19 WorldClim bioclimatic variables99 included. Retained bioclimatic variables and selected Maxent\u2019s regularization multipliers were used to predict the potential distribution of the common sage for the present time, mid-Holocene (6000 YBP), LGM , and last interglacial . For the LGM, three models were used: CCSM4, MIROC, and MPI-ESM-P. The obtained environmental suitability models were visualized in QGIS version 2.18.12. (QGIS Development Team (2020). QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org).To assess the geographical span of suitable environmental niches during the historical periods, environmental niche modelling (ENM) was performed as implemented in MAXENT ver. 3.3.3\u00a0k.Supplementary Information."}
+{"text": "Widespread uptake of vaccines is necessary to achieve herd immunity. However, uptake rates have varied across U.S. states during the first six months of the COVID-19 vaccination program. Misbeliefs may play an important role in vaccine hesitancy, and there is a need to understand relationships between misinformation, beliefs, behaviors, and health outcomes. Here we investigate the extent to which COVID-19 vaccination rates and vaccine hesitancy are associated with levels of online misinformation about vaccines. We also look for evidence of directionality from online misinformation to vaccine hesitancy. We find a negative relationship between misinformation and vaccination uptake rates. Online misinformation is also correlated with vaccine hesitancy rates taken from survey data. Associations between vaccine outcomes and misinformation remain significant when accounting for political as well as demographic and socioeconomic factors. While vaccine hesitancy is strongly associated with Republican vote share, we observe that the effect of online misinformation on hesitancy is strongest across Democratic rather than Republican counties. Granger causality analysis shows evidence for a directional relationship from online misinformation to vaccine hesitancy. Our results support a need for interventions that address misbeliefs, allowing individuals to make better-informed health decisions. Vaccination is the lynchpin of the global strategy to fight the SARS-CoV-2 coronavirus3. Surveys conducted during February and March 2021 found high levels of vaccine hesitancy with around 40\u201347% of American adults hesitant to take the COVID-19 vaccine5. However, populations must reach a threshold vaccination rate to achieve herd immunity 8. Evidence of uneven distributions of vaccinations9 raises the possibility of geographical clusters of non-vaccinated people10. In early July 2021, increased rates of the highly transmissible SARS-CoV-2 Delta variant were recorded in several poorly vaccinated U.S. states9. These localized outbreaks will preclude eradication of the virus and may exacerbate racial, ethnic, and socioeconomic health disparities.The COVID-19 pandemic has killed over 4.9 million people and infected 241 million worldwide as of October 202111. Some factors are linked to COVID-19 vaccine hesitancy, with rates in the U.S. highest among three groups: African Americans, women, and conservatives12. Other predictors, including education, employment, and income are also associated with hesitancy13. A number of studies discuss the spread of vaccine misinformation on social media14 and argue that such campaigns have driven negative opinions about vaccines and even contributed to the resurgence of measles16. In the COVID-19 pandemic scenario, widely shared misinformation includes false claims that vaccines genetically manipulate the population or contain microchips that interact with 5G networks18. Exposure to online misinformation has been linked to increased health risks19 and vaccine hesitancy20. Gaps remain in our understanding of how vaccine misinformation is linked to broad-scale patterns of COVID-19 vaccine uptake rates.Vaccine hesitancy covers a spectrum of intentions, from delaying vaccination to outright refusal to be vaccinated21. EUA was then\u00a0given to\u00a0two other vaccines in early 2021. Initially, vaccines were selectively administered with nationwide priority being given to more vulnerable cohorts such as elderly members of the population. As vaccines became available to the entire adult population22, adoption was driven by limits in demand rather than in supply. It is therefore important to study the variability in uptake across U.S. states and counties, as reflected in recent surveys24.The Pfizer-BioNTec COVID-19 vaccine was the first to be given U.S. Food and Drug Administration Emergency Use Authorization (EUA)\u00a0on December 10th 2020Here we study relationships between vaccine uptake, vaccine hesitancy, and online misinformation. Leveraging data from Twitter, Facebook, and the Centers for Disease Control and Prevention (CDC), we investigate how online misinformation is associated with vaccination rates and levels of vaccine hesitancy across the U.S. We also use Granger Causality analysis to investigate whether there is evidence for a directional association between misinformation and vaccine hesitancy.17, which were collected between January 4th and March 25th from the Twitter filtered stream API using a comprehensive list of keywords related to vaccines (see Supplementary Information). We leveraged the Carmen library29 to geolocate almost 1.67\u00a0M users residing in 50 U.S. states, and a subset of approximately 1.15\u00a0M users residing in over 1,300 counties. The larger set of users accounts for a total of 11\u00a0M shared tweets. Following a consolidated approach in the literature28, we identified misinformation by considering tweets that contained links to news articles from a list of low-credibility websites compiled by a politically neutral third\u00a0party (see details in the Supplementary Information). We measured the prevalence of misinformation about vaccines in each region by (i)\u00a0calculating the proportion of vaccine-related misinformation tweets shared by each geo-located account; and (ii)\u00a0taking the average of this proportion across accounts within a specific region. The Twitter data collection was evaluated and deemed exempt from review by the Indiana University IRB (protocol 1102004860).Our key independent variable is the mean percentage of vaccine-related misinformation shared via Twitter at the U.S. state or county level. We used 55\u00a0M tweets from the CoVaxxy dataset9. Vaccine hesitancy rates are based on Facebook Symptom Surveys provided by the Delphi Group24 at Carnegie Mellon University. Vaccine hesitancy is likely to affect uptake rates, so we specify a longer time window to measure this variable, i.e., the period January 4th\u2013March 25th 2021. We computed hesitancy by\u00a0inverting the proportion of individuals \u201cwho either have already received a COVID vaccine or would definitely or probably choose to get vaccinated, if a vaccine were offered to them today.\u201d See Supplementary Information for further details.Our dependent variables include vaccination uptake rates at the state level and vaccine hesitancy at the state and county levels. Vaccination uptake is measured from the number of daily vaccinations administered in each state during the week of 19\u201325 March 2021, and measurements are derived from the CDCThere are no missing vaccine-hesitancy survey data at the state level. Observations are missing at the county level because Facebook survey data are available only when the number of respondents is at least 100. We use the same threshold on the minimum number of Twitter accounts geolocated in each county, resulting in a sample size of N\u2009=\u2009548 counties.Our multivariate regression models adjust for six potential confounding factors: percentage of the population below the poverty line, percentage aged 65\u2009+\u2009, percentage of residents in each racial and ethnic group , rural\u2013urban continuum code , number of COVID-19 deaths per thousand, and percentage Republican vote (in 10 percent units). Other covariates, including religiosity, unemployment rate, and population density, were also considered  as well as logged versions of misinformation (to correct positive skew). These results are presented in Supplementary Information Tables -S8.j-th observation is assumed to be \u03c32/wj where wj is the weight. The weights are set equal to the size of the sample from which the average is calculated. We estimate weighted regression with the aweights command in Stata 16. In addition, because counties are nested hierarchically within states, we use cluster robust standard errors to correct for lack of independence between county-level observations.We conduct multiple regression models predicting vaccination rate and vaccine hesitancy. Both dependent variables are normally distributed, making weighted least squares regression the appropriate model. Data are observed (aggregated) at the state or county level rather than at the individual level. Analytic weights are applied to give more influence to observations calculated over larger samples. The weights are inversely proportional to the variance of an observation such that the variance of the We investigate Granger causality between vaccine hesitancy and misinformation by comparing two auto-regressive models. The first considers daily vaccine hesitancy rates 31 on y help forecast hesitancy rates x. We assume geographical regions to have equivalence and independence in terms of the way misinformation influences vaccine attitudes. Thus, we use the same parameters for The variable 30. However, in our case, there are several reasons why this is not appropriate. First, we have missing time\u00a0windows in some of our regions. Second, our assumptions of equivalence and independence for regions may not be accurate. For these reasons, we use a bootstrap method to estimate the expected random distribution of p-value of our Granger Causality analysis is then given by the proportion of trials . Investigating covariates known to be associated with vaccine uptake or hesitancy, we find that an increase in the mean amount of online misinformation is significantly associated with a decrease in daily vaccination rates per million . These two factors alone explain nearly half the variation in state-level vaccination rates, and are themselves moderately correlated  and thus dropped for parsimony.Looking across U.S. states, we observe a negative association between vaccination uptake rates and online misinformation , and between political partisanship and hesitancy , while the percentage of Hispanic or Latinx residents\u00a0is negatively associated . The percentage of residents below the poverty line is also positively associated with vaccine hesitancy .To investigate vaccine hesitancy, we leverage over 22\u00a0M individual responses to daily survey data provided by Facebook24 see . ReportsN\u2009=\u2009548  for which we are able to measure both variables is \u2009548 see . Politic\u2009548 see . Using a\u2009548 see . This ma31. We find that misinformation helps forecast vaccine hesitancy, weakly at state level (p\u2009=\u20090.0519) and strongly at county level (p\u2009<\u20090.001; see Our results so far demonstrate an association between online misinformation and vaccine hesitancy. We investigate evidence for directionality in this association by performing a Granger Causality analysis35. We did not observe significant differences in the top sources shared in Republican vs. Democratic majority states.Finally, Fig.\u00a0Our results provide evidence for the problem of geographical regions with lower levels of COVID-19 vaccine uptake, which may be driven by online misinformation. Considering variability across regions with low and high levels of misinformation, the best estimates from our data predict a\u2009~\u200920% decrease in vaccine uptake between states, and a\u2009~\u200967% increase in hesitancy rates across Democratic counties, across the full range of misinformation prevalence. At these levels of vaccine uptake, the data predict SARS-CoV-2 will remain endemic in many U.S. regions. This suggests a need to counter misinformation in order to promote vaccine uptake.20 found that exposure to online misinformation can increase vaccine hesitancy. Our work serves to provide evidence that those findings, which were obtained under controlled circumstances, scale to an ecological setting. Due to the fact that vaccine hesitancy and misinformation are socially reinforced, both ecological and individual relationships are important in demonstrating a causal link36. However, we are still unable to rule out confounding factors, so uncertainty remains about a causal link and further investigation is warranted.An important question is whether online misinformation drives vaccine hesitancy. Our analyses alone do not demonstrate a causal relationship between misinformation and vaccine refusal. Our work is at an ecological scale and vaccine-hesitant individuals are potentially more likely to post vaccine misinformation. However, at the individual level, a recent study25. Our results indicate that there is a geographical component to this spread, with opinions on vaccines spreading at a local scale. While social media users are not representative of the general public, existing evidence suggests that vaccine hesitancy flows across social networks37, providing a mechanism for the lateral spread of misinformation offline among those connected directly or indirectly to misinformation spreading online. More broadly, our results provide additional insight into the effects of information diffusion on human behavior and the spread of infectious diseases38.Public opinion is very sensitive to the information ecosystem and sensational posts tend to spread widely and quickly40. Our results are also limited to a small period of time. Finally, other factors might also influence vaccination hesitancy levels, including accessibility to vaccines, changes in COVID-19 infection and death rates, as well as legitimate reports about vaccine safety41.A limitation of our findings is that we are not measuring the exposure, by geographical region, to misinformation on Twitter but rather the sharing activity of a subset of users. Besides, our analyses are based on data averaged over geographical regions. To account for group-level effects we present a number of sensitivity analyses, and note that our findings are consistent over two geographical scales. Our source-based approach to detect misinformation at scale might not capture the totality of misleading and harmful content related to vaccines, and many low-credibility sources publish a mixture of false and true information42. While people have a constitutional right to free speech, it is important to maintain an environment where individuals have access to good information that benefits public health.Associations between online misinformation and detrimental offline effects, like the results presented here, call for better moderation of our information ecosystem. COVID-19 misinformation is shared overtly by known entities on major social media platforms43. We also provide aggregated measurements of online misinformation shared by geolocated Twitter users. Results at the state and county level can be fully reproduced using the STATA scripts provided in the repository. Due to Twitter\u2019s terms of use and service, we can only release IDs of the tweets present in our dataset, which can be reconstructed using the Twitter API. The IDs are accessible in the public dataset associated with the CoVaxxy project17 from the Observatory on Social Media at Indiana University.All measurements of vaccine uptake and vaccine hesitancy rates as well as socioeconomic, political, and demographic variables at the state and county level are publicly available in the online repository associated with this paperSupplementary Information."}
+{"text": "Familial hypocalciuric hypercalcemia (FHH) is a rare autosomal dominant disease, which requires differential diagnosis from relatively common primary hyperparathyroidism (PHPT) in order to avoid unnecessary surgery.A 16-year-old female had been followed by the department of psychosomatic medicine at our institution. Throughout the follow-up period, her plasma calcium levels were high, plasma Pi levels were relatively low, and plasma intact PTH was relatively high. She was referred to our department to determine the cause of her hypercalcemia. Her 24\u00a0h urinary calcium excretion was as low as 100\u00a0mg/day, and calcium creatinine clearance ratio was below 0.01. Moreover, she had a family history of hypercalcemia . The genetic testing for her family revealed that she, her brother, and her father were definitively diagnosed with FHH type 1 due to the heterozygous calcium-sensing receptor mutation (NM_00388:4:c.164C\u2009>\u2009T:p.Pro55Leu).We experienced a 16-year-old female with FHH, in whom genetic testing identified the heterozygous calcium-sensing receptor mutation (NM_00388:4:c.164C\u2009>\u2009T:p.Pro55Leu) as pathogenic, permitting a definitive diagnosis of FHH type 1. The genetic testing for calcium sensing receptor is beneficial to distinguish asymptomatic primary hyperparathyroidism from FHH. Hypercalcemia is a condition causing disorders in multiple organs, including bone, brain, and heart . The cauUrinary calcium excretion and calcium creatinine clearance ratio (CCCR) levels are widely used for differential diagnosis of FHH and PHPT; even so, it is sometimes difficult to distinguish the two diseases \u20136. ApproCASR gene and belongs to a G protein-coupled receptor family that regulates parathyroid hormone (PTH) secretion, vitamin D synthesis, and calcium absorption and resorption [GNA11 gene and mediates CaSR signaling [CASR, GNA11, or AP2S1 genes cause FHH type 1 (FHH1), FHH type 2 (FHH2), and FHH type 3 (FHH3), respectively [CASR gene [To date, FHH-related mutations have been identified in genes related to the calcium sensing system that regulates calcium homeostasis . The calsorption , 5. G-prignaling , 5. Adapignaling , 5. Lossectively . ApproxiCASR mutation p.Pro55Leu, which permitted the definitive diagnosis of the disease. In this case with no symptom, family history and genetic test for CASR was beneficial for the diagnosis of FHH type1.Here we report a case of FHH1 in which genetic testing identified the heterozygous A 16-year-old female had been followed by the department of psychosomatic medicine at our institution for borderline personality disorder. Throughout the follow-up period, her plasma calcium levels were high, plasma Pi levels were relatively low, and plasma intact PTH (PTHi) was relatively high    and PTHi were 11.5  mg/dL, 3.1  mg/dL, and 51  pg/mL, respectively  was performed and a pathogenic CASR mutation (CASR: NM_000388.4: exon2: c.164C\u2009>\u2009T: p. Pro55Leu) was noted in her father (I-1), brother (II-1), and the proband (II-3)  and father (I-1) had hypercalcemia Fig.\u00a0A, we pro and fathWe report here a case of hypercalcemia with family history and low CCCR with hypovitaminosis D, in which genetic testing permitted definitive diagnosis of FHH1.FHH is a very rare benign inherited condition that typically does not require parathyroidectomy. Patients with FHH usually have no symptoms and are often diagnosed by chance during routine blood examination. Weakness, fatigue, issues with concentration, constipation, polyuria, headache, and polydipsia have been reported by some people with FHH. The calcimimetic drugs are sometimes effective to improve hypercalcemic symptoms such as muscle aches, anorexia, polydipsia and constipation . Rarely,CASR mutation p.Pro55Leu was found to be pathogenic in the patient\u2019s father (I-1), brother (II-1) and the proband (II-3). This mutation has previously been found in several individuals with FHH including one Japanese [In the current study, a heterozygous Japanese , 8\u201316; iJapanese , 16. ConCASR p.Pro55Leu mutation, in which only genetic testing allowed definitive diagnosis of FHH1 and thus avoided unnecessary surgery. The patient and family commented that they were very relieved to have found the cause of the disease. To diagnose with FHH by a genetic test is important to avoid unnecessary surgical treatment.In conclusion, we report a case of FHH1 due to Clinical data from the corresponding author will be available upon request."}
+{"text": "PGRN) is a secreted glycoprotein encoded in humans by the GRN gene, located on chromosome 17q21. Several nonsense and missense pathogenetic GRN mutations have been described.Progranulin .The younger sister presented at the age of 64 with inspiratory stridor, dysphonia and exercise-induced dyspnea. Transnasal fiberoptic laryngoscopy showed bilateral adduction of the vocal cords at rest and paradoxical further adduction of the vocal cords during forced inspiration, suggesting the hypothesis of an adductor laryngeal dystonia. The older sister presented at the age of 63 with a rapidly progressive corticobasal syndrome. The only clinical feature common to both sisters was a dysexecutive syndrome. The c.893G\u2009>\u2009A mutation in exon 9 of GRN p.R298H mutation, which is first detected in two members from the same family, showing an extremely different phenotypes. Moreover, we report the first case of an FTD-associated mutation presenting with inspiratory stridor and dysphonia linked to adductor laryngeal dystonia, thus expanding the clinical spectrum of GRN-related disorders.Our report supports the pathogenicity of the The online version contains supplementary material available at 10.1007/s00415-022-11285-7. PGRN) is a secreted glycoprotein encoded in humans by the GRN gene, located on chromosome 17q21. PGRN is a growth factor involved in numerous processes  and implicated also in tumorigenesis [Progranulin , whereas homozygous ones lead to the development of neuronal ceroid lipofuscinosis (NCL) [GRN pathogenic mutations introduce a premature stop codon that triggers nonsense-mediated loss of GRN mRNA and subsequent loss of 50% of plasma PGRN levels leading to haploinsufficiency. [To date, several nonsense and missense pathogenetic is (NCL) . Most ofGRN mutation with extremely different clinical phenotypes and family history of dementia and behavioral disorders.We herein describe neurological features of two sisters carrying a Patient 1 is a 65-year-old Caucasian woman followed at the Movement Disorder outclinic of the University of Salerno, Italy. Symptoms onset occurred at 64\u00a0years with dysphonia, exercise-induced dyspnea and inspiratory stridor, which was mainly nocturnal (Supplementary Audio file). Since stridor was her main complaint, she was referred to our Movement Disorders outclinic in order to consider a diagnosis of multiple system atrophy (MSA). Two paternal aunts were diagnosed with dementia and behavioral disorders. Her father died at 54\u00a0years due to complications of diabetes and did not present during lifetime any cognitive or behavioral symptoms. Her mother died in old age and did not present any neurological symptoms.Patient 2 is a 68-year-old Caucasian woman that presented at the age of 63 with severe depression of mood, unresponsive to several antidepressants, and severe apathy.18F-FDG PET, 3-Tesla brain MRI, chest CT, transnasal fiberoptic laryngoscopy, electromyography (EMG) and polysomnography. To better assess dysphonia, voice quality was evaluated by the acoustic voice quality index (AVQI), a tool also validated in Italian  version 03.01 in Italian [Patient 1 performed DaT-SCAN, brain  Italian , that qu Italian . The voiPatient 2 underwent neuropsychological evaluation and brain MRI one year after the onset of behavioral symptoms.GRN gene, including the intron\u2013exon boundaries, were PCR-amplified, and sequenced on an ABI 3500 Genetic Analyzed . To evaluate the evolutionary amino acidic conservation was performed the bioinformatics analysis. In silico analyses were performed to evaluate the pathogenic role of the c.893G\u2009>\u2009A using PholyPhen2 (http://genetics.bwh.harvard.edu/pph2/), Mutation Taster (http://www.mutationtaster.org/). MAPT, C9orf72, FUS, TARDBP, VCP and CHMP2B genes were also analyzed by Sanger Sequencing.All 13 exons of the At our first evaluation, one year after symptoms onset, patient 1 presented frontal release signs , laryngeal stridor, moderate dysphonia, tetrahyperreflexia and left Babinski sign. No extrapyramidal signs, muscle weakness or atrophy were observed. The patient was clinically re-evaluated 6\u00a0months after the first assessment, and her neurological examination was unchanged. She underwent a comprehensive neuropsychological assessment, showing a dysexecutive syndrome with significantly impaired inhibitory control (Stroop test) and planning (copy of the Rey\u2019s complex figure). Mini-Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) scores were in the normal range. Moreover, memory, visuo-spatial and language domains were preserved; no psychiatric symptoms were present. A single-domain non-amnesic mild cognitive decline was diagnosed.As for patient 2, at onset of behavioral symptoms, her neurological examination was normal. One year later, she developed memory deficits and a clearly asymmetric parkinsonism with dysarthria, reduction of verbal fluency, spastic laughter and ideomotor apraxia. Levodopa therapy was tried, with poor response. Her neuropsychological examination revealed a severe dysexecutive syndrome and a multidomain cognitive decline. At the age of 65\u00a0years, she was anarthric and had developed severe dystonia on the left side of the body and in the cranial district. She was bedridden since the age of 66\u00a0years. She was diagnosed with corticobasal syndrome. At last examination at the age of 68, she presented severe dementia with few residual non-verbal emotional reactions, severe rigidity prevalent on the left side of the body, severe dysphagia requiring a semi-liquid diet.18F-FDG PET showed hypometabolism in the medial frontal cortex  was observed. Stroboscopy showed mild bilateral vocal tremor with a good lamina propria expansion without glottal gaps. During the forced inspiratory maneuver (abduction task), a paradoxical further adduction of the vocal cords was seen with airflow limitation due to glottic respiratory space reduction.A transnasal fiberoptic laryngoscopy was performed to investigate the nature of the laryngeal stridor and exercise-induced dyspnea; at rest it showed bilateral adduction of the vocal cords in a paramedian position. No vocal fold structural lesion was found, and complete glottic closure during full phonation of Fig.\u00a0An illustration of the patient's AVQI is shown in Needle EMG, performed in the bulbar, thoracic regions and at least two muscles innervated by different roots and peripheral nerves for each limb, showed widespread fasciculation potentials in the lumbosacral district and isolated fasciculation potentials in the cervicobrachial district, without chronic motor unit changes or reduced central activation. Central Motor Conduction, measured in the ulnar and posterior tibial nerves, resulted normal.Brain MRI, performed in patient 2 one year after the onset of behavioral symptoms, was normal. Since she was rapidly bedridden, no further imaging studies were performed.GRN was found in heterozygosis in both sisters  in only one of the sisters. The presence of mutations in the MAPT, C9orf72, FUS, TARDBP, VCP and CHMP2B genes was also excluded in both sisters.The c.893G\u2009>\u2009A variant in exon 9 of ers Fig.\u00a0a. This vers Fig.\u00a0b. FunctiGRN p.R298H mutation was first described in 2010 in a single patient affected by FTLD with an unknown family history [In this paper, we describe two sisters with quite different neurological phenotypes related to a rare GRN mutation. Sanger sequencing revealed in both sisters a missense variant (c.893G\u2009>\u2009A) not present in a control cohort of 300 healthy individuals of the same geographic area. Among rarely reported missense mutations,  history Functional prediction analysis by PolyPhen2 (http://genetics.bwh.harvard.edu/pph2/) and Mutation Taster (http://www.mutationtaster.org/) revealed that this variant has a probable damaging role.The p.R298H is highly conserved across species. . The mutation was detected in only one patient with pathologically confirmed FLTD with ubiquitin-positive inclusions and unknown family history. [The p.R298H variant was already described by Yu et al.  as potenhistory. . [The same variant was also reported by Karch et al.  in one F. GRN p.R298H variant in FTD, since it was first found in two Italian sisters with FTD and family history of behavioral disturbances and dementia.Our data support the pathogenic role of Moreover, our findings suggest that this mutation may be associated with an extremely variable phenotype. In fact, the older sister showed a rapidly progressive corticobasal syndrome and was bedridden within three years from onset. A corticobasal syndrome with asymmetric akinetic rigid parkinsonism, asymmetric upper limb dystonia and cortical signs  similar to that observed in patient 2 was reported by Karch et al. in association with the same mutation. GRN mutations. Indeed, inspiratory stridor is a typical feature of multiple system atrophy, where it is considered a diagnostic red flag, but our patient did not present parkinsonian or cerebellar signs, despite a moderately reduced DaT uptake in the right putamen. In MSA, the pathogenesis of stridor is debated, and the two mechanisms most likely involved are the degeneration of the ambiguous nucleus, found in some pathological studies, and the paradoxical muscular activity of the laryngeal adductor muscles during inspiration, found by EMG examination during sleep [In the younger sister, the main complaints were stridor and exercise-induced dyspnea. To the best of our knowledge, stridor has never been described in patients with frontotemporal dementia or ng sleep . In our ng sleep . Indeed,ng sleep A study of patients with a clinical diagnosis of ADLD and Abductor laryngeal dystonia was performed to assess the neural correlates of abnormal sensory discrimination by means of fMRI. In both groups, abnormal temporal discrimination thresholds were related with brain activation during symptom production in the left primary sensorimotor cortex and with resting brain activation in the left anterior cingulate cortex. ADLD patients showed negative correlations between abnormal temporal discrimination thresholds and symptom-related brain activation in the left middle/inferior gyrus, posterior cingulate cortex and bilateral SMA, while the left superior frontal gyrus and precuneus were positively correlated during the resting state . We can Moreover, the EMG findings of patient 1 met the El Escorial criteria for a \u201cclinically possible ALS\u201d, suggesting a subtle MN degeneration associated with FTLD, confirming the wide phenotypic variability of FTLD mutations , and reqStridor has shown a high positive predictive value for a diagnosis of MSA, but it cannot be considered pathognomonic for this disease. Our patient was referred to us with a suspicion of MSA, but both clinical features and positive family history, albeit with different clinical manifestations, prompted us to search for an alternative diagnosis. Our paper expands the spectrum of clinical symptoms of FLTD with the first ever described case of stridor associated with a FLTD mutation. This rare GRN mutation seems to be associated with a variable phenotype at onset, but also with a different disease progression. Additional genetic studies in larger Italian cohort are required to clarify the role of this variant in the development of disease.Supplementary file1 (MP4 28387 KB)Supplementary file2 (MP4 526 KB)Below is the link to the electronic supplementary material."}
+{"text": "Background and Objectives: Ureteral stent insertion passively dilates the ureter. Therefore, it is sometimes used preoperatively before flexible ureterorenoscopy to make the ureter more accessible and facilitate urolithiasis passage, especially when ureteroscopic access has failed or when the ureter is expected to be tight. However, it may cause stent-related discomfort and complications. This study aimed to assess the effect of ureteral stenting prior to retrograde intrarenal surgery (RIRS). Materials and Methods: Data from patients who underwent unilateral RIRS for renal stone with the use of a ureteral access sheath from January 2016 to May 2019 were retrospectively analyzed. Patient characteristics, including age, sex, BMI, presence of hydronephrosis, and treated side, were recorded. Stone characteristics in terms of maximal stone length, modified Seoul National University Renal Stone Complexity score, and stone composition were evaluated. Surgical outcomes, including operative time, complication rate, and stone-free rate, were compared between two groups divided by whether preoperative stenting was performed. Results: Of the 260 patients enrolled in this study, 106 patients had no preoperative stenting (stentless group), and 154 patients had stenting (stenting group). Patient characteristics except for the presence of hydronephrosis and stone composition were not statistically different between the two groups. In surgical outcomes, the stone-free rate was not statistically different between the two groups (p = 0.901); however, the operation time for the stenting group was longer than that of the stentless group . There were no differences in the complication rate between the two groups (p = 0.523). Conclusions: Among surgical outcomes for RIRS with a ureteral access sheath, preoperative ureteral stenting does not provide a significant advantage over non-stenting with respect to the stone-free rate and complication rate. The ureteral stent is an irreplaceable tool for urologists. First described by Herdman in 1949, and later developed in the current \u2018double-J\u2019 shape by Thomas Hepperlen and Roy Finney in the 1970s, ureteral stents are commonly used to relieve obstruction of the ureter, prevent complications following upper urinary tract procedures, and provide a scaffold for healing of the ureter. Although a ureteral stent placement serves as the most minimally invasive method for draining urine from the kidney to the bladder, it has some drawbacks, including infection, pain, encrustation, dislodgement, hematuria, and irritative voiding symptoms such as frequency and urgency .Since Marshall designed the first flexible ureterorenoscope in 1964, there have been ongoing technological improvements. Technological advances focused on reducing the diameter of the scope while increasing the deflection angle, and it was mainly used for diagnostic purposes. However, during the 1990s, a deflection system with a larger working channel of 3.6 Fr was introduced, and advancements in laser technology using holmium: YAG as a flexible lithotripter became widely employed for retrograde intrarenal surgery (RIRS) in the treatment of upper urinary tract stones during the late 1990s. With its improved stone-free rate (SFR) and low complication rate, treatment indications for RIRS have significantly expanded, and it is now recommended as the first- or second-line treatment for all categories of kidney stones, including stones larger than 20 mm, although multisession treatment may be required according to current treatment guidelines .RIRS requires repetitive scope insertion to fragment and extract urinary stones; thus, to aid access to the proximal ureter and renal pelvis and to lower intrarenal pressure, a ureteral access sheath (UAS) is widely used during RIRS. However, sometimes ureteral access is not possible\u2014previous studies have reported 8.8% to 20% failure rates for UAS insertion . For theIn this regard, some studies have analyzed the relationship between RIRS and preoperative ureteral stenting, reporting that preoperative ureteral stent placement improves the SFR ,12,13. HWe retrospectively reviewed patients who had RIRS for ureteral or renal stones from January 2016 to May 2019. The decision to treat the stone surgically was made according to EAU guidelines, which include symptomatic ureteral stones, growing renal stones, obstruction caused by stones, and infection . The decFirstly, we collected cases in which RIRS was performed for a proximal ureter or renal stone. For an accurate comparison of surgical outcomes of RIRS, we excluded cases that were not unilateral RIRS  and included only cases carried out for renal stones by checking low-dose non-contrast stone CT imaging routinely performed within 3 days of surgery and the surgical records. However, stones that were ureteral stones before stent placement but were found to be located inside the kidney after stent placement were included .Patient characteristics, including age, sex, body mass index (BMI), presence of hydronephrosis, and treated side, were obtained. Hydronephrosis was identified using preoperative computed tomography (CT) diagnostic imaging. Maximal stone length (MSL) and modified Seoul National University Renal Stone Complexity (mS-ReSC) score were set as stone characteristics. MSL was measured via CT imaging in bone windows/level settings and the mS-ReSC score was assigned according to the number of sites involved in the renal pelvis (#1), superior and inferior major calyceal groups (#2\u20133), and anterior and posterior minor calyceal groups of the superior (#4\u20135), middle (#6\u20137), and inferior calyx (#8\u20139). If the stone was in the inferior sites , one additional point per site was added to the original score . However, the requirement for written informed consent of subjects was waived due to the anonymization of patient data and the retrospective study design.In cases that used preoperative stenting, cystoscopic ureteral stent insertion  was performed under local anesthesia by various urological department residents. As usual, the ureteral stent was inserted using a cystoscope and guidewire in the lithotomy position.\u00ae Wire Guide; Cook Medical, Bloomington, IN, USA) was introduced as a safety guidewire into the renal pelvis in a retrograde fashion. If preoperative ureteral stenting was performed, a hydrophilic guidewire was inserted through the previously inserted ureteral stent. A dual-lumen ureteral catheter  was advanced over the safety guidewire; retrograde pyelography was performed; and then a stiff guidewire  was placed next to the safety guidewire. An 11/13-Fr  was advanced into the proximal ureter over the stiff guidewire. A flexible uretero-reno videoscope was inserted through the UAS. Four kinds of scopes were used, as follows: FLEX-XC digital flexible video ureterorenoscope , URF-V and URF-V2 flexible video ureteroscopes (Olympus Corp.), and LithoVue\u2122 single-use digital flexible ureteroscope (Boston Scientific). Lithotripsy was performed with a holmium: YAG laser lithotripter  using 200-micron laser fibers. Depending on the size and hardness of the stone, fragmentation and dusting methods were utilized appropriately. Large, fragmented stones were extracted with a 1.9-Fr Zero Tip\u2122 Nitinol Stone Basket (Boston Scientific). Stone dust particles were not removed, as they were expected to drain naturally. A 6-Fr double-J ureteral stent was routinely placed after the RIRS procedure and maintained for 1 to 2 weeks in all patients. In all cases, surgical procedures were performed by the same experienced single surgeon (J.Y.L.). After removal of the postoperative stent on an outpatient basis, follow-up non-contrast CT was performed at 1 to 3 months, and the presence or absence of residual stones confirmed the stone-free status.RIRS was performed under general anesthesia as follows. The patient was placed in the lithotomy position, and a 0.035\u2032\u2032 flexible hydrophilic-coated guidewire , whereas the other group had a double-J ureteral stent placed prior to RIRS (stenting group). Patient demographics, stone characteristics, and surgical outcomes were compared between the two groups. Surgical outcomes included operative time, postoperative complications categorized according to the Clavien\u2013Dindo classification system , and SFRt-test was used for statistical comparisons of continuous demographic variables. The Shapiro\u2013Wilk test was performed to check the distribution of continuous variables, and the Man n\u2013Whitney test was performed if the normal distribution was not met. Pearson\u2019s chi-squared test with Yates\u2019s correction for continuity was used to compare categorical variables. Univariate and multivariate logistic regression with a binomial method were performed to analyze factors affecting the SFR. All computations were performed using R version 4.2.1 .Data are presented as the mean \u00b1 standard deviation unless otherwise indicated. Dichotomous and categorical variables are presented as actual numbers and as percentages of the total population. Student\u2019s two-sample Included in this study were 260 patients who underwent unilateral RIRS for intrarenal stone. The patients were divided into two groups: 106 patients who had no stent insertion before RIRS were classified as the stentless group, and 154 patients who had preoperative ureteral stent insertion were classified as the stenting group.p = 0.015 and p = 0.025, respectively). The rate of hydronephrosis present on preoperative CT scans was higher for the stenting group than for the stentless group (62.3% vs. 46.2%). Calcium oxalate stones were the most commonly occurring stone type in both groups; however, uric acid stones were more common in the stenting group than in the stentless group (28.6% vs. 15.1%)  .p =0.001). However, the SFR and postoperative complication showed no statistical difference between the two groups   . In the p < 0.001, both groups). A multivariate analysis corroborated a shorter MSL (p = 0.006) and lower mS-ReSC score (p < 0.001) as independent significant factors for the SFR , which was adequate for the surgical indication of RIRS.Although Law et al. reported in their meta-analysis that preoperative stenting improves SFR in the ureterorenoscopic treatment of renal stones , the comWe also collected only cases performed by a single, sufficiently skilled surgeon to exclude the effect of differences between operators. In our medical center, four kinds of flexible uretero-reno videoscopes were used and three of which were re-usable , whereas one was disposable for single use (LithoVue\u2122). The type of scope was not distinguished when collecting data according to a systematic review that found no significant differences in surgical outcomes including SFR, complication rate, operation time, and hospital stay between the use of reusable and disposable flexible ureteroscopes for stone surgeries .p = 0.353), and the stenting group also had a higher proportion than in their study (59.2% vs. 46.1%).Logistic regression modeling revealed that preoperative stenting was not the independent predictor of SFR in our study, which was consistent with the previous studies ,29. ThisThe finding of no difference in postoperative complication rates with and without stenting was consistent with that of a meta-analysis by Law et al. . Upon rep = 0.008) [The present study found that operative time was longer in the stenting group. The same results were found in some studies and Lumma et al. suggested that this could be attributed to stent extraction prior to RIRS ,26. In c= 0.008) . They atThe data for this study was limited to January 2016 through May 2019. As we have gained sufficient experience with safe access sheath insertion and digital flexible ureteroscopy, we have seen fewer cases of failed ureteral entry or ureteral injury on the first attempt. Therefore, we have gradually reduced preoperative ureteral stenting in our institution, which can cause unnecessary hematuria, UTIs, and urolithiasis in patients scheduled for surgery ,31. In aAnother limitation of this retrospective study is that ureteral stent insertion was not randomized. Assimos et al., in their study, presented a predictive model for preoperative ureteral stent placement. In general, clinicians preferred to place preoperative ureteral stents in cases with high comorbidities such as high ASA score, solitary kidney, anticoagulant use, and Crohn\u2019s disease . We perfA well-designed randomized controlled trial is needed to address these shortcomings and validate the results. However, despite these limitations, we aimed to eliminate confounding factors from previous studies through more detailed study design, which might help strengthen existing guidelines to prevent unnecessary discomfort and additional costs from preoperative ureteral stent insertion.Among patients who underwent RIRS with a UAS, those who had preoperative ureteral stenting did not show a significant difference in SFR and complication rate compared with those who did not. Therefore, except when necessary to resolve obstructive uropathy or renal colic, routine preoperative stenting is not needed to improve surgical outcomes of RIRS."}
+{"text": "Thrombocytopenia, hemorrhage and platelet transfusion are common in patients supported with venoarterial extracorporeal membrane oxygenation (VA ECMO). However, current literature is limited to small single-center experiences with high degrees of heterogeneity. Therefore, we aimed to ascertain in a multicenter study the course and occurrence rate of thrombocytopenia, and to assess the association between thrombocytopenia, hemorrhage and platelet transfusion during VA ECMO.N\u2009=\u200916) study on transfusion practices in patients on VA ECMO, in which a retrospective cohort (Jan-2018\u2013Jul-2019) focusing on platelets was selected. The primary outcome was thrombocytopenia during VA ECMO, defined as mild (100\u2013150\u00b7109/L), moderate (50\u2013100\u00b7109/L) and severe (<\u200950\u00b7109/L). Secondary outcomes included the occurrence rate of platelet transfusion, and the association between thrombocytopenia, hemorrhage and platelet transfusion, assessed through mixed-effect models.This was a sub-study of a multicenter  patients developed a thrombocytopenia, of which a significant part severe . One or more platelet transfusions were administered in 226 patients (54%), whereas 207 patients (49%) suffered a hemorrhagic event during VA ECMO. In non-bleeding patients, still one in three patients received a platelet transfusion. The strongest association to receive a platelet transfusion was found in the presence of severe thrombocytopenia . After including an interaction term of hemorrhage and thrombocytopenia, this even increased up to an OR of 110 (95% CI 34\u2013360).Of the 419 patients included, median platelet count at admission was 179\u00b710Thrombocytopenia has a higher occurrence than is currently recognized. Severe thrombocytopenia is strongly associated with platelet transfusion. Future studies should focus on the etiology of severe thrombocytopenia during ECMO, as well as identifying indications and platelet thresholds for transfusion in the absence of bleeding.Trial registration: This study was registered at the Netherlands Trial Registry at February 26th, 2020 with number NL8413 and can currently be found at https://trialsearch.who.int/Trial2.aspx?TrialID=NL8413.The online version contains supplementary material available at 10.1186/s13054-023-04612-5. To compare the different subgroups of thrombocytopenia severity, as well as for the subgroup analyses comparing either bleeding versus non-bleeding or transfused versus non-transfused patients, either a Mann\u2013Whitney To assess the association between thrombocytopenia, hemorrhage and platelet transfusion, mixed-effects models were used. Platelet transfusion was considered the chronological effect of either hemorrhage or thrombocytopenia, and thus used as the dependent outcome. To define the unadjusted effect, a reduced model was applied to assess the effect of either hemorrhage or severity of thrombocytopenia on receiving a platelet transfusion, solely correcting for ECMO duration and center. To further adjust for confounding, an advanced model was developed using center and duration as random effect, as well as an a priori defined set of confounders as fixed effects. These confounders were identified in the literature and included: sex, age, history of cardiovascular disease, SOFA score at day of ECMO initiation, cannulation site , daily aPTT, a thrombotic complication during ECMO and anticoagulation type (reference: unfractionated heparin). In addition to this advanced model, a final model was created including an interaction term combining hemorrhage and thrombocytopenia. This interaction term included thrombocytopenia with hemorrhage, thrombocytopenia without hemorrhage, and hemorrhage without thrombocytopenia. Odds ratios (OR) were presented with their 95% confidence interval (95% CI).Missing data were assessed after data collection. Patterns in missing data were analyzed  and variables containing more than 50% missing values were excluded from the dataset, which did not result in the exclusion of any of the pre-defined covariates of interest. Missing data were not imputed, since the employed mixed-effect models use maximum-likelihood estimation to handle missing data.9/L [119\u2013253\u00b7109/L]. An overview of baseline characteristics can be found in Table N\u2009=\u2009356, 86%), and over half of the patients was cannulated using a surgical technique . The nadir platelet count at the first day of ECMO showed an average 49\u00b7109/L [6\u2013102\u00b7109/L] decrease when compared to the last known value before cannulation.Of the total of 433 patients, 419 were eligible for further analyses , 50\u2013100\u00b7109/L in 159 patients (38%), and 100\u2013150\u00b7109/L in 60 patients (14%). An overview of the degree of thrombocytopenia is shown in Fig.\u00a09/L (29%). With the exception of the yet severely thrombocytopenia patients, the platelet course showed an initial decrease in platelet count, followed by a stabilization and further platelet recovery, as shown in Fig.\u00a0Nearly all patients developed a thrombocytopenia during ECMO 398/418, 95%), of which two third already during the first day . During ECMO, lowest platelet count was\u2009<\u200950\u00b7108, 95%, o9/L, patients that developed a severe thrombocytopenia during ECMO had a higher lactate level, higher SOFA score and lower platelet count before ECMO initiation . In addition, with a median of 6\u00a0days [4\u20139], the total days of ECMO were longest in patients with a severe thrombocytopenia during ECMO (adjusted P\u2009<\u20090.01). Unadjusted 28-day mortality was highest in patients that suffered a severe thrombocytopenia, significantly lower than patients with other degrees of thrombocytopenia (P\u2009<\u20090.01).In comparison with the patients with a nadir platelet count\u2009>\u2009100\u00b7109/L, whereas half of the centers listed a threshold of 50\u00b7109/L. One third of the centers stated different thresholds for bleeding versus non-bleeding patients, resulting in an increased threshold for platelet transfusion in the presence of bleeding. The proportion of patients that received a platelet transfusion ranged from 25 to 92% among the centers .Almost all centers used unfractionated heparin as standard anticoagulation, with the exception of one center that used bivalirudin in all patients. Platelet transfusion thresholds varied from 10 to 100\u00b710N\u2009=\u2009146/179, 82%). When the nadir platelet count during ECMO decreased, the number of days with a transfusion as well as the total amount transfused increased  received one or more platelet transfusions during ECMO, at a median of 2\u00a0days 1\u20133] of the total ECMO duration of 5\u00a0days [3\u20138]. Per day with a transfusion, one unit of platelets [1\u20132] was administered, adding up to a total of 4 units [2\u20137]. Of the patients that developed a severe thrombocytopenia, 4 out of 5 received a platelet transfusion during ECMO (\u20133 of theN\u2009=\u2009150); however, in non-bleeding patients, still 36% was transfused (N\u2009=\u200976). Majority of these patients (48/76) had a severe thrombocytopenia during ECMO. Bleeding patients, however, received platelets on more occasions and received higher amounts per transfusion event.Almost half of the patients 207/419, 49%) suffered a hemorrhagic complication during ECMO. Bleeding patients had a lower platelet count prior ECMO cannulation and during ECMO, when compared to those not bleeding . The OR for transfusion in the presence of hemorrhage decreased slightly to 2.39 (95% CI 1.9\u20133.0).Lastly, the model was performed using an interaction term of hemorrhage and thrombocytopenia , the occurrence of transfusion-related complications such as transfusion-associated circulatory overload or transfusion-related acute lung injury, as well as HIT. Lastly, no data on platelet function or drugs possibly influencing platelet function were collected, as protocols lack standard use of those tests, and differences between centers in the usage and type of function testing were considered too large.The occurrence of thrombocytopenia is considerably higher than currently recognized in VA ECMO. Severe thrombocytopenia is an important factor for platelet transfusion, also in the absence of bleeding. It is clear that future research in VA ECMO should focus on the etiology of thrombocytopenia, including the influence of medication during ECMO, as well as evaluating the indications and thresholds for platelet transfusion in bleeding and non-bleeding patients on VA ECMO.Additional file 1. Data definitions events under ECMO. Transfusion questionnaire. Flowchart. Figure S1. Platelet course. Table S1. Transfusion per center: platelet transfusion and occurrence rates. Table S2. Baseline demographics, stratified by transfusion status. Table S3. Platelet course, transfusion and complications, stratified by transfusion status. Table S4. Transfusion products as stratified per depth of thrombocytopenia. Table S5. Baseline demographics, stratified by hemorrhage. Table S6. Platelet course, transfusion and complications, stratified by hemorrhage. Table S7. Advanced model including interaction term."}
+{"text": "This is a peer-review report submitted for the paper \u201cFinding Potential Adverse Events in the Unstructured Text of Electronic Health Care Records: Development of the Shakespeare Method\u201dThis paper  investigWhat is the accuracy of the new method, the Shakespeare method, for identifying attributed and unattributed potential AEs? The previous study showed the process of this method in the literature . This paToo many keywords. I would suggest that the authors reduce some of the keywords.In the \u201cConclusions\u201d subsection, I would suggest the paragraphs be reorganized to improve them."}
+{"text": "Cholesterol plays an essential role in maintaining the rigidity of cell membranes and signal transduction. Various investigations confirmed empirically that the dysregulation of cholesterol homeostasis positively correlates with tumor progression. More specifically, recent studies suggested the distinct role of cholesterol in ovarian cancer cell proliferation, metastasis and chemoresistance. In this review, we summarize the current findings that suggest the contribution of cholesterol homeostasis dysregulation to ovarian cancer progression and resistance to anti-cancer agents. We also discuss the therapeutic implications of cholesterol-lowering drugs in ovarian cancer. Ovarian cancer (OC) is the fifth most lethal gynecologic malignancy and accounts for 4% of all cancer-related deaths in women . The medAs one of the main energy sources, lipids are involved in various extracellular and cellular biological functions, such as components of cell membranes, messengers and signaling molecules involved in generating and maintaining the biological functions of the body ,9,10,11.OC is among the cancers that are highly impacted by serum cholesterol levels . CholestThe two main sources of cholesterol are dietary (exogenous) and de novo, mainly by, the liver but also by adrenal gland, intestine, and gonads . In the In the de novo pathway, synthesis begins with the conversion of mitochondrial acetyl-CoA from hepatic mitochondria into 3-hydroxy-3-methylglutaryl (HMG)-CoA by 3-hydroxy-3-methylglutaryl-CoA synthase (HMGCS) and HMG-CoA reductase (HMGCR) . This enThe balance of this complex process of cholesterol metabolism is maintained through the \u201csterol-sensitive system\u201d . First, Aberrant cholesterol metabolism has been proposed as a metabolic hallmark in various cancers, including ovarian cancer . Given tThe aberrant expression of epidermal growth factor activates SCAP- mediated SREBP-2 activation, leading to elevated LDLR expression and consequently the cellular uptake of cholesterol ,46,47. TIn ovarian cancer, dysregulated lipid metabolism has been associated with cancer cell proliferation, tumor progression, metastasis and resistance against anti-cancer agents ,50. It iThe alteration of cholesterol metabolism has been shown to induce cell proliferation in ovarian cancer ,55. The Various studies have suggested that aberrant lipid metabolism including cholesterol metabolism positively correlates with metastasis in OC ,62,63. FAccumulating evidence suggests the involvement of aberrant lipid metabolism in inducing resistance to anti-cancer drugs . For exaSREBP2 is a transcription factor that mainly regulates the expression of the genes encoding enzymes associated with cholesterol metabolism, such as LDLR, FDFT1 and HMGCR . Zheng eLastly, emerging studies have suggested the involvement of cholesterol metabolites in ovarian cancer resistance . The uprThe alteration of cholesterol metabolism is associated with different signaling pathways, such as cholesterol biosynthesis and cholesterol uptake . One of Alternatively, since cholesterol biosynthesis requires high levels of energy and precursors, cancer cells mainly rely on cholesterol absorption . NiemannWe analyzed the correlation of the abovementioned genes with PFS only in patients with stage IV ovarian cancer, within a 60-month follow-up threshold. Importantly, we noticed significant improvements in PFS in OC patients who expressed lower levels of NPC1L1 . SimilarBased on evidence of the involvement of aberrant cholesterol metabolism in cancer progression, various researchers are focusing on repurposing existing hypocholesterolemic drugs to treat cancer. Different classes of cholesterol-lowering drugs are available, such as HMG-CoA reductase inhibitors (statins), anti-PCSK9  and NPC1L1 inhibitors (ezetimibe) . VariousAs mentioned above, the upregulation of PCSK9 is one of the main factors that elevates serum cholesterol. Both evolocumab and alirocumab are monoclonal antibodies against PCSK9, which inhibit PCSK9 from binding to the LDLR ,103,104.Ezetimibe is an inhibitor of NPC1L1 and blocks intestinal cholesterol absorption . EzetimiAltogether, these data suggest that the impact of hypocholesterolemic drugs in ovarian cancer is poorly understood and requires further investigation. It is worth mentioning that other lipid-lowering drug classes, such as fibrates, niacin, bempedoic acid, volanesorsen (anti-apo CIII) and sequestrants including cholestyramine, lodalis, Ethyl eicosapentaenoic acidomega 3, mipomersen and pelacarsen ,116,117 It has been reported that sexual steroid hormones play a pivotal role in tumor progression in various cancers . In OC, It Is Important to note that dysregulated lipid metabolism can also impact the production and metabolism of estrogen and progesterone, which may have implications for OC development . EstrogeLipids, particularly cholesterol, serve as building blocks for steroid hormones, including estrogen and progesterone. Cholesterol is converted into pregnenolone, which is then further metabolized to produce various steroid hormones, including estrogen and progesterone. Dysregulated lipid metabolism, such as increased cholesterol synthesis or altered cholesterol transport, can influence the availability of cholesterol for hormone synthesis . In OC, Understanding the interplay between dysregulated lipid metabolism, estrogen and progesterone production/metabolism and the response to hormone-based therapies is an active area of research in ovarian cancer that requires further investigation to elucidate any specific association mechanisms and identify potential therapeutic targets, in order to improve treatment outcomes in hormone-dependent ovarian cancer.As stated above, cholesterol is a critical precursor in the synthesis of various substances, including bile acids . The livThe relationship between lipid pathways and ovarian cancer is an area of ongoing research. Lipids play important roles in various cellular processes, including cell signaling, energy storage and membrane structure. The dysregulation of lipid metabolism has been implicated in the development and progression of various cancers, including ovarian cancer. It is important to note that the understanding of the specific mechanisms linking lipid pathways to ovarian carcinogenesis is still evolving, and more research is needed to fully elucidate their roles. The complex interplay between lipid metabolism and ovarian cancer involves multiple factors and pathways, and aberrant cholesterol metabolism appears to be an essential factor in ovarian cancer progression. Therefore, further studies are required to fully understand the impact of cholesterol on ovarian cancer and to elucidate the involved mechanistic pathways that modulate cell proliferation, metastasis and drug resistance. Understanding of the various target pathways will form the foundation of the investigation into hypocholesterolemic drugs and provide a pivotal therapeutic approach to slow the progression of ovarian cancer."}
+{"text": "Acute ST-segment elevation myocardial infarction (STEMI) is a serious cardiovascular disease. High thrombus burden is an independent risk factor for poor prognosis of acute myocardial infarction. However, there is no study on the correlation between soluble semaphorin 4D (sSema4D) level and high thrombus burden in patients with STEMI.This study aimed to investigate the relationship between sSema4D level and the thrombus burden of STEMI and further explore its effect on the main predictive value of the occurrence of major adverse cardiovascular events (MACE).From October 2020 to June 2021, 100 patients with STEMI diagnosed in our hospital\u2019s cardiology department were selected. According to the thrombolysis in myocardial infarction(TIMI)score, STEMI patients were divided into high thrombus burden groups (55 cases) and non-high thrombus burden groups (45 cases) 0.74 patients with stable coronary heart disease (CHD) were selected as stable CHD group, and 75 patients with negative coronary angiography (CAG) were selected as control group. Serum sSema4D levels were measured in 4 groups. The correlation between serum sSema4D and high-sensitivity C-reactive protein (hs-CRP) in patients with STEMI was analyzed. The relationship of serum sSema4D levels between the high and non-high thrombus burden group was evaluated. The effect of sSema4D levels on the occurrence of MACE was explored in one year after percutaneous coronary intervention.P\u2009<\u20090.05) with a correlation coefficient of 0.493. The sSema4D level was significantly higher in the high versus non-high thrombus burden group , P\u2009<\u20090.05). Moreover, MACE occurred in 19 cases in high thrombus burden group and 3 cases in non-high thrombus burden group. The results of Cox regression analysis showed that sSema4D was an independent predictor of MACE .Serum sSema4D level was positively correlated with hs-CRP level in STEMI patients (The sSema4D level is associated with coronary thrombus burden and is an independent risk factor for MACE. Acute myocardial infarction (AMI) is the most common acute and severe cardiovascular disease, and has become a main cause of sudden death in adults owing to its acute onset, rapid progression, and high mortality rate . The AmeSema4D/CD100 is a homodimeric protein that belongs to the semaphore family of axon-directing proteins. Members of the semaphore protein family recently received increasing attention because of their diverse functions in the immune system. Sema4D is the first semaphore with immune functions in T cell priming, antibody production, and intercellular adhesion play an important role . Sema4D The relationship between sSema4D levels and chronic heart failure and coronary heart disease had been reported worldwide , but no Participants A total of 100 patients with STEMI admitted to our hospital\u2019s cardiology department between October 2020 and June 2021 were included. According to the TIMI score , STEMI pThe inclusion criteria were as follows: 18\u201375 years of age; having met the diagnostic criteria of the \u201c2019 Chinese Society of Cardiology guidelines for the diagnosis and management of patients with ST-segment elevation myocardial infarction\u201d ; The exc0 level, no thrombus ; 1 level, the lumen development was blurred ; 2 level, the length of thrombus was 1 / 2 of the diameter of the blood vessel ; 3level, the length of thrombus was 1 / 2\u20132 times the diameter of blood vessel ; 4 level, thrombus length\u2009>\u20092 times vessel diameter ; 5 level, completely occluded. High thrombus load is usually defined as greater than 2 level.Stable CHD usually includes three conditions, namely chronic exertional angina, ischemic cardiomyopathy, and stable course phase after acute coronary syndrome (ACS) , 18.There was no obvious stenosis in the main coronary artery or its main branches.All patients were administered aspirin 300\u00a0mg and clopidogrel 300\u00a0mg preoperatively. Postoperatively, the patients were administered nitrates, aspirin, clopidogrel, atorvastatin, \u03b2-blockers, and angiotensin-converting enzyme inhibitors according to the modern treatment of STEMI \u3002.General patient information was also obtained. Immediately after admission, 2 mL of cubital venous blood was drawn from the patient, placed in a dry lithium heparin blood collection tube, and sent to our hospital\u2019s biochemical laboratory for testing. Before surgery, 3 mL of blood was drawn from all patients and placed in dry lithium heparin blood collection tubes, centrifuged at 3000 \u00d7 g for 15\u00a0min, and the supernatant was carefully collected in EP tubes with a pipette. The EP tubes were numbered, grouped together, and stored at -80\u00a0\u00b0C for testing. After the serum samples were collected uniformly, serum sSema4D levels were detected by a double-antibody one-step sandwich enzyme-linked immunosorbent assay  performed in strict accordance with the manufacturer\u2019s instructions. Other routine laboratory tests were sent to our hospital\u2019s biochemical laboratory .Two datasets (GSE34198 and GSE48060) from the Gene Expression Omnibus (GEO) database were retrieved. The raw data were downloaded as MINiML files. The microarray data were normalized by the normalize quantiles function of the preprocessCore package in R software. Box plots were drawn using the \u201csva\u201dR package, and the \u201cComBat\u201d package was used to draw the principal component analysis (PCA) plot. The \u201csva\u201d package  in R is All patients underwent electrocardiography immediately after admission and at 2\u00a0h postoperative. Each echocardiogram was reviewed in the outpatient clinic at 1 month postoperative and each patient followed up for more than 1 year after treatment. The occurrence of MACE and the time were recorded. MACE was considered as non-fatal ischemic or hemorrhagic stroke, non-fatal myocardial infarction, hospitalized unstable angina pectoris, unplanned revascularization including PCI and coronary artery bypass grafting (CABG), and cardiac death. Patients who died.of non-cardiovascular diseases were divided into loss to follow-up.P\u2009<\u20090.05 was considered statistically significant [The data were analyzed by SPSS version 23.0 statistical software. Quantitative normally distributed data are expressed as mean\u2009\u00b1\u2009standard deviation . There was no significant difference in serum sSema4D levels between the stable CHD group  , P\u2009>\u20090.05) and the control group  , P\u2009>\u20090.05) , P\u2009<\u20090.001), with a correlation coefficient of 0.493 , hs-CRP , P\u2009<\u20090.001) and sSema4D , P\u2009<\u20090.001) were higher in the high versus non-high thrombus burden group, while the left ventricular diastolic dysfunction (LVDd) , P\u2009<\u20090.05) and ST-segment.The univariate analysis revealed no statistically significant intergroup differences in age, sex, hypertension, diabetes, smoking history, liver function , renal function (creatinine level), low-density lipoprotein cholesterol, cardiac troponin I (cTNI), creatine kinase-MB (CK-MB), or other clinical indicators. The levels of total cholesterol, N-terminal precursor B-type brain natriuretic peptide (NT-proBNP), P\u2009<\u20090.001) were lower in the high versus non-high thrombus burden group , 7, P\u2009<\u20090.P\u2009<\u20090.001; Table\u00a0A multivariate binary logistic regression analysis showed that sSema4D was an independent risk factor for a high thrombus burden in STEMI (odds ratio(OR), 1.938; 95% confidence interval(CI), 1.442\u20132.604; P\u2009<\u20090.05); the AUC of sSema4D was 0.888, while the cutoff value was 19.968. At this time, its specificity of predicting STEMI thrombus burden was 78.2%, while the sensitivity was 93.3%.Receiver operating characteristic curves were drawn Fig.\u00a0; Table\u00a03P\u2009<\u20090.05). In the multivariate Cox regression analysis, we found that sSema4D and ejection fraction were independent factors affecting the prognosis of STEMI  , LVDd, ST-segment regression rate, and sSema4D level were prognostic factors in STEMI patients . We infer that sSema4D, like hs-CRP, mediates the inflammatory response and participates in the arteriosclerosis process of STEMI [Activation of the inflammatory response is an important pathological change in cardiovascular and cerebrovascular diseases, while atherosclerotic plaque formation and rupture are closely related to myocardial cell and post-stent injuries . It is pof STEMI . In manyof STEMI , anti-neof STEMI  and coroof STEMI , elevateof STEMI  while thof STEMI . Interleof STEMI , 41. Theof STEMI , 29.HoweIn the past, cTnI was known to have high sensitivity and specificity for the diagnosis and evaluation of slight myocardial injury. Nageh  and otheOn the one hand, hs-CRP can activate the complement system, release harmful terminal products, and damage the myocardium. However, as an inflammatory factor, it has a chemotactic effect on fibrin, which can lead to thrombosis . During P\u2009<\u20090.05), that is, the lower the expression level of sSema4D, the better the fallback situation and the better the prognosis of patients. However, Chinese studies on ST-segment regression after reperfusion therapy primarily used resting electrocardiograms, which are mostly intermittent recordings that cannot continuously observe ST-segment changes or provide long-term monitoring of arrhythmias; therefore, their usefulness is limited.In recent years, with increasing clinical research, reperfusion at the cellular level in patients after PCI can be identified with a change in the occlusion site; that is, the presence of a ST-segment change on the electrocardiogram can predict prognosis. In this experiment, the comparison of ST-segment regression rate values between the high and non-high thrombus burden groups of STEMI patients was investigated at 2\u00a0h postoperative. It was concluded that the fallback ratio of non-high thrombus burden group was larger , P\u2009<\u20090.001). This is because sSema4D is involved in biological processes such as cell migration and angiogenesis. Its expression level decreases with STEMI coronary remodeling, participates in the pathogenesis of myocardial ischemia, and can have a synergistic effect with serum inflammatory factors, which together lead to disease deterioration. Moreover, it is directly involved in the cardiac signaling pathway or indirectly involved in the myocardial-independent pathway. In this study, the shortest time to MACE events was 1 year, which is relatively limited; moreover, its sample size is small and unable to fully reflect the long-term occurrence of MACE. During the follow-up process, patients reported having a vague memory of the specific timing of the occurrence of MACE events; sSema4D may be more effective than myocardial markers in the STEMI with high thrombus load, providing new ideas for the prevention and treatment of STEMI with high thrombus load and becoming a potential therapeutic target. Thus, the results may exhibit deviations. In future studies, we will further investigate the specific mechanism by which sSema4D affects coronary thrombus burden.This study also found that sSema4D was an important risk factor for MACE after PCI that was closely related to prognosis ; moreover, it was an independent predictor of MACE in the Cox regression analysis .This study revealed that sSema4D in STEMI patients was significantly expressed in the high thrombus burden group, . It can be inferred that sSema4D is a potential therapeutic target for patients with a high thrombus burden and may serve as a new inflammatory marker for coronary atherosclerosis."
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