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+{"text": "Karstadt makes an important point regarding the need for more adequate long-term carcinogenicity testing of the artificial sweetener acesulfame K. The issues raised in her letter stimulated me to offer some additional considerations.a) agents that are slightly carcinogenic at any dose; b) low or extremely low doses of a carcinogenic agent of any kind; or c) mixtures of small doses of carcinogenic agents.As reported in a previous paper , one of Epidemiologic and experimental studies are fundamental in the identification and quantification of diffused carcinogenic risks, but they must be designed and conducted to be as powerful as possible with adequate methodology. In the case of experimental studies, it is not sufficient to follow the standard protocol used in ordinary experiments. Instead, it is necessary to conduct studies that may be defined as \u201cmega-experiments,\u201d using a vast number of animals  in order to express a marked difference in the variation of effects, and exposing the animals in all phases of development to allow the agent to express its full carcinogenic potential.It is based on this rationale that the European Ramazzini Foundation performed a mega-experiment on 1,800 rats and demonstrated that, in our experimental conditions, aspartame is a multipotential carcinogenic agent .The results of our study , 2006 atIn examining the raw data of our study, the First, the Second, if the statistically significant increased incidence of lymphomas/leukemias observed was indeed caused by an infected colony, one would expect to observe an increased incidence of lymphomas/leukemias not only in females but also in males. The Finally, in support of the hypothesis regarding the safety of aspartame, the Interestingly, the same scrutiny applied to our study has not been applied to a recent abstract published by The safety\u2014in particular, the noncarcinogenicity\u2014of today\u2019s most widely diffused artificial sweeteners and their blends is largely based on studies conducted decades ago. I second Karstadt\u2019s nomination of acesulfame K for further study; however, I add that it should be evaluated using a long-term mega-experiment."}
+{"text": "A recent study provided evidence that farmed rabbits in China harbor a novel hepatitis E virus (HEV) genotype. Although the rabbit HEV isolate had 77\u201379% nucleotide identity to the mammalian HEV genotypes 1 to 4, their genomic organization is very similar. Since rabbits are used widely experimentally, including as models of infection, we investigated whether they constitute an appropriate animal model for human HEV infection.Forty-two SPF rabbits were divided randomly into eleven groups and inoculated with six different isolates of rabbit HEV, two different doses of a second-passage rabbit HEV, and with genotype 1 and 4 HEV. Sera and feces were collected weekly after inoculation. HEV antigen, RNA, antibody and alanine aminotransferase in sera and HEV RNA in feces were detected. The liver samples were collected during necropsy subject to histopathological examination.Rabbits inoculated with rabbit HEV became infected with HEV, with viremia, fecal virus shedding and high serum levels of viral antigens, and developed hepatitis, with elevation of the liver enzyme, ALT. The severity of disease corresponded to the infectious dose , with the most severe hepatic disease caused by strain GDC54-18. However, only two of nine rabbits infected with HEV genotype 4, and none infected with genotype 1, developed hepatitis although six of nine rabbits inoculated with the genotype 1 HEV and in all rabbits inoculated with the genotype 4 HEV seroconverted to be positive for anti-HEV IgG antibody by 14 weeks post-inoculation.These data indicate that rabbits are an appropriate model for rabbit HEV infection but are not likely to be useful for the study of human HEV. The rabbit HEV infection of rabbits may provide an appropriate parallel animal model to study HEV pathogenesis. Hepatitis E virus (HEV) is transmitted between humans by the fecal-oral route and causes an acute, self-limiting hepatitis with high morbidity in young adults. Hepatitis E is an important public health concern in many developing countries in Asia and Africa and occurs sporadically in some industrialized countries. HEV is a small, non-enveloped virus with a single-stranded, positive-sense RNA genome of approximately 7.2 kb containing three open reading frames (ORFs), ORF1, ORF2 and ORF3, where ORF3 partially overlaps ORF2 Since the first non-human strain of HEV was isolated from a pig in the United States in 1997 and designated swine HEV As the presumed natural reservoir of HEV genotypes 3 and 4, pigs are able to amplify the virus but only develop subclinical disease in vitro culture system and a small animal model of HEV infection would be extremely valuable. This study investigated the experimental infection of specific pathogen-free (SPF) rabbits with HEV isolated from rabbits, to study systematically HEV pathogenesis and replication of rabbit HEV in its natural host. Furthermore, we also studied the infection of rabbits with HEV genotypes 1 and 4 to characterize the clinical course associated with cross-species HEV infection.At present, the advancement of HEV research has been hampered to by the lack of an effective The animal experimentation was approved by the Committee of Laboratory Animal Welfare and Ethics, National Institute for the Control of Pharmaceutical and Biological Products. The regulation for the review committee of laboratory animal welfare and ethics and protocol for the review on laboratory animal welfare and ethics, National Institute for the Control of Pharmaceutical and Biological Products, were followed.3, 4.69\u00d7101, 6.35\u00d7103, 2.59\u00d7104, 6.40\u00d7104, 6.93\u00d7104 and 6.74\u00d7106 genome equivalents per mL, respectively. The second-generation infectious stock of GDC54-18 was recovered from the feces of a rabbit experimentally-infected with the GDC54 HEV strain during the acute phase of infection. The virus suspensions were stored at \u221270\u00b0C for later use. The HEV RNA content of virus samples were determined prior to inoculation by reverse transcription-polymerase chain reaction (RT-PCR), as described previously The rabbit strain of HEV was originally recovered from serum samples from rabbits bred in two farms in Gansu province, China 6 genome equivalents of RH1 or 1.14\u00d7107 genome equivalents of RH4 Two strains of HEV isolated from humans were used for challenge. A genotype 4 strain (RH4) was collected from the feces of an individual with acute hepatitis E in Beijing, China and a genotype 1 strain (RH1) was collected from the feces of patients with sporadic hepatitis E in the Xinjiang Autonomous Region, China Forty-two 7-week old SPF rabbits weighing between 800 and 1000 g were obtained from the National Institute for the Control of Pharmaceutical and Biological Products, in Beijing, China. Prior to inoculation, all rabbits were confirmed negative for anti-rabbit HEV antibodies and antigens by an enzyme-linked immunosorbent assay (ELISA).ad libitum.Rabbits were divided randomly into eleven groups and inoculated intravenously with different doses of the various HEV isolates . Each raBlood samples and feces were collected weekly following virus challenge. Serum samples were tested for levels of the liver enzyme, alanine aminotransferase (ALT), HEV antigen and anti-HEV antibodies using standard methods, as described below. Serum and feces samples were tested weekly for HEV RNA by real-time PCR, as reported previously No gross pathological lesions were observed in the livers during necropsies. Liver samples collected at necropsy were fixed in 10% neutral buffered formalin and processed for routine histological examination. Each specimen was embedded in paraffin and cut into 3 \u00b5m serial sections, stained with hematoxylin\u2013eosin and subjected to histopathological examination by light microscopy.All rabbits were monitored weekly after inoculation for 14 weeks. ALT concentrations in sera were measured on the day of collection using an automated analyzer  according to the manufacturer's instructions. Biochemical evidence of hepatitis was recorded when the serum ALT concentration exceeded the baseline ALT level by more than two-fold, as defined by a peak ALT value that was equal to or greater than twice the pre-challenge values. These criteria were based on the correlation between the level of ALT increase and liver pathology described in a previous study using a non-human primate as an animal model of hepatitis All serum samples were tested for the presence of HEV antigen as described previously To determine the genomic dose of each inoculum, RNA was extracted from sera and fecal suspensions using a spin-column kit and the procedures of Kinghawk Biopharmaceutical Company  and evaluated by a real-time fluorescence RT-PCR assay with primers based on the conserved regions of HEV ORF3, as reported previously Clinical signs, such as diarrhea and jaundice, were not observed; however, some rabbits decreased food consumption for part of the duration of the study. Some rabbits suffered accidental death during blood collection by heart puncture.Prior to inoculation, all rabbits were seronegative for HEV. All control rabbits remained seronegative throughout the study. Anti-HEV IgG was detected in six of nine rabbits inoculated with the RH1 strain HEV (group RH1) and in eight of nine rabbits inoculated with the RH4 strain (group RH4) by 14 weeks post-inoculation (wpi) . All rabAll rabbits were negative for HEV antigen prior to inoculation and no control rabbits seroconverted throughout the study. The mean S/CO values of antigenemia differed between the rabbits inoculated with genotypes 1 and 4 and rabbits inoculated with the non-passaged rabbit HEV isolates (p<0.0001) . HEV antPre-inoculation samples taken from all rabbits were negative for HEV RNA and all uninoculated control rabbits remained negative throughout the experiment. Rabbit HEV RNA was detected variably in serum and fecal samples from rabbits in the various groups .Viremia was detected in sera from 1 to 3 wpi in two of nine RH4 group rabbits inoculated with genotype 4 HEV, with one animal intermittently positive thereafter. No viral RNA was detected in any rabbit of the RH1 group inoculated with HEV genotype 1. Viremia was detected only three times among any of the six groups of rabbits inoculated with the non-passaged rabbit HEV isolates, including one of two rabbits in the GDC9 group at 8 wpi, one of two in the RC12 group at 10 wpi and one of two in the GDC46 group at 10 wpi. For the 1GDC54-18 group rabbits, viremia was detected from 1 to 2 wpi and 5 to 6 wpi. HEV RNA was first detected in the serum of two of four rabbits in the 2GDC54-18 group at 1 wpi and was intermittently positive thereafter, up to 11 wpi.Fecal shedding of viruses was not detected in group RH1. For group RH4 rabbits, HEV RNA was first detected in feces from two of the nine rabbits at 2 wpi and one them was intermittently positive thereafter. All of the groups inoculated with the non-passaged rabbit HEV isolates were intermittently positive for HEV RNA in feces, except group RC12. For the group 1GDC54-18 rabbits, HEV RNA was shed in the feces first at 1 wpi and lasted until 4 to 11 wpi. Three of the four group 2GDC54-18 rabbits were HEV RNA positive at 4 wpi and from 6 to 8 wpi.The viruses recovered from selected experimentally-infected rabbits were sequenced to confirm that they were derived from the original inoculum, with nucleotide identity 99\u2013100% to the original inoculum.No instant elevations in serum ALT-levels were observed in the four control rabbits, nine rabbits each in groups RH1 and RH4 during the entire study . HoweverNo statistical data on the pathological signs of HEV infection in the liver can be reported because liver biopsies were not performed and no rabbits were regularly necropsied during the entire study. Some rabbits that showed elevated ALT levels when they were necropsied at the end of the study or died due accidentally and all control rabbits were investigated for liver pathology. Multifocal lymphohistiocytic infiltrates were distributed irregularly in the liver of a rabbit from group RH4  and locaThe lack of an effective cell culture system and a small animal model has hampered the progress of HEV research. Experimental infections of pigs and non-human primates with HEV have been well documented 3 copies/reaction 1 genome equivalents) seroconverted at 4 wpi without antigenemia or HEV RNA in feces. In contrast, rabbits in the GDC54 inoculated group developed more severe clinical symptoms following challenge with a larger virus inoculum . Nonetheless, we infected rabbits successfully with HEV isolated from rabbits and proved that they are a suitable animal model for the study of rabbit HEV.Six original isolates of rabbit HEV were used to infect 12 SPF rabbits. Based on a 304 bp region of ORF2, the rabbit HEV strains used in this study shared 84\u201399% identity and are 73\u201377, 70\u201376, 75\u201382, 71\u201377, and 53\u201365% identical to the corresponding regions of human HEV genotypes 1, 2, 3, 4, and avian HEV, respectively Because the virulence of the first-generation rabbit HEV was limited, we used virus recovered from the feces of rabbits inoculated with the GDC54 strain of HEV as a second passage inoculum for the further studies. The second passage HEV GDC54-18 virus inoculum was used to infect another eight rabbits, using two different doses. As expected, challenging with higher doses of virus in terms of genome equivalents caused a more severe hepatitis. Serum ALT levels, which are indicative of recent liver damage and suggestive of an acute infection, peaked at 10 wpi  with a v7 genome equivalents) of all test animals, seven rabbits seroconverted at 14 wpi. In summary, seroconversion, viremia, fecal virus shedding or serum liver enzyme elevation were observed in two of nine RH4 group rabbits, suggesting evidence of virus infection and hepatitis. The results indicate that HEV genotype 4 have the ability to cross species and infect rabbits. As we have reported previously, genotype 1 and 4 HEV can infect monkeys and all animals were infected and developed severe hepatitis Another objective of this study was to determine whether human HEV could infect rabbits. So far, no evidence has proved that human genotypes 1 and 2 HEV can infect pigs, although genotypes 3 and 4 can infect both human and pigs. In addition to pigs, many other animals have been shown to be susceptible to infection with genotypes 3 and 4 HEV and can serve as a reservoir of HEV that can amplify the virus and transmit to humans In summary, we infected rabbits successfully with rabbit HEV and the clinical picture in these animals was similar to HEV infection of monkeys. It seems that rabbits constitute a valuable model to study the mechanisms of HEV replication and pathogenesis. These studies indicate the rabbit model has potential for the development of HEV vaccines and clinical therapies for the treatment of infection."}
+{"text": "To the editor: Hepatitis E virus (HEV) is a positive-sense single-stranded RNA virus  (Rattus norvegicus), black (Rattus rattus), and cotton (Sigmodon hispidus) rats, by using ELISA with antigens derived from G1 HEV. These results suggest that HEV or HEV-like virus infections occur in wild rats. However, the virus genome has not been detected, and the source of the infection was confirmed in few cases; thus far, it is not clear whether the anti-HEV IgG was induced by HEV or other HEV-like viruses. The detection of a partial genome of G1 HEV from wild rats in Nepal was reported in 2002  and 1 derived from the supernatant of a hepatocarcinoma cell line, PLC/PRF/5, that was injected with the pig specimen. The infectivity of these samples was confirmed by experimental infections of cynomolgus monkeys  were used in this study. These rats, which are bred to be immunodeficient, are known to be susceptible to rat HEV, but it is unknown if they are susceptible to other types of HEV. All rats were negative for G3 HEV RNA and anti-HEV antibodies, as determined by nested reverse transcription PCR (4 copies of G3 HEV; the 3 rats in group 2 were injected with the cell culture supernatant sample, which contained 4 \u00d7 106 copies of G3 HEV. Serum samples were collected weekly for examination of HEV RNA and anti-HEV IgG and IgM and were also used to determine alanine aminotransferase values. Fecal samples were collected every 3 days to detect HEV RNA. The animals were humanly killed by exsanguination 91 days postinjection, liver tissues were collected, and a 10% tissue suspension was prepared as described (The 6 rats were randomly assigned to 2 groups, injected intravenously with 500 \u00b5L of an HEV sample suspension through the tail vein, and monitored for 3 months. The 3 rats in group 1 were injected with the sample derived from pig feces, which contained 5 \u00d7 10For groups 1 and 2, all serum samples collected 1\u201313 weeks postinjection were negative for HEV RNA and anti-HEV IgG and IgM. HEV RNA also was not detected in fecal samples or liver tissues. Alanine aminotransferase elevation was not observed in any serum samples.In conclusion, even by using samples with high titers of HEV RNA in injection experiments, we were unable to cause infection with G3 HEV in immunodeficient nude rats. We found no evidence that rats are susceptible to infection with G3 HEV."}
+{"text": "In this work, we propose a multilevel and multiparametric approach in order to model the growth and progression of oral squamous cell carcinoma (OSCC) after remission. OSCC constitutes the major neoplasm of the head and neck region, exhibiting a quite aggressive nature, often leading to unfavorable prognosis.We formulate a Decision Support System assembling a multitude of heterogeneous data sources , aiming to capture all manifestations of the disease. Our primary aim is to identify the factors that dictate OSCC progression and subsequently predict potential relapses of the disease. The discrimination potential of each source of data is initially explored separately, and afterwards the individual predictions are combined to yield a consensus decision achieving complete discrimination between patients with and without a disease relapse. Moreover, we collect and analyze gene expression data from circulating blood cells throughout the follow-up period in consecutive time-slices, in order to model the temporal dimension of the disease. For this purpose a Dynamic Bayesian Network (DBN) is employed which is able to capture in a transparent manner the underlying mechanism dictating the disease evolvement, and employ it for monitoring the status and prognosis of the patients after remission.By feeding as input to the DBN data from the baseline visit we achieve accuracy of 86%, which is further improved to complete discrimination when data from the first follow-up visit are also employed.Knowing in advance the progression of the disease, i.e. identifying groups of patients with higher/lower risk of reoccurrence, we are able to determine the subsequent treatment protocol in a more personalized manner. The research for this paper has been conducted in compliance with the Helsinki Declaration; the protocol of the study has been edited in compliance to the Good Clinical Practice and approved by the following Ethical Committees: Comitato Etico Unico per la Provincia di Parma and Ethical Committee of Centro M\u00e9dico Oncol\u00f3gico MD Anderson Espa\u00f1a. Written, informed consent was obtained from each patient prior to study participation. All patients have been diagnosed with OSCC and have reached remission, after successful therapeutic intervention . Thereafter, we collect clinical, imaging and gene expression data, both from the primary tumor as well as from circulating blood cells, at the baseline state of the patient. The clinical data contain standard measurements and laboratory markers from the patient's medical record as well as pathology and risk factor data referring to the organism as a whole  of missing values are omitted from our analysis, whereas the values of the features with less percentage of missing values are imputed with the modes and means, in the case of nominal and numeric features, respectively. Another important issue present in our dataset is that the enrolled patients are unevenly distributed in the classes of relapsers and non-relapsers, resulting in considerable class imbalance. For this purpose, we employ the Synthetic Minority Oversampling Technique (SMOTE) , which uEspecially for the case of genomic data (both tissue and blood ones), the initial feature vector consisting of 45,015 gene expression values, is subject to certain steps in order to enhance the quality of the raw input data, that is subsequently employed for data analysis; to this end, redundant and control genes are removed, as well as genes with low data quality or high percentage of missing values. The outcome is a set of 33,491 high quality genes that are further analyzed in order to procure a limited subset of genes that are mostly differentially expressed between relapsers and non-relapsers. For this purpose we employ the Significance Analysis of Microarrays (SAM) algorithm , which pAs soon as the feature vectors from each source of data are assembled, we either feed them directly as input to the classification algorithms, or employ a feature selection algorithm in order to discard redundant or correlated features and maintain a more informative subset, thus, facilitating the classification task as well. For this purpose two feature selection algorithms have been employed, namely the Correlation-based Feature Subset selection (CFS)  and the Next, we examine the performance of six popular classification algorithms  towards For evaluation purposes, we employ two techniques, namely 10-fold cross validation and leave-one-patient-out. During 10-fold cross validation the dataset is split into 10 stratified sets, whereby 9/10 are used for training and the remaining 1/10 is used for testing; after a full rotation, the results over the 10 testing sets are averaged in order to procure the overall performance of the algorithm. The leave-one-patient-out technique is quite similar to 10-fold cross validation, where the number of folds is equal to the number of patients in the dataset. Specifically all patients but one are used for training and the remaining one is used for testing in a round-robin manner. The evaluation metrics used to compare the employed classification schemes are: sensitivity, specificity, accuracy, the Kappa statistic, as well as the Area Under Curve (AUC), which is an evaluation index obtained from the Receiver Operating Curve (ROC) analysis.In the second part of our analysis (i.e. Disease Evolution Monitoring), we aim to predict the probability of a patient to develop a relapse during the follow-up period; hence, we introduce the time dimension and estimate the approximate timing that a relapse is more likely to occur. For this purpose, we employ time-course gene expression data, extracted at predefined time-intervals during the follow-up period from circulating blood cells. Data from 23 patients are analyzed, out of which 11 have already been diagnosed with a disease relapse and the remaining 12 are disease free. The flowchart of the Disease Evolution Monitoring analysis is depicted in Figure Initially, the gene expression data obtained from 45,015 genes are filtered in order to omit duplicate and control genes as well as the ones that are of low quality; it should be noted that all microarray experiments have been conducted using the same platform, the same array design and the same feature extraction software version in order to exclude unwanted data perturbations other than biological variability. Next, we identify the genes that are mostly differentially expressed between relapsers and non-relapsers; in addition, we extract a personalized gene subset  aiming to capture patient-specific perturbations of the disease progression within its molecular basis. Both the aforementioned inputs are fed as input to a Dynamic Bayesian Network (DBN)  in orderAfter applying some basic filtering steps upon the gene expression data , we are left with a set of 33,491 genes, that are fed to the next steps of our analysis. The SAM algorithm  is subseIn addition, we extract a patient-specific genetic indicator denoting the progression of the disease for a specific patient; for each patient we compare the gene expression values before treatment (cancerous profile) and during the first stages of remission (cancer-free profile). The outcome is a limited set of differentially expressed genes representative for each patient, allowing for personalized modeling of the disease evolvement. The expression of these genes from all succeeding follow-up visits is compared in turn with the cancerous and the cancer-free profile, calculating the correlation and the Euclidean distance; these metrics provide, respectively, a qualitative and quantitative measure of the patient's prognosis. In the case of the Euclidean distance a weighted variant is employed which takes into account the significance of each gene in the personalized genetic signature. This weighting factor is proportional to the differential expression of each gene between the cancerous and the cancer-free profile.In the next step of our analysis, we employ a DBN aiming to identify potential relapses of the disease, as well as the approximate time-frame of the relapse. DBNs are basically temporal extensions of Bayesian Networks, as shown in the provisional DBN architecture of Figure In order to estimate the best performing DBN architecture, i.e. the dependencies among the employed variables within the same time-slice (intra-slice dependencies), as well as across successive time-slices (inter-slice dependencies), we employ two search algorithms, namely the Greedy and the Simulated Annealing. Based on the trained model, we are able to conjecture the values of all variables from future time-slices, including of course the probability of reoccurrence, by using the junction tree algorithm for inferencing from the DBN. In order to formulate and fine-tune the DBN network, as well as for inferencing from the trained model, the miniTUBA system has been used . Due to In the sections that follow, we present the results obtained in our effort to stratify the patients in high and low risk groups based on the reoccurrence probability. Initially, each source of data is treated independently and subsequently all single-classifier decisions are combined using a multi-type classifier. In the first step, each source of data is subject to preprocessing, and then the resulting feature vector is fed to the target classifier either unaltered or after applying certain feature selection algorithms, i.e. CFS and wrapper. As for the actual classification task, we have examined the performance of the following classifiers: Bayesian Networks, Naive Bayes, Artificial Neural Networks, Support Vector Machines, Decision Trees and Random Forests.Table The employment of the CFS algorithm for feature selection maintains the following features as most discriminatory: smoker, tumor thickness, lymphoplasmacytic rection, perineural invasion, num mitoses HPF, surgical margins, p53 stain and N staging. The employment of the wrapper algorithm has pinpointed the following features for each classification algorithm: BN: ecog status, cholesterol, grade differentiation and N staging; NB: allergies, cholesterol, depth invasion, lympphoplasmacytic rection and N staging; ANN: ecog status, cholesterol, depth of invasion and N staging; SVM: ecog status, smoking duration and N staging; DT: depth of invasion, p16ink4a stain and N staging; RF: quantity of cigarettes, galvanic current, eating habits, BMI, depth invasion and N staging.In Table The employment of the CFS algorithm for feature selection resulted in a refined feature vector containing the following features: extra-tumor spreading, extra-nodal spreading, texture (lymph node), site (lymph node), side (lymph node) and number of lymph nodes. Afterwards, the employment of the wrapper feature selection algorithm has resulted in the following lists of features for each classification algorithm: BN: extra-tumor spreading and site (lymph node); NB: extra-tumor spreading and site (lymph node); ANN: extra-tumor spreading and site (lymph node); SVM: extra-tumor spreading, floor of the mouth invasion and site (lymph node); DT: extra-tumor spreading, perineural infiltration (tumor), bone infiltration (tumor) and site (lymph node); RF: extra-tumor spreading and site (lymph node).Using the gene expression values acquired from the cancerous tissue, we aim to identify those genes that are mostly differentially expressed between relapsers and non-relapsers; for this purpose we employ the SAM algorithm and set the fold-change threshold to 1.8, thus, yielding a set of 9 genes, shown in Table Subsequently, the retained genes are employed as part of a series of classification schemes in order to discriminate the patients into high and low risk groups, in terms of reoccurrence probability. The results obtained with all employed classification schemes are shown in Table The utilization of the CFS algorithm for feature selection, subsequently retains the following features: TCAM, SOD2, AMDHD1, AY358224, PHACTR1, AK026836 and RPRM. Afterwards, the employment of the wrapper algorithm coupled with a different target classification algorithm each time has retained the following features: BN: TCAM1, AMDHD1, AY358224, PHACTR1 and RPRM; NB: TCAM1, SOD2, AMDHD1, PHACTR1 and RPRM; ANN: TCAM1, SOD2, AMDHD1, PHACTR1, SLC5A12, AK026836 and RPRM; SVM: TCAM1, SOD2, FCAR, AMDHD1, AY358224 and PHACTR1; DT: AMDHD1, AY358224, PHACTR1, AK026836 and RPRM; RF: SOD2, FCAR, AMDHD1, AY358224, PHACTR1 and RPRM.In the literature, there have been identified several genes that are descriptive of the development and the prognosis of oral cancer, therefore, we have additionally integrated these genes with the ones identified in our work, in order to gain enhanced generalization capability and explore the overall discriminative potential of the resulting unified gene set. The literature derived set consists of 28 genes ,24 whoseIt is noteworthy that even though both gene sets perform quite satisfactory, the union of the two gene sets significantly ameliorates the obtained results, enhancing the generalization capability of the classification procedure.Same as with the tissue genomic data, for the blood genomic data as well, initially we employ the SAM algorithm in order to identify the genes that are mostly differentially expressed between relapsers and non-relapsers; the obtained gene subset is shown in Table The discriminative potential of the gene subset is evaluated either by providing it directly as input to a series of classification algorithms, or by applying certain feature selection algorithms, prior to the classification task. The results obtained when the 11 genes are used for classification without performing feature selection are shown in Table The employment of the CFS algorithm maintains the following genes as most significant: A_24_P221960, THC2399272, BM683433, OXCT2, A_24_P230388, A_32_P57247 and AL566369. Next, the employment of the wrapper algorithm maintains the genes that are specifically tuned to achieve the best performance using a specific classification algorithm. Those genes are for BN: A_24_P221960, THC2399272, BM683433 and OXCT2; for NB: THC2410448, A_24_P221960, BM683433, OXCT2, A_32_P57247 and A_24_P942151; for ANN: A_24_P221960, THC2399272 and CN391963; for SVM: THC2410448, OXCT2 and A_24_P942151; for DT: A_24_P221960; for RF: A_24_P221960 and THC2399272.Next, we employ the best performing classification schemes identified using each source of data separately, and merge the individual predictions using a weighted majority voting algorithm, achieving perfect discrimination between patients with and without disease relapse. Table For the second part of our analysis, we employ gene expression values extracted from blood samples which have been collected in predefined time-intervals during the follow-up period. After the raw values  have been accordingly preprocessed, concluding in 33,491 high quality genes, we employ the SAM algorithm for time-course gene expression data in order to identify the genes that exhibit the most differential expression pattern over the follow-up. These retained genes Table  are subsThe aforementioned genes serve as input in order to formulate the architecture of the DBN, used subsequently for monitoring the evolvement of the disease over the follow-up. Specifically, we search among thousands of possible architectures using the Simulated Annealing and the Greedy algorithm in order to identify the best-performing ones, i.e. the ones yielding the highest results. It should be noted that no restrictions have been imposed on the nodes of the network, in order to obtain the network whose interactions yield the highest performance. In Figure For the evaluation of the trained DBN model, we have employed the leave-one-patient out technique, and based on the individual predictions we have calculated the overall results, that are shown in Table In this work we have collected and analyzed a broad set of heterogeneous data from various sources, i.e. clinical, imaging tissue genomic and blood genomic, in order to capture the progression of the disease during remission and predict potential disease relapses. For this purpose a twofold analysis has been performed: i) Baseline Data Analysis which employs solely data obtained at the baseline visit aiming to identify a potential disease relapse and ii) Disease Evolution Monitoring, that explores gene expression from genes obtained at predefined intervals over the follow-up coupled with a personalized genetic signature in order to capture the temporal progression of the disease and hence predict the approximate timing of a potential relapse.The Baseline Data Analysis outcome is compared with the works presented in the literature review, as shown in Table We observe that the currently proposed methodology exhibits superior results compared to the other methods presented in the literature, nevertheless, direct comparison cannot be performed since different datasets have been employed in each case. It should be noted that the currently employed dataset contains a considerable number of patients, i.e. 86, compared to the other ones in the literature. A significant advantage of the current work is the multitude of data that has been employed  whereby a separate classifier has been trained from each source of data, as well as an overall one that combines the individual classifiers. On the other hand, the other methodologies in the literature exploit mostly genomic data coupled with information from the clinical profile of the patients.In addition, the modular architecture of the current work allows for inspecting the \"verdict\" based on each source of data separately and hence conjecturing about the contribution and validity of each type of data. Of course an overall decision is calculated which weighs the individual predictions yielding a more accurate consensus outcome. Alternatively, the combination of all sources of data could be achieved by pooling all data in a single dataset (i.e. the bag of features approach); however, with this approach the intersection of patients across all sources of data would have to be used, and given the uneven distribution of patients this would conclude in a rather limited dataset. In terms of validation, the current work has been evaluated using 10-fold cross validation and the leave-one-patient-out technique, in order to assess thoroughly the achieved performance. The results obtained with the leave-one-patient-out technique are in complete accordance with the ones obtained using 10-fold cross validation, therefore, the former ones are not included in the manuscript.The best performing classification schemes based on each source of data are summarized in Table The features maintained from each source of data can be inspected independently, however, it should be noted that it is their combination that yields the results presented previously. Based on the clinical data, the best performing classification scheme involves a Decision Tree where the initial input has been stripped off from redundant features using the wrapper feature selection algorithm, maintaining the following features as most significant: depth of invasion, p16ink4a stain and N staging. Especially the depth of invasion and the N staging constitute major factors affecting the disease prognosis. For the case of imaging data the Naive Bayes classifier coupled with the wrapper algorithm for feature selection employs the combination of extra-tumor spreading and (lymph node) site, thus, achieving the highest performance. Next, we move on to the genomic data (from tissue and blood) where the best performing gene subsets are shown in Table Both for the clinical and the imaging input vector, the employment of the wrapper algorithm yields the feature subset with the highest discriminating ability; in accordance with our findings, the wrapper algorithm has been reported in the literature to often outperform other feature selection algorithms due to the fact that it is tuned to the target classification algorithm . HoweverAn important aspect of the current work that should be highlighted, is the fact that for the tissue genomic data, we have merged the gene subset identified using the current dataset with a set of genes pinpointed in the literature as highly correlated and descriptive of oral cancer reoccurrence. As shown in the Results section, the union of the two gene subsets achieves superior performance compared to the individual sets. Besides the performance amelioration, it is very important that in this manner we consolidate information from other data resources, thus, achieving enhanced generalization capability.As for the Disease Evolution Monitoring, a substantial advancement is the incorporation of the time dimension, thus, capturing the temporal nature of the disease. This is particularly interesting from a medical point of view, since we are able to conjecture the approximate timing that a potential relapse is more likely to occur. Moreover, the ability of DBNs to capture time-varying parameters, resembles better the actual disease progression and facilitates the modeling of the evolvement over the follow-up period. The employment of a DBN which features a transparent architecture allows for inspecting the interplay among the involved parameters and therefore, reasoning is provided for each decision. DBNs have been elsewhere used in other domains in order to capture time-varying events, yielding quite satisfactory and accurate results.As shown in the DBN architecture depicted in Figure The relatively small association of the extracted genes with literature findings related to cancer and more specifically oral cancer, was expected since blood gene expression has been scarcely studied, and therefore few and sporadic references exist in the literature. However, given the fact that even a subset of the currently extracted genes have been identified in cancer related pathways, is quite encouraging and further supports the proof-of-concept that is intended with the approach of Disease Evolution Monitoring in the current work. In any case, the relatively small set of patients, compared to the initially enormous number of genes was a hindrance in the first place, yet the preliminary results obtained using the leave-one-patient-out technique are quite satisfactory.In this work we have presented a multiscale and multiparametric approach for modeling the progression of oral cancer during remission. Specifically, our approach consist of two main analyses, i) Baseline Data Analysis where clinical, imaging, tissue genomic and blood genomic data were employed in order to predict a potential disease relapse and ii) Disease Evolution Monitoring aiming to capture the progression of the disease based on gene expression data acquired from circulating blood cells, and subsequently exploit this information in order to predict if and roughly when a relapse is more likely to appear. This information can be used to stratify the patients into high and low risk groups according to the relapse probability; hence, the treatment protocol can be either intensified or relaxed accordingly. Moreover, it is very important to unify our study with findings from similar studies alongside with further verification of the results in order to achieve enhanced generalization capability and conjecture meaningful and solidified corollaries.The proposed approach encompasses heterogeneous sources of data that are expected to assess the disease status from several aspects and therefore, can be proven very helpful towards studying complex diseases, such as cancer.The authors declare that they have no competing interests.KPE and YG conceived, designed and implemented the study, DIF supervised the study and provided substantial advice and guidance during all phases. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6947/12/136/prepub"}
+{"text": "Oral verrucous carcinoma (OVC) is a low grade variant of oral squamous cell carcinoma, and oral verrucous hyperplasia (OVH) is a benign lesion without malignant features. However, pathologists are sometimes presented with borderline lesions and are indecisive as to diagnose them as benign or malignant. Thus, these lesions are tentatively termed oral verrucous lesions (OVLs). HuR is an ARE mRNA-binding protein, normally localized in the nucleus but cytoplasmic exportation is frequently observed in cancer cells. The present study aimed to elucidate whether expression of the HuR protein facilitates the diagnosis of true malignant lesions. Clinicopathological features were evaluated, and immunohistochemical analysis for p53, Ki67 and HuR proteins was performed in 48 cases of OVH, OVC and OVL, and the outcomes were correlated using appropriate statistical analysis. The association of these three proteins in relation to malignant transformation was analyzed after a 3-year follow-up of 25 OVL cases. The basal characteristics  of all cases had no significant association with the types of lesions. Gingiva (39.4%) was the common site for all lesions. Distribution of the examined proteins had a significant association with the lesions. As compared with the OVLs, the number of immunostained-positive cells was significantly higher in the OVCs and lower in the OVH cases. During follow-up, 24% of the OVLs underwent malignant transformation for which high HuR expression and a diffuse staining pattern in the epithelium were observed. Taken together, the high degree of HuR expression with diffuse staining pattern in the epithelium may be an effective diagnostic tool that determines the potential of OVLs for malignant transformation. Verrucous carcinoma was first described by Ackerman as a variant of squamous cell carcinoma of the oral cavity . ClinicaHuR is a ubiquitously expressed mRNA-binding protein. Intracellularly, HuR is localized predominantly in the nucleus but it shuttles between the nucleus and the cytoplasm. The export of HuR is mediated by its association with transportin 1 (Trn1) and transportin 2 (Trn 2) via the shuttling sequence termed\u2019HNS\u2019 in the hinge region and by its association with pp32, APRIL and SET \u03b1/\u03b2 protein, which includes the nuclear export signal recognized by the export receptor chromosome maintenance region 1 (CRM1) \u20137. AU-riThe present study examined 6 cases of OVH, 17 cases of OVC and 25 OVLs. All samples were collected from Hokkaido University Hospital between 1985 and 2010. Diagnosis was based on histological examination. Cases with epithelial verrucous hyperplasia without cellular dysplasia were diagnosed as OVH whereas OVCs were characterized by verrucous proliferation of squamous epithelium with wide and elongated rete ridges exhibiting a pushing border invasion into the underlying connective tissue with epithelial dysplasia. However, for OVLs we used the following diagnostic criteria: epithelial hyperplasia with hyperkeratosis and a verrucous surface, non-invasion of the hyperplastic epithelium into the lamina propria compared with adjacent normal mucosal epithelium, and lesions with varying degrees of epithelial dysplasia. The cases with OVLs were further followed up for 3 years to evaluate their potential for malignant transformation. Detailed demographic and clinical data for the cases of OVH, OVC and OVLs are listed in 2O2 for 5 min followed by blocking solution [1% BSA in phosphated-buffered saline (PBS)] for 30 min. The immunohistochemical detection of HuR was carried out using anti-HuR monoclonal antibodies  in PBS in blocking solution in a humidified chamber at 4\u00b0C overnight. The sections were then subjected to Simple Stain Max PO (M)  at 37\u00b0C for 30 min. Careful rinses were performed with several changes of PBS between the stages of the procedure. Visualization was carried out using the ChemMate EnVision kit/HRP , and the sections were counterstained with hematoxylin. The same tissues were immunostained with monoclonal antibodies against p53 and Ki-67 (1:100). The stained slides were examined by light microscopy, and the positive cell distribution in the different levels of the epithelium was observed. As shown in The paraffin blocks of the specimens were cut in 5-\u03bcm sections and examined immunohistochemically. Sections were deparaffinized in xylene, rehydrated in graded alcohol and subjected to antigen retrieval by heat treatment in Tris-EDTA (TE) buffer. To inhibit endogenous peroxidase activity, the slides were then immersed in 3% HNuclear staining was considered as positive for p53 and Ki67 proteins, whereas cytoplasmic staining was considered as positive for HuR. The sections were initially scanned at low power, at least 3 high-power fields were then chosen randomly, and at least 1,000 cells were counted in level 1 of the epithelium for each case. The labeling indices (LIs) of cytoplasmic or nuclear staining of each antibody were defined as a ratio of immunostaining-positive cells to the total number of cells counted. Normal oral epithelium (NE) and oral squamous cell carcinoma (SCC) were used as negative and positive controls, respectively.The Chi-square test was applied to compare the association of the patient basal characteristics  and the distribution pattern of the target proteins  in all lesions . The mean LIs of the three examined proteins were compared by the Student\u2019s paired t-test. The association between the expression of proteins and malignant transformation of 25 OVLs was also assessed by Chi-square analysis. SPSS for Windows release 17.0 (SPSS) was used for statistical analysis. A value of P<0.05 was considered to indicate a statistically significant result.The basal characteristics  of 48 patients and their association with 3 different types of oral lesions  are presented in The mean LI (percentage of positive staining) of the examined proteins was analyzed in all lesions. The mean LI of the OVLs was significantly greater than that of the OVHs and lower than that of the OVCs . The LI Six out of 25 (24%) OVLs underwent malignant transformation, and all of them had a high HuR LI (60%) and positive staining at level 2 (66.7%) of the epithelium . This asOVC and OVH are commonly diagnosed by morphological analysis, and expression levels of p53 and Ki67 are usually considered as biomarkers to facilitate the diagnosis of OVC. The p53 gene is reported to be the most frequent target for genetic alterations leading to cancer, and its mutation has been demonstrated in epithelial neoplasms. Although the p53 protein is expressed constitutively in all cells, it normally cannot be detected by immunohistochemical methods due to its short half-life and quick disintegration. Mutated p53 on the other hand, being more stable, accumulates in the cell and is readily detected by immunohistochemistry . OverexpKi67 is a proliferation-associated protein that is believed to play a critical role in regulation of the cell cycle. The protein is expressed strictly during the active parts of the cell cycle, including the G1, S and G2/M phases, but not in resting cells in the G0 phase \u201317. ImmuOral verrucous borderline lesions (OVLs) are a much greater diagnostic and therapeutic challenge. Lesions showing mild to moderate dysplasia with few positive signals for p53 and Ki67 make diagnosis difficult, and pathologists often hesitate to categorize them as benign or already transformed malignant lesions considering the possible extensive surgical exposure of the cancer patient, whereas benign lesions are treated conservatively. In the present study, OVLs showed mild to moderate epithelial dysplasia , and expIt would be a great achievement in cancer diagnosis if we could evaluate the histochemical parameters for the risk of carcinoma in OVLs. Cytoplasmic HuR expression has been implicated in the malignancy of colon, ovarian, breast, salivary gland, uterine, larynx and prostate cancers and has been postulated to contribute to the cancerous malignant phenotype \u201325. UndeAs expected, OVCs showed diffuse cytoplasmic expression of HuR  throughout the epithelium, whereas expression was rarely noted in the OVHs  and distributed in the lower one-third of the epithelium (level 1). Although HuR expression was restricted to level 1 in more than half (64%) of the OVLs, the expression was extended to the lower two-thirds (level 2) of the epithelium in the remaining 36% cases which is comparable to the expression of HuR in OVCs (35.3%). Notably, HuR expression in OVLs  was 2.4-fold lower than that in the OVCs, whereas in OVHs the expression was 42.7-fold lower. This considerable difference between OVHs and OVLs compared to OVCs provide additional information regarding the role of HuR in cancer development of OVL cases.We further followed up the OVLs to understand the risk of malignant transformation. All 25 OVL cases were divided into high and low group based on their LIs for the three examined protein, and their association with cancer development was assessed. As shown in"}
+{"text": "Woven coronary artery is relatively rare and can be complicated in both acute and chronic phases. A few case reports have been published until now. Herein we report a case with right woven coronary artery managed with drug-eluted stent implantation without complication. Woven coronary artery (WCA) disease is an extremely rare congenital anomaly with unexplained etiology . In this54-year-old male with tightening chest pain and palpitation on exertion for two months was admitted to cardiology outpatient clinic two years ago. Resting electrocardiogram showed q waves and extra systoles on D3 and aVF. Echocardiography showed akinesia at inferior and posterior walls, and ejection fraction was 44%. We performed coronary angiography by using the Judkins technique from right femoral artery. There were plaques at LAD and %50 stenosis at proximal Circumflex arteries, the lesions were considered to be insignificant , and theWoven coronary artery (WCA) is a very rare congenital anomaly which can affect both RCA and LAD and may lead to acute coronary syndromes in some circumstances . It was This anomaly may be accepted as a benign disease. Some reports showed no cardiovascular adverse events during 5-year follow-up \u20135. NeverIn conclusion, WCA may cause myocardial ischemia and damage. All interventional cardiologists might keep this anomaly in mind and should beware of the possible consequences of WCA in catheterisation laboratory. PCI or coronary artery bypass grafting may be treatment options for this anomaly."}
+{"text": "Development of homogenous metal matrix nanocomposites with uniform distribution of nanoreinforcement, preserved matrix nanostructure features, and improved properties, was possible by means of innovative processing techniques. In this work, Al-SiC nanocomposites were synthesized by mechanical milling and consolidated through spark plasma sintering. Field Emission Scanning Electron Microscope (FE-SEM) with Energy Dispersive X-ray Spectroscopy (EDS) facility was used for the characterization of the extent of SiC particles\u2019 distribution in the mechanically milled powders and spark plasma sintered samples. The change of the matrix crystallite size and lattice strain during milling and sintering was followed through X-ray diffraction (XRD). The density and hardness of the developed materials were evaluated as function of SiC content at fixed sintering conditions using a densimeter and a digital microhardness tester, respectively. It was found that milling for 24 h led to uniform distribution of SiC nanoreinforcement, reduced particle size and crystallite size of the aluminum matrix, and increased lattice strain. The presence and amount of SiC reinforcement enhanced the milling effect. The uniform distribution of SiC achieved by mechanical milling was maintained in sintered samples. Sintering led to the increase in the crystallite size of the aluminum matrix; however, it remained less than 100 nm in the composite containing 10 wt.% SiC. Density and hardness of sintered nanocomposites were reported and compared with those published in the literature. In the n 100 nm  and formOn the one hand, the use of powder metallurgy processing techniques such as ball milling, mechanical milling/alloying  resultedThe SPS was used to prepare fully dense and high strength pure aluminum ,33,34,35In a very recent work , the aut\u03b2 (45\u201355 nm) of 97.5% purity, supplied by Nanostructured and Amorphous Materials  were used in this investigation. The chemical composition and particle size distribution of the aluminum powder are presented in Aluminum powder of 99.88% purity, supplied by , and SiCrB is the full width at half-maximum (FWHM) of the diffraction peak after instrument correction, k is constant ; \u03bb is wavelength of the X-ray radiation (\u03bb = 0.15405 nm); L and \u03b7 are crystallite size and lattice strain, respectively; and \u03b8 is the Bragg angle. Al-SiC nanocomposite powders containing 1, 5 and 10 wt.% SiC were prepared through mechanical milling; one of the most important techniques used to synthesis nanocomposite powders at the solid state . It invor is related to the measured width of the peak (obsB) and peak broadening caused by factors except the crystallite size effect (iB), frequently called instrumental broadening factor and calculated using a fully annealed sample, through the following formula:The BThe above Equations (1) and (2) are frequently used to evaluate crystallite size change and strain accumulation ,46,47 inThe prepared nanocomposite powders were consolidated through spark plasma sintering using a fully automated equipment , model HP D 5. The powder was directly charged into a 20 mm graphite die through which the current was passed. The sintering temperature was measured using a thermocouple inserted in the graphite die through a drilled hole. A graphite sheet was used to minimize friction between the die walls and the powder as well as to ease the ejection of the sample after the sintering has been completed. Samples were sintered at 600 \u00b0C for 10 min under an applied pressure of 50 MPa using a heating rate of 200 \u00b0C/min.3) and quantified according to Archimedes principle. Vickers microhardness of the spark plasma sintered samples was measured using a digital microhardness tester . All measurements were conducted by applying the same conditions: a load of 100 gf, a time of 12 s. The data reported were the average of 10 values.The density of the sintered samples was measured using an Alfa Mirage electronic densimeter, model MD-300 s (accuracy of 0.001 g/cmThe evolution of particles\u2019 morphology of Al-5 wt.% SiC powder milled for different times is presented in A similar behavior was observed in nanocomposite powders containing 1 and 10 wt.% of SiC where a milling time of 24 h deceased the size of Al particles as shown in Comparing A typical X-ray mapping of Al-5 wt.%SiC nanocomposite powder milled for 9 and 24 h is presented in X-ray diffraction spectra of the as-received un-milled Al powder and Al-SiC nanocomposite powders milled for 24 h are presented in 4C3. This is may be because, from one side, the composites are prepared through powder technology and not melt technology; and from the other side the milling decreases the intensity of peaks [4C3 in mechanically milled Al-SiC nanocomposites had been confirmed by other researchers even at high milling speeds of 320 rpm [The XRD spectra of the milled Al-SiC nanocomposite powders did not reveal the formation of secondary brittle phases such as Alof peaks . Therefoof peaks . The abs 320 rpm  and 500  320 rpm . Further 320 rpm ,52,57.The change of the matrix crystallite size as a function of milling time is presented in In addition to reducing the particle size as discussed above, milling decreased the crystallite size of the \u03b1-aluminum. It can be clearly seen, in i.e., 0.77 was slightly higher than the lattice strain of 0.43 reported by Bathula and co-workers [Strain in the aluminum matrix as a function of milling time is presented in It is evident from Analysis of the evolution of the \u03b1-aluminum crystallite size as a function of milling time showed that a milling time of 12 h is suitable time to reach a nanostructured matrix. However, uniform distribution of SiC nanoparticles was achieved at 24 h of milling. Therefore, the Al-SiC nanocomposite powders milled for 24 h were spark plasma sintered at fixed sintering conditions to investigate their densification and characterize their microstructure features.i.e., the relative density reached 100%. The nanocomposites displayed less densification compared with the monolithic pure metal. The addition of 1 wt.% SiC decreased the relative density to 99.59%. The increase of SiC to 5 wt.% decreased the relative density to 97.14%. Further increase of SiC content to 10 wt.% decreased the relative density to 94.64%. The decrease of the relative density of the nanocomposites with the increase of SiC content is in agreement with the fact that nanostructured samples are likely to be more porous [The relative density of samples sintered at 600 \u00b0C for 10 min under an applied pressure of 50 MPa using a heating rate of 200 \u00b0C/min is presented in e porous . This isSintering increased the crystallite size of pure aluminum from 298 to 366 nm. As for the nanocomposites, the crystallite size increased from 140 to 298 nm for sample containing 1 wt.% SiC, 50 to 144 for sample containing 5 wt.% SiC, and 32 to 66 nm for sample containing 10 wt.% SiC. This shows that the higher the SiC content, the lower the crystallite size of the aluminum matrix. Therefore, it can be concluded that the presence and amount of SiC contributed to the inhibition of grain growth. The crystallite size of the aluminum matrix is within the range reported by other researchers for pure aluminum and Al-based nanocomposites reinforced with SiC or other reinforcements and processed through spark plasma sintering or other methods as summarized in The hardness of samples sintered at 600 \u00b0C for 10 min under an applied pressure of 50 MPa using a heating rate of 200 \u00b0C/min is presented in The hardness of the Al-10 wt.% SiC nanocomposite was higher than the hardness of other nanocomposites reinforced either with CNTs or SiC. These include Al2124 + 1 wt.% CNTs , Al6061 In summary, milling of the Al-SiC nanocomposites led to uniform distribution of the SiC reinforcement and reduced the crystallite size of the aluminum matrix to less than 140 nm. The sintered nanocomposites maintained the uniform distribution of SiC particles and displayed good densification. SiC inhibited grain growth of the matrix and increased the hardness of the composites. The influence of sintering parameters particularly heating rate, compaction pressure, sintering temperature and time, on the structure and mechanical properties of spark plasma sintered Al-SiC nanocomposites will be the subject of future studies.Al-SiC nanocomposites containing 1, 5, and 10 wt.% SiC were successfully synthesized by mechanical milling and consolidated through spark plasma sintering. Milling of the powders for 24 h led to uniform distribution of SiC nanoparticles and decreased the crystallite size of the \u03b1-aluminum phase from around 300 to 140, 50, and 32 nm, in nanocomposites containing 1, 5, and 10 wt.% SiC, respectively. This yielded a nanostructured aluminum matrix. The uniform distribution of SiC achieved by mechanical milling was maintained in sintered samples. Sintering led to the increase in the crystallite size of the aluminum matrix, however, it remained less than 100 nm in the composite containing 10 wt.% SiC. The nanocomposite reinforced with 10 wt.% SiC had a relatively high Vickers hardness value of 171.53 HV."}
+{"text": "Aspergillus niger phyA2 gene could significantly improve phosphorus bioavailability to poultry and livestock. However, little information has been reported about the effects of phytase transgenic maize on the Asian corn borer (ACB), Ostrinia furnacalis (Guen\u00e9e). This study provides valuable information about the physiological, biochemical and gut microflora functional diversity changes of ACBs fed phytase transgenic maize. The weights, survival rates, in vivo protein contents, activities of two detoxification enzymes and three antioxidant enzymes of ACBs fed phytase transgenic maize exhibited no significant differences to those fed non-transgenic maize. Functional diversities of the gut microflora communities of ACBs were not affected by different fodder treatments, but significant differences were observed between different generations of ACBs. Our study provides useful information about the biochemical responses and gut microflora community functional diversities of ACBs fed phytase transgenic maize firstly and the results will help to assess the potential effects of phytase transgenic maize on other target and non-target arthropods in the future.Transgenic maize hybrids that express the  In plants, phosphorus is mainly stored in oilseeds and cereal grains in the form of phytate6. However, phytate-P is absorbed at low levels in monogastric animals due to the absence of the phosphatase enzyme, phytase, in their digestive tract. To avoid the arrested growth and development of animals that is caused by phosphorus deficiency, two approaches have been developed. One approach is to add inorganic phosphate into animal feeds; however, this leads to severe phosphorus pollution of surface water and groundwater7. The other approach is to add phytase to the feed; however, this is prohibitively expensive. To reduce the cost, phytase transgenic maize was previously developed by Chen et al.8. Phytase activity in the kernels of this transgenic maize is approximately 50-fold higher than that of non-transgenic maize8. Therefore, using phytase transgenic maize as the major ingredient in animal feed could not only decrease feed costs but also be beneficial to the environment.Phosphorus is an essential nutrient for most living organisms; including mammals, terrestrial insect herbivores, bacteria and algae9. However, the debate about the safety of GM crops has never stopped. Before entering the market, a whole series of animal and environmental experiments must be carried out10. The potential effects on non-target organisms are considered to be one of the most important aspects of this assessment. The Asian corn borer (ACB), Ostrinia furnacalis (Guen\u00e9e), is the main maize crop pest in Asia. The larvae of ACBs take the maize kernels as one of the major food. As the phytase was specifically expressed in the kernels of phytase transgenic maize8, ACBs are directly exposed to high concentrations of phytase proteins when they take kernels of phytase transgenic maize as the food source. Besides, ACBs could serve as prey that transfer potential effects to other organisms at higher trophic levels. Thus, we use ACB as an indicator for the potential ecological effects assessment of phytase transgenic maize on the non-target organisms.According to the ISAAA report in 2016, genetically modified (GM) maize was the second most popular GM crop in the world, with over 60.6\u2009million hectares grown globally11. In addition, the development and nutritional utilization of the ACB and Helicoverpa armigera were also tested by feeding the insects on phytase transgenic and non-transgenic maize12. The combined results from these studies have shown that phytase transgenic maize has no obvious impact on the seasonal changes of carabid beetles, or on the development and nutritional utilization of the ACB and H. armigera species12.Several aspects of the potential ecological effects of phytase transgenic maize on non-target organisms have been investigated. Using the pitfall trap method, the effects of phytase transgenic maize on the seasonal changes of carabid beetles were analysed13. Biolog EcoPlates, which contain a large number of ecologically relevant structurally diverse carbon substrates, are commonly used to determine the CLPPs of culturable bacteria14. In comparison with alternative approaches, Biolog EcoPlates method has many advantages, such as simplicity, convenience and one experiment could produce adequate information about functional diversity of bacteria. Many published studies have used Biolog EcoPlates to determine the functional diversities of microbial communities16.The functional diversity of a microbial community is its potential activities, including numbers, types and rates, in utilizing a suite of substrates. Understanding the functional diversity of a microbial community is an essential prerequisite to uncover the roles of microbial communities in different environments. Community-level physiological profiles (CLPPs) can be useful for evaluating the functional diversities of microbial communities and they have been widely used to differentiate microbial communitiesrd instar between ACB larvae fed phytase transgenic maize and control maize12. These include larval survival rate, duration of the 1st and the 2nd instar larvae, fresh weight and nutritional utilization. These indices at the early stage of ACB development could not reflect the full impacts of phytase transgenic maize on ACBs. In this study, the effects of phytase transgenic maize on ACB were investigated more comprehensively. The survival rate and fresh weight of ACBs at the beginning of the 5th instar were analysed. Importantly, the biochemical effects and the functional diversities of gut microbial communities of ACBs fed phytase transgenic maize were investigated firstly in this study. The results contribute significantly to elucidate the biological effects of phytase transgenic maize on ACBs and provide new clues to evaluate the effects of GM crops on other arthropods.Up to date, only a few parameters have been compared at or before the early 3th instar, ACB larvae fed phytase transgenic maize and control lines (Liyu 35 and Zhengdan 958) were collected to calculate survival rate and fresh weight. As shown in Table\u00a0P value\u2009>\u20090.05). Moreover, the two indices were similar in three generations under all fodder treatments , acetylcholinesterase (AChE), catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) were tested to determine the biochemical responses of the ACBs after feeding with either phytase transgenic or non-transgenic maize.P value\u2009>\u20090.05) (Table\u00a0As shown in Table\u00a05) Table\u00a0.Table 2CP value\u2009>\u20090.05)  and 63.61\u201375.45\u2009U/g pro, respectively (Table\u00a05) Table\u00a0. These r18. With more species and greater abundances of gut microflora, more types and greater amounts of carbon sources will be utilized in the plate cells. In general, AWCD of a certain microbial community correlates with the growth of the cultured microbes. Two phases of AWCD changes were observed in this study, a linear increasing stage and a stationary phase  value is an important index of overall metabolic activity for a microbial communityase Fig.\u00a0. Two wayase Fig.\u00a0. These rnd and the 3rd ACB generations utilized more carbon substrates than the 1st generation , Simpson index (D) and McIntosh index (U), were calculated to evaluate the species richness, dominance and homogeneity of the gut microbial communities of ACBs under different fodder treatmentsDuncan\u2019s new multiple range test of data collected in each generation revealed that the three indices H, D and U of ACB fed phytase transgenic maize were similar to those fed control maize Table\u00a0. However22. Based on the AWCD values after 96\u2009h incubation, the single carbon source utilization patterns of the ACB intestinal microbial communities after the three different fodder treatments were analysed by PCA. In total, eight principal components (PCs) were extracted. The proportion and cumulative proportion of the eight PCs were shown in Table\u00a0st generation of ACBs that were fed phytase transgenic and non-transgenic maize kernels were distributed on the positive axis of PC1, while the 2nd and the 3rd generations of ACBs that were fed the three different fodders were distributed on the negative axis  variables and the metabolic features of different microbial communities can be presented as relative positions in a 2-D figurexis Fig.\u00a0. These rBacillus thuringiensis (Bt) toxins, protease inhibitors and lectins26. Phytase transgenic maize is the first transgenic maize developed in China that has obtained a security certificate8. The use of phytase transgenic maize can not only improve the phytate-P utilization of livestock, but can also reduce the cost of fodder production. Although phytase is only over-expressed in the kernels of phytase transgenic maize and these plants do not carrying the insect-resistant trait, they may still affect the growth and development of any living organisms. These may include terrestrial insect herbivores, bacteria and algae; because phosphorus is necessary for the growth and development of all these organisms27. Moreover, the nutritional characteristics of crops may be altered after genetic transformation28 and this might directly or indirectly affect the fitness of organisms that use these plants as food sources. Consequently, evaluation of the potential effects of phytase transgenic maize on non-targeted organisms is important. The ACB is a major threat to maize production in Asia and mainly feeds on maize kernels at its larval stage. It could be used as an indicator for the assessment of the ecological effects of GM crops on non-target organisms. This study provides the first detailed information on the physiological and biochemical responses, as well as the changes in the functional diversity of gut microflora, in ACBs consuming phytase transgenic maize.With the advent of plant biotechnology, increasing numbers of transgenic crops with various properties, such as insect resistance, herbicide tolerance and improved quality, have been developed and planted in several countries. The potential environmental impacts of these GM crops should be investigated before they enter the market. Previous studies have mainly focused on the environmental risks of insect-resistant transgenic crops expressing 29, laying hens30 and roosters31. However, studies have shown that the growth and development rates of some insects, including Choristoneura occidentalis, Acheta domesticul and Manduca sexta, are accelerated when they are fed diets that contain high concentrations of phosphorus33. The published papers concentrated on the potential effects of phytase transgenic maize on herbivorous insects, including ACB, H. armigera and carabid beetles showed no significant differences between phytase transgenic maize and its isogenic maize12. Diets contain high concentrations of phytase transgenic maize did not affect the survival, fresh weight, nutrition utilization and the duration of the 1st and the 2nd instars of ACBs and H. armigera12. To fully appreciate the effects of phytase transgenic maize on ACBs, we examined multiple physiological, biochemical and gut microflora parameters in ACBs fed diets contain phytase transgenic maize at the 4th or the 5th instar. Three generations were used for each study to guarantee repeatability. In agreement with the previous study12, the survival rates and fresh weights of ACBs were unaffected with consumption of phytase transgenic maize  and Zhengdan 958 (non-GM maize) were grown in the transgenic experimental station of the Shandong Academy of Agricultural Sciences in Jinan, China. Before tassels began shedding, the tassels and husks were bagged to prevent cross-pollination and pathogen invasion. Approximately 3 d after silks emerged from the husks, they were self-pollinated to produce pure seeds. The immature maize kernels were collected at 15 d after artificial pollination. Once the maize kernels were mature, they were collected and then ground into a fine powder to make maize meal. The fodders  used for the larvae culture were prepared the same as that described by Zhang ACB larvae were acquired from the Institute of Plant Protection, Shandong Academy of Agricultural Sciences. Fodders containing phytase transgenic maize, Liyu 35 or Zhengdan 958 were cut into small cubes (~1.0\u2009g) and then cubes were individually placed into wells of a 24-well cell culture plate . Neonates were transferred individually to the surface of fodder cubes with a soft brush. Then the cell culture plates were sealed with lids.12.After pupation, the ACB was transferred into a 200\u2009mL beaker at 26\u2009\u00b1\u20091\u2009\u00b0C, with a 70\u2009\u00b1\u200910% relative humidity and a 16\u2009h light: 8\u2009h dark photoperiod. The inner surface of the beaker was covered with A4 paper onto which the ACBs could lay their eggs and the rims of the beakers were sealed with pieces of cotton with a 0.25\u2009mm sieve diameter. Adult female and male ACBs were paired for breeding and were fed with immature maize kernels and supplemented with a cotton ball that had been pre-soaked in a 3% sugar solution. The eggs produced were promptly removed until the adults diednd and the 3rd generations being offspring of the 1st generation.Three generations of ACBs fed phytase transgenic maize and non-transgenic maize (Liyu 35 and Zhangdan 958) were used in this experiment, with the 2th instar stage. Each larva was gently touched with a writing brush tip and if there was no observed reaction, it was considered to be dead. Once a larva was died, it was removed immediately from the culture plate. The living ACB larvae at the beginning of the 5th instar were counted and weighted to calculate the survival rate and the average fresh weight, respectively. For each fodder treatment, 100 larvae per group were tested. Three replicates were performed for each treatment.Both the survival rate and larval fresh weight were measured at the beginning of the 5th instar stage were used for each fodder test and each test was repeated three times. In total, 27 larvae were tested for each fodder treatment. In each group, nine ACBs at the beginning of the 5th instar stage were weighed and placed into a physiological saline solution at a weight ratio of 1\u2009g tissue: 9\u2009g physiological saline and then homogenized. The homogenates were centrifuged for 10\u2009min at 2,500\u2009rpm and the resulting supernatants were stored for later analysis.Nine larvae at the beginning of the 52O2) as a substrate. The formation of H2O2\u2013ammonium molybdate complex was monitored by the change in absorbance at 405\u2009nm. One unit of CAT activity was defined as the amount of CAT that decomposes 1\u2009\u00b5M H2O2 per second per mg protein. The POD and SOD activities were evaluated using guaiacol and nitroblue tetrazolium assays, respectively. POD activity was assayed by the change of absorbance at 470\u2009nm after guaiacol oxidation in the presence of H2O2 as described previously36. One unit of POD activity was considered as that which oxidized 1\u2009mg substrate per minute per mg protein. The activity of SOD was determined spectrophotometrically by measuring its ability to inhibit the reduction of nitroblue tetrazolium (NBT) according to the method of Beyer and Fridovich37. The amount of SOD required to inhibit 50% of the initial reduction of NBT under light was defined as one unit. GST and AChE were also measured using a Micro Glutathione S-transferase (GST) Assay Kit and a Micro Acetylcholinesterase (AchE) Assay Kit, respectively, according to the manufacturer\u2019s instructions from the Beijing Solarbio Science & Technology . GST assay used 1\u2013chlopro\u20132,4\u2013dinitrobenzene (CDNB) as a substrate, its activity was determined by monitoring the increase in the absorbance at 340\u2009nm due to GSH\u2013CDNB conjugate accumulation. The amount of GST that catalyses the conjugation of 1\u2009\u00b5mol/L GSH with CDNB per minute per mg protein was regarded as one unit. The activity of AchE was determined by measuring the increase in the absorbance at 412\u2009nm caused by the formation of 5-thio-2-nitrobenzoic acid (TNB). One unit of AchE was defined as the amount that catalyses the formation of 1\u2009nmol TNB per minute per mg protein.The total protein contents of the ACB was determined using a total protein assay kit . The enzyme activity of CAT was measured using a CAT assay kit (Ultraviolet)  with hydrogen peroxide  value. AWCD\u2009=\u2009\u2211(Ai\u2009\u2212\u2009AA1)/n, where Ai indicates the absorbance for the ith cell, AA1 indicates the absorbance for cell A1 and n indicates the number of substrates. When Ai\u2009\u2212\u2009AA1 was negative, 0 was used in the formula.The metabolic capabilities and functional diversity of the gut microbial communities were assessed by Biolog assays 40. We used three diversity indices, H, D and U, to evaluate the species richness, dominance and homogeneity, respectively, using the following three equations41:The diversity index analysis of the gut microbial community was calculated after 96\u2009h incubationi indicates the proportion of the relative absorbance ratio of the ith cell to the sum of the plate total relative absorbance; ni indicates the relative absorbance of the ith cell42.and43. The significances of different fodder treatments in three generations were tested using two-way ANOVA with GraphPad Prism 5.0 software . The relationships among different samples based on discriminative carbon sources were determined using PCA analyses .All the data obtained in this study were analysed statistically. The significances of different fodder treatments in one generation were analysed by multiple comparisons of the Duncan\u2019s new multiple range testSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4"}
+{"text": "Mycobacterium tuberculosis and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from M. tuberculosis culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-\u03baB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against M. tuberculosis in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development.Macrophages constitute the first line of defense against  Mycobacterium bovis Bacillus Calmette-Guerin (BCG), is unable to provide significant protection against pulmonary TB, with the exception of the most severe forms of TB in early childhood [M. tuberculosis (Mtb) will provide a better understanding of host-pathogen interactions and can facilitate the development of effective vaccines.Tuberculosis (TB) is a leading cause of human mortality and infectious disease-related morbidity worldwide . The emehildhood . Althoughildhood , vaccineMacrophages constitute the first line of defense against mycobacteria. They are critical in linking innate and adaptive immunity and serve as the host cell niche that allow Mtb to survive . Upon myIn this study, we identified a novel macrophage-activating mycobacterial protein from Mtb culture filtrate proteins (CFPs) by multidimensional fractionation and then investigated its immunoreactivity. We found that a recombinant of this newly identified Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and to express CD80 and CD86 co-stimulatory molecules and MHC class I/II molecules through TLR4, MyD88, and TRIF. Rv2882c-activated macrophages induced a significant expansion of the effector/memory T cell population. Moreover, Rv2882c exhibited short-term protective efficacy in a BCG prime-boost vaccination in a mouse model.All animal procedures were approved by the Institutional Animal Care and Use Committees of Chungnam National University (Permit Number: CNU-00284). All animal experiments were performed in accordance with Korean Food and Drug Administration (KFDA) guidelines.M. bovis BCG (Tokyo strain) was kindly provided by Korean Institute of Tuberculosis (KIT). All mycobacteria were grown in 7H9 medium supplemented with 0.5% glycerol, 0.05% Tween-80 , 10% oleic acid, albumin, dextrose, and catalase .Mtb H37Rv (ATCC 27294) and H37Ra (ATCC 25177) were purchased from American Type Culture Collection . -/-; B6.129-Tlr2tm1Kir/J) and C57BL/10 TLR4 knockout  mice were purchased from the Jackson Laboratory  The mice were maintained under barrier conditions in a biohazard animal room at the Medical Research Center of Chungnam National University, Daejeon, Korea. The animals were fed a sterile commercial mouse diet with ad libitum access to water under standardized light-controlled conditions (12-h light and 12-h dark periods). The mice were monitored daily, and none of the mice showed any clinical symptoms or illness during this experiment.Specific pathogen-free female C57BL/6 mice (6 weeks old) were purchased from Charles River Laboratories , and 5- to 6-week-old C57BL/6J TLR2 knockout (TLR2BMDMs were flushed through the femurs of C57BL/6 mice with Dulbecco's modified Eagle's medium (DMEM) . Erythrocytes were lysed by applying RBC lysis buffer  for 3 min at room temperature. After washing the cells and preparing a single-cell suspension, total cells were suspended in DMEM containing 10% fetal bovine serum, 50 ng/mL mouse macrophage colony stimulating factor (M-CSF), and 1% antibiotics (Welgene). The cells were then placed in 100-mm plates and incubated for 7 days at 37\u00b0C in 5% CO2. The medium was replaced every 3 days during a 7-day incubation.2, and 1 mM MgCl2) for 15 min at 37\u00b0C. Then, the lung cells and aggregates were filtered through a 40-\u03bcm cell strainer in Dulbecco\u2019s phosphate-buffered saline (PBS) using a sterile 1-mL syringe. The spleens were mashed on a 40-\u03bcm cell strainer in RPMI medium (Welgene). The erythrocytes were lysed using RBC lysis buffer for 2 min at room temperature. High-gradient magnetic-activated cell sorting (MACS)  was used to fractionate the T cell subsets. The cells were incubated with anti-mouse CD4 Ab-coated magnetic microbeads (Miltenyi Biotec) and then were positively selected on paramagnetic columns  according to the manufacturer\u2019s instructions.Lungs were isolated under sterile conditions, cut into 0.5-cm pieces, and agitated in 5 mL cell dissociation buffer , 1 mM CaClE. coli O111:B4 was purchased from InvivoGen . Endotoxin filter (END-X) and endotoxin removal resin (END-X B15) were acquired from the Associates of Cape Cod . Anti-phosphorylated ERK1/2 monoclonal Ab, anti-ERK1/2 monoclonal Ab, anti-phosphorylated p38 monoclonal Ab, anti-p38 monoclonal Ab, anti-phosphorylated JNK monoclonal Ab, anti-JNK monoclonal Ab, anti-phosphorylated I\u03baB-\u03b1 monoclonal Ab, anti-I\u03baB-\u03b1 monoclonal Ab, and anti-tubulin polyclonal Ab were obtained from Cell Signaling Technology . Anti-TLR2, anti-TLR4, and anti-histidine (His) antibodies (Abs) were purchased from Santa Cruz Biotechnology . HRP-conjugated anti-mouse IgG Ab and HRP-conjugated anti-rabbit Ab were obtained from Calbiochem . Phycoerythrin (PE)-conjugated mAbs directed against IL-10, IL-12p70, CD80, CD86, and MHC class II, and allophycocyanin-conjugated mAb directed against F4/80 were purchased from eBioscience . Mouse TNF-\u03b1, MCP-1, IL-6, IL-10, IL-12p70, IFN-\u03b3, and IL-2 ELISA kits were obtained from eBioscience.Recombinant M-CSF was purchased from Peprotech . Fluorescein isothiocyanate (FITC)-annexin V/PI kits were purchased from BD Biosciences . LPS from Mtb H37Rv (ATCC 27294) was grown for 6 weeks at 37\u00b0C as surface pellicles in Sauton\u2019s medium, and then the CFPs were prepared as previously described . The CFPfrr) was amplified by PCR using genomic DNA from Mtb H37Rv (ATCC 27294) as the template and the following primers: forward, CATATGATTGATGAGGCTCTCTTCGAC-3\u20325\u2032- and reverse, AAGCTTGACCTCCAGCAGCTCGCCTTC-3\u20325\u2032-. The resulting PCR product was inserted into the pET-22b (+) vector  after digesting both using the NdeI and HindIII restriction enzymes. The recombinant protein was prepared as previously described [To produce the recombinant Rv2882c protein, the corresponding gene (escribed . To remo6 cells/mL) to investigate the cytotoxic effect of Rv2882c. After 24 h of treatment, the harvested BMDMs were washed using PBS, stained with FITC-Annexin V and PI (BD Biosciences), and then analyzed using a FACSCanto flow cytometer (BD Biosciences).Rv2882c (10 \u03bcg/mL) was added to isolated BMDMs cultured in 12-well plates  or Rv2882c (10 \u03bcg/mL), or left unstimulated, for 12 h in the presence of GolgiPlug (BD Biosciences). The samples were first blocked for 15 min in 10% (vol/vol) normal goat serum and stained at 4\u00b0C in flow cytometry buffer (PBS/2% BSA) with APC-conjugated F4/80 antibodies for 30 min at 4\u00b0C. Cells stained with the appropriate isotype-matched immunoglobulin were used as negative controls. The cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) according to the manufacturer\u2019s instructions. Intracellular cytokines were detected using fluorescein-conjugated antibodies (eBiosciences) in a permeabilization buffer. Cell surface staining was performed using specifically labeled fluorescent-conjugated mAbs, and the staining intensity was determined using flow cytometry , after which the data were analyzed using FlowJo data analysis software (BD Biosciences), as recently described .7) were lysed with lysis buffer  Triton X-100; 10% (vol/vol) glycerol; 1 mM PMSF; 1 \u03bcg/mL each of aprotinin, leupeptin, and pepstatin; 1 mM Na3VO4; and 1 mM NaF). Twenty micrograms of the His-tagged Rv2882c protein and cell lysate were mixed and incubated at 4\u00b0C for 6 h, and then Ni-NTA agarose beads were added  according to the manufacturer\u2019s instructions. The beads were mixed with the cell lysate diluted in lysis buffer and rocked for 2 hr at 4\u00b0C. After incubation, the mixtures of beads and cell lysates were centrifuged at 150 g for 1 min at 4\u00b0C, and then the beads were washed three times with buffer and collected by centrifugation. After removing the supernatant, the beads were boiled in SDS-PAGE sample buffer, and the bound proteins were analyzed by immunoblotting with anti-TLR2, anti-TLR4, and anti-His Abs.BMDMs  was added to the cultures at a final concentration of 0.1% (vol/vol) as a solvent control. The BMDMs were washed using PBS and pretreated with the inhibitors in DMEM medium containing glutamine for 1 h prior to treatment with Rv2882c for 24 h. The inhibitors were used at the following concentrations, as determined by careful titration: U0126 (10 \u03bcM), SB203580 (20 \u03bcM), SP600125 (10 \u03bcM), and Bay11-7082 (5 \u03bcM). The viability of BMDMs was assessed using an MTT assay.+ T cells for 3 days. To determine the bacterial burden, a fraction of the macrophage cultures was immediately lysed using 0.1% saponin. The supernatants and cell lysates were serially diluted and plated on 7H10 agar plates supplemented with 10% OADC enrichment medium to determine the bacterial burden per well.BMDMs were infected with Mtb at a multiplicity of infection of 1:1 (bacteria to BMDMs) for 4 h. Then, the infected BMDMs were treated with amikacin (200 \u03bcg/mL) for 2 h, followed by two washes with PBS. The infected BMDMs were treated with Rv2882c (10 mg/mL) or LPS (100 ng/mL) for 24 h, and then co-cultured with CD44 CFU/0.2 mL/subcutaneous) vaccination, 5 mice per group were subcutaneously immunized on their backs with 10 \u03bcg of Ag85B and Rv2882c emulsified with dimethyl dioctadecylammonium bromide  and 25 \u03bcg monophosphoryl lipid A  in a total volume of 0.2 mL two times over a 4-week interval. After 6 weeks, the vaccinated mice were infected with Mtb H37Ra (ATCC 25177). Briefly, following anesthetization with a xylazine:zoletil (9:1) mixture, 5 mice per group were intratracheally infected with 50-\u03bcL suspensions at infectious dose of 106 CFU of H37Ra per mouse lung.After the BCG  statistical software . The data in the graphs are expressed as the mean values \u00b1 SD; *p < 0.05, **p < 0.01, and ***p < 0.001 were considered statistically significant.All experiments were repeated at least three times, with consistent results. The significance of differences between two groups was determined by unpaired Student's Escherichia coli, and its purity was confirmed by SDS-PAGE. The purified protein had a molecular mass of approximately 23 kDa and reacted with anti-His antibody (Ab)  . Endotoxody (Ab) . To deteTo confirm whether recombinant Rv2882c induced macrophage activation and stimulated pro-inflammatory cytokine production in macrophages, cytokine levels in the culture supernatants of BMDMs treated with Rv2882c at 1, 5 or 10 \u03bcg/mL for 24 h were determined. LPS was used as a positive control. Rv2882c significantly increased the production of TNF-\u03b1, IL-6, IL-12, and MCP-1, but not IL-10, in a dose-dependent manner . In cont-/-, and TLR4-/- mice, and then treated them with Rv2882c for 24 h. The expression of surface molecules such as CD80 and CD86 and production of pro-inflammatory cytokines were significantly reduced in the Rv2882c-treated BMDMs from the TLR4-/- mice, but not from the WT or TLR2-/- mice  T cell population compared to LPS- or Ag85B-treated macrophages, and the population of na\u00efve (CD62Lhigh/CD44low) T cells in the spleen and lungs was significantly decreased in co-cultures with Rv2882c-treated macrophages  and spleen (-2.1 log10) compared to that of the non-vaccinated group (P < 0.05). In lungs, the Rv2882c-boosted mice had significantly lower bacterial counts than the Ag85B-boosted mice did. Th1-mediated immune responses play an important role in protective immunity against Mtb [in vitro with Ag85B or Rv2882c, and the cytokines produced were determined by ELISA. The stimulating antigens specifically induced cytokine production in cells from the corresponding antigen-boosted mice  using Phenyl Sepharose. The primary fractions were divided and concentrated into seven fractions. Each of the primary fractions was further fractionated by hydroxyapatite chromatography (HAT). The eluates were pooled into five to nine fractions based on the protein band pattern and were concentrated. A third fractionation was performed using DEAE ion-exchange chromatography.(TIF)Click here for additional data file.S2 Fig5/well) activation was not due to endotoxin contamination in the protein preparation, Rv2882c (10 \u03bcg/mL) was (B) denatured by heating for 1 h at 100\u00b0C, (C) digested with Proteinase K (10 \u03bcg/mL) for 1 h at 37\u00b0C, or (D) pretreated with polymyxin B (50 \u03bcg/mL) for 1 h prior to stimulating the BMDM cultures. After 24 h, the quantities of TNF-\u03b1 and IL-6 in the culture medium were measured by ELISA. All data are expressed as the mean values \u00b1 SD (n = 3); ***p < 0.001 = a significant difference compared to the Rv2882c-treated BMDMs, as determined by unpaired Student\u2019s t-test. Treatments with no significant effect are indicated by n.s.(A) The amount of residual LPS in the RpfE preparation was estimated using the Limulus amoebocyte lysate (LAL) test according to the manufacturer\u2019s instructions. To ensure that Rv2882c-induced BMDM (1 \u00d7 10(TIF)Click here for additional data file.S3 Fig5/well) were stimulated with LPS (100 ng/mL), Rv2882c (10 \u03bcg/mL), heat denaturated LPS (100 ng/mL) or heat denaturated Rv2882c (10 \u03bcg/mL) for 24 h. The BMDMs analyzed for the expression of surface markers using flow cytometry. The cells were gated on the F4/80+ BMDMs. The BMDMs were stained with anti-CD86 and anti-MHC class II antibodies. The percentage of cells that are positive is shown in each panel. (B) BMDMs were treated with Rv2882c (10 \u03bcg/mL), Rv2882c (10 \u03bcg/mL) mixed with polymyxin B, LPS (100 ng/mL), and LPS (100 ng/mL) mixed with polymyxin B in time course. The mixture was prepared by reacting Rv2882c (10 \u03bcg/mL) or LPS (100 ng/mL) with polymyxin B (50 \u03bcg/mL) for 1 h prior before treatment. Cell lysates were subjected to SDS-PAGE and immunoblotted using Abs specific to phospho-p38 (p-p38), p38, phospho-ERK1/2 (p-ERK1/2), ERK1/2, phospho-JNK (p-JNK), JNK, phospho-I\u03baB- (p-I\u03baB-), and I\u03baB-. \u03b1-tubulin was used as the loading control for cytosolic fractions.(A) BMDMs (1 \u00d7 10(TIF)Click here for additional data file.S4 FigMaterials and Methods. The lung and spleen cells (5 \u00d7 106/well) were treated with Rv2882c (10 \u03bcg/mL) or Ag85B (10 \u03bcg/mL) for 5 days. The levels of IFN-\u03b3 in the culture supernatants were determined by an ELISA. The data are shown as mean \u00b1 SD (n = 5); *p < 0.05, **p < 0.01, or ***p < 0.001: a significant difference between treated and untreated groups, as determined by one-way ANOVA. Treatments without a significant effect are indicated by n.s.The cells were prepared as described in (TIF)Click here for additional data file.S5 Fig6 CFUs of Mtb H37Ra. The bacterial loads in the lungs and spleens were determined at 3, 6, 10, 15, 20, and 26 weeks.The mice were intratracheally infected with 10(TIF)Click here for additional data file."}
+{"text": "Histoplasma capsulatum produces diverse clinical manifestations, ranging from an influenza-like illness to a cavitary lung disease to life-threatening dissemination affecting multiple major organ systems [H. capsulatum is wider spread than originally considered.Human infection with the pathogenic fungus  systems , 2. Long systems , 4, new  systems , 6, and  systems . In the H. capsulatum exists as a mold and generates two types of conidia or spores-macroconidia and microconidia. These forms are asexual ovoid structures produced at the tips of hyphae. The portal of entry is the lung, and microconidia are considered to be the form that reaches the alveoli and terminal bronchioles of the lung because of their small size (2\u20135 \u03bcm). Exposure of the mold phase to 37 \u00b0C induces an orderly change in gene expression, driving conversion of spores into yeast cells that are typically 2\u20134 \u03bcm in diameter [This fungus is considered to be dimorphic; it grows both as a mold and a yeast. The phase depends on temperature. At 25 \u00b0C, diameter . It is tH. capsulatum is soil laden with bird excreta or bat guano. Contact with the fungus usually requires disturbing the soil, thus aerosolizing spores. Droppings from several avian species have been implicated in supporting the growth of the fungus; these include starlings, blackbirds, pigeons, and, less commonly, oilbirds (found in South America) and grackles [H. capsulatum was recovered from the soil, thus establishing incontrovertibly that infection is acquired from the environment. The fungus was isolated from soil containing chicken excreta surrounding a chicken coop [H. capsulatum particles has been estimated to reach 105 per gram of soil [The habitat of grackles . It was ken coop . The org of soil . Of note of soil .H. capsulatum has been notoriously difficult to isolate directly from the soil, although occasionally using a mineral oil flotation process has achieved success. Rather, the simplest and perhaps most reliable method is to inoculate mice intraperitoneally with an emulsion of the soil, wait approximately 15 to 30 days, and culture several organs, including liver, spleen, and lungs, for the presence of the fungus [Despite numerous attempts to culture the fungus from environmental reservoirs, e fungus . The mouH. capsulatum? To this day, the answer to this question remains elusive. Attempts have been made in the past, but this work has long been neglected. What we do know is that a carbohydrate from chicken excreta, not otherwise identified, produced excellent growth and sporulation of H. capsulatum. Other constituents in excreta that promote growth of the fungus include salts, particularly phosphate, which is enriched in excreta compared to the surrounding soil, and nitrogen. The nitrogenous substances that fuel growth remain unknown, although urea has been suggested but not definitively documented [What is it about bird excreta or bat guano that fosters the presence of cumented , 11.Numerous reports of epidemics or outbreaks of histoplasmosis in the US and Canada have been described over the years, dating back to the late 1930s \u201316. An oCoccidioides sp. The second outbreak resulted from construction of a new natatorium on the campus of Indiana University\u2013Purdue University Indianapolis (IUPUI). In total, approximately 200,000 individuals were infected as a consequence of the two outbreaks.The outbreak that developed in Montreal in the 1960s is an exception to the rule that, prior to the 1970s, most outbreaks developed in rural areas, as might be expected for an infection associated with bird or bat excreta. Given that many of these episodes originally were reported in rural areas, the number of individuals infected often has been fewer than 100. Since the 1970s, the setting for epidemics has shifted from rural to urban . With a H. capsulatum had on the health of Indianapolis residents. Although outbreaks of histoplasmosis typically do not cause a high degree of mortality or morbidity and much of the disease is self-limited, these two were accompanied by more than 300 hospitalizations and at least 15 deaths. There were more than 40 cases of disseminated histoplasmosis. It is important to note that these outbreaks preceded the AIDS epidemic and the introduction of new biologicals such as tumor necrosis factor-\u03b1 antagonists that are major risk factors for disseminated histoplasmosis. The reason for the devastating impact is not clear, but the risk factors that were identified included ages greater than 54 and immunosuppression. The scope of these two epidemics establish the importance of the urban setting, wind, and immune status when considering the impact of histoplasmosis on a population.Another important element of these two episodes is the detrimental impact H. capsulatum in the city and its neighborhoods that caused this increase in cases [Indianapolis did suffer another epidemic in the late \u201880s to the early \u201890s. However, this time, many of the cases developed in AIDS patients. This outbreak was not traced to a specific demolition or construction site. Rather, it might have been foci of in cases . The majHistoplasma outbreaks is a cave [An often neglected but important source of s a cave . Bats haBats are not only found in caves but can be present in tunnels. A recent outbreak in the Dominican Republic highlighted this association . A dam hH. capsulatum require individuals to be in proximity to areas that are being excavated or where soil is disrupted. One outbreak occurred in a hotel in Acapulco, Mexico in which cleaning of air ducts and the use of stairwells was associated with the spread of spores that caused illness in 21 students who were vacationing over spring break [H. capsulatum spores. Other outbreaks related to air handling systems include one in which room air conditioners spread spores that had been swept off a roof of the building.Not all the exposures to ng break . Anotherng break . The likH. capsulatum is not a reportable disease, and therefore, tracking the number of cases is a difficult task. Usually, histoplasmosis comes to our attention when there are outbreaks, whether they involve a few or a few thousand individuals. Once considered a rural disease of the Midwest and Southeast, the pattern of outbreaks has shifted to involve urban areas. Concomitantly, recognition of the disease as one of the Americas is simply no longer true. The geographic extent of the disease has broadened considerably, with an increasing number of reports from Asia. Occupational workers must be provided with the appropriate knowledge regarding the risks when they are tasked with environmental remediation for a site that potentially contains H. capsulatum spores. Unfortunately, no safe soil disinfectant is available that will kill spores; hence, the best protection for workers is the proper protective equipment and wetting of the soil to minimize aerosols.Infection with"}
+{"text": "The following information is missing from the Competing Interest statement: BDH and CW received funding from the Shell Social Investment Fund. This funding began in 2011, after completion of this study, and did not support the work reported in this article.Additionally, the authors are providing the available individual-level data underlying the results as Supporting Information file S1 Dataset(CSV)Click here for additional data file."}
+{"text": "The sheep meat sector in southern Italy, based mainly on light milk-fed lambs, requires technical innovations to improve the production system, the product quality, and enhance the consumption of lamb meat. To fulfill these requirements, this investigation aimed to implement feeding strategies to reduce the cost and energy level of diets for dairy breed lambs slaughtered at an older age than the light lambs, applying a feed restriction at 75% and/or including an inexpensive and local byproduct, such as durum wheat bran (DWB), as a fiber source. The proposed feeding plans were suitable to increase the slaughter age of lambs up to 120 days and produce lean carcasses that, compared to those from 90-day-old lambs, were heavier and with improved meat quality in terms of major water retention and tenderness. The dietary inclusion of DWB limited the fat content and improved the health properties of lamb meat with regard to its antioxidant capacity and fatty acid profile, whereas it reduced lambs\u2019 growth when associated with feed restriction.p = 0.04). The incidence of fat tissue in the hind leg increased (p < 0.0001) from 90L (5.82 and 5.45% with DWB0 and DWB20) to 120R (8.80 and 8.43% with DWB0 and DWB20) and 120L lambs (10.7 and 11.8% with DWB0 and DWB20). Older lambs\u2019 meat, compared to that of 90L lambs, showed analogous levels of intramuscular fat, higher water retention, tenderness and lightness, and a more intense red colour. In meat from 120-day-old lambs, DWB intake tended to reduce the fat level (p = 0.009) and increased polyphenol content  in 120R and 120L lambs; p = 0.02), antioxidant capacity , and the presence of n-3 polyunsaturated fatty acids (FA) , thereby improving the meat\u2019s health properties. The panelists perceived the effects of DWB inclusion as well as the feeding level with triangle tests.This experiment aimed to investigate the possibility to increase the carcass weight of dairy breed lambs and produce moderate-fat meat by applying inexpensive feeding strategies based on restriction and through the use of a fibrous byproduct such as the durum wheat bran (DWB). Sixty-five 45-day-old lambs of the Valle del Belice breed, divided into 6 groups, were fed alfalfa hay supplemented with concentrate feeds including DWB at 0% or 20% , supplied ad libitum (L) or restricted at 75% (R), and slaughtered at 90 or 120 days of age. The groups were as follows: DWB0-90L (n = 14), DWB20-90L (n = 14), DWB0-120R (n = 10), DWB20-120R (n = 9), DWB0-120L (n = 9), DWB20-120L (n = 9). The diet did not affect feed intake, growth or carcass weight of lambs fed ad libitum, whereas 120-day-old lambs fed DWB associated to restriction showed the lowest weight gain (105 vs. 170, 185 and 190 g/day in DWD20-120R, DWB0-120R, DWB0-120L and DWB20-120L;  The development of efficient and productive livestock farming systems implies the introduction of innovations related to processes and/or products. The sector of sheep meat in southern Italy currently needs to be renewed with regard to both the production system and the product type. Indeed, in this area, as in other Mediterranean countries, sheep meat is obtained mainly from carcasses lighter than 7 kg of milk-fed lambs of dairy breeds slaughtered at about 30 days of age ,2,3, andIn this context, the production of lamb meat and the relative consumption could be enhanced by implementing production models that allow the slaughter age to be raised to obtain heavier and more muscled carcasses than those of traditional light lambs and meet the consumers\u2019 preferences with regard to meat quality.Several studies have indicated that heavier lamb carcasses show a better conformation , and higHowever, findings from other authors ,11 have The feeding regime is definitely one of the aspects to be carefully modulated on order to achieve an acceptable meat quality in terms of fatness and preserve adequate levels of technical-economic efficiency of the production system. In particular, diets designed for lambs of dairy breeds slaughtered at an older age to obtain heavier carcasses than those of light suckling lambs should be able to reduce feeding costs, limit fat deposition, and ensure appreciable nutritional and organoleptic properties for consumers.Therefore, in order to reduce feeding costs, this experiment proposed the use of one of the main byproducts in the milling industry, durum wheat bran (DWB). This byproduct is cheap and easily available in southern Italy because durum wheat is a staple crop in Mediterranean regions, where is used for high-quality end products such as pasta, couscous and bourghul. While grain protein content, color and gluten strength are considered the most important features needed for use in pasta and bread production , DWB shoAccordingly, the present study aimed to investigate the possibility of raising the carcass weight of lambs of dairy breeds and producing medium-fat meat by increasing the slaughter age and applying inexpensive feeding strategies based on restriction and the use of a fibrous byproduct such as DWB.The experiment was carried out in the period April\u2013June at the private Bulfara farm, located in Alimena, in the province of Palermo, Sicily, Italy . At weaning, 68 lambs of the Valle del Belice breed, aged about 35 days, were individuated and assigned to two homogeneous groups according to sex, type of birth (single and twin), age and live weight.Both groups were divided into two subgroups of 17 lambs. Each of these four subgroups was transferred in an indoor straw-bedded pen equipped with two linear feeders of 3.7 m, with access to an outdoor paddock where a water trough was placed. There, the groups were fed immediately with the experimental diets and housed until the lambs were 90 days old.The experimental period started after 10 days of adaptation to the new housing conditions and feeding treatments, when the lambs averaged 44 \u00b1 6 days of age and 16.2 \u00b1 2.7 kg of live weight.The experimental diets, fed ad libitum (L), included alfalfa pelleted hay as a common forage basis, supplemented with one of two isoenergetic concentrates consisting of faba bean and barley grains in the form of a coarsely ground meal, and differing in the presence of DWB at 0% or 20%  . Both haAfter 45 days of ad libitum feeding, 7 lambs homogeneously selected from each subgroup, corresponding to 14 lambs for each experimental diet, were slaughtered at an average age of 90 days (90L). In contrast, for 30 days until slaughter at 120 days of age, the four subgroups, each consisting of 10 lambs, received the same diets ad libitum (120L) or restricted (120R) at 75% of the ad libitum intake recorded for the 120L subgroups in the previous week. In this phase, from 90 to 120 days of age, each subgroup was housed under the same conditions as those described for the previous phase. At the end of this period, all the lambs were slaughtered, with the exception of three lambs  that were removed from the experiment due to their poor health status not attributable to feeding treatments.On the basis of this experimental design, the factors involved were feeding plan , defined by the feeding level, ad libitum (L) or restricted at 75% (R), age to slaughter, 90 or 120 days, and diet, without or with 20% DWB in the concentrate ; accordingly, the six experimental groups were as follows: DWB0-90L (n = 14), DWB20-90L (n = 14), DWB0-120R (n = 10), DWB20-120R (n = 9), DWB0-120L (n = 9), DWB20-120L (n = 9).The lambs were managed during the experiment according to the European Directive 2010/63/EU on the protection of animals used for scientific purposes, complying with the Italian Legislative Decree 26/2014, and were slaughtered according to the European Union Regulations (Council Directive 93/119 EEC) on the protection of animals at the time of slaughter or killing.During the experimental period, the weight of feeds offered to lambs from each pen and those refused by the same lamb group were recorded daily to calculate the group feed intake. Samples of the offered feeds  were collected every two weeks and analyzed following AOAC  methods Samples of concentrates and their ingredients were also analysed in triplicate for the content of ferulic acid and total phenolic acids, expressed in \u03bcg/g DM. Extraction of phenolic acids from feeds samples 250 mg) was processed according to the procedure reported by Laddomada et al. 0 mg was .Feeds extracts were also prepared in duplicate according to L\u00f3pez-Andr\u00e9s et al.  and usedThe fatty acids (FA) of feeds extracted from 50 mg samples followed the one-step extraction and transesterification procedure , with C2During the entire experiment, lambs were regularly weighed at two-week intervals to evaluate the rate of growth and the feed conversion.The lambs were weighed at the end of the experiment, then kept fasting from solids for 12 h, after which they were transported to a commercial slaughterhouse and weighed again before slaughter. After removing skin, feet and gastrointestinal tract, the hot carcasses were kept at room temperature (>10 \u00b0C) for 6 h, chilled to 4 \u00b0C for 24 h, and then weighed. The weight of gastrointestinal content was estimated by weighing the full and empty gastrointestinal tract and used for calculation of empty body weight. Afterwards, the chilled carcasses were dissected to separate and weigh the head, internal organs and the right half of the carcass. The perirenal and pelvic fat, hind leg and Longissimus dorsi (LD) muscle were removed from the right half of carcass and then weighed. The hind leg was dissected in its tissue components  to determine their incidence and the meat-to-bone ratio. The LD muscle was cut into three parts: proximal , intermediate  and distal (Longissimus lumborum muscle).The pH was measured on intermediate LD samples 24 h after slaughter (ultimate pH), using a Hanna FC 200 pHmeter equipped with a penetrating probe . 2 + b* 2) \u00d7 0.5), and hue angle  was calculated as (H = arctg b*/a*) [A colorimetric analysis was performed in duplicate on the section of the intermediate LD samples and the perirenal fat after 1 h of exposition at ambient temperature, with a Minolta Chroma Meter CR300  using the illuminant C. Results are expressed as lightness , redness , and yellowness , according to the CIE L* a* b* system [g b*/a*) .The LD samples were vacuum packed and frozen at \u221220 \u00b0C for subsequent analysis. The weights of the frozen intermediate LD samples and the corresponding meat thawed at 4 \u00b0C for 24 h were used to determine thawing loss. Cooking loss was measured on the same intermediate LD samples wrapped in polyethylene bags, cooked in a water bath at 75 \u00b0C for 40 min, cooled for 1 h, and then reweighed to determine moisture loss.The Warner-Bratzler (WB) shear force was measured on three cylinders of cooked meat with a 12.7 mm diameter using an Instron 5564 .After being frozen, the proximal LD samples were freeze-dried and ground and then analysed to determine moisture, fat and ash content according to the AOAC methods . ProteinLyophilized samples of LD meat of 36 male lambs were used to prepare the aqueous extracts in duplicate according to the procedure described by Luciano et al.  to measu2SO4 catalysis) directly. The FAME were recovered in 1.5 mL hexane and 1 \u03bcL of each sample was injected by auto-sampler into an HP 6890 gas chromatography system equipped with a flame ionization detector . Separation of FAME was performed using a capillary column 100 m in length with an internal diameter of 0.25 mm and film thickness of 0.25 \u03bcm . Gas chromatography conditions and identification of each FA were as described by Bonanno et al. [The fatty acid (FA) composition was determined on lyophilized LD meat from 120-day-old male lambs (n = 18). The extraction of the fat and the preparation of the FA methyl esters (FAME) were performed according to O\u2019Fallon et al. . Brieflyo et al. . Each ino et al.  as folloo et al. : HPI =  procedure of SAS 9.2 software .Parameters related to in vivo performance of lambs were analysed separately for the two growth periods, from 45 to 90 and from 90 to 120 days of age. The daily feed intake recorded for the lambs of each subgroup was analyzed with a model that for the 45\u201390-day period included the effect of the diet (2 levels: DWB0 and DWB20), and for the 90\u2013120-day period included the effects of feeding level , diet and the interaction FL \u00d7 diet. The individual growth parameters of lambs were analysed with the same models, including the effect of sex as well.The post-mortem parameters related to slaughter performance and carcass and meat assessment were statistically processed according to a model comprising the feeding plan , diet, sex and the interaction FP \u00d7 diet. For total polyphenols, TEAC, and FA composition; the model did not include the effect of sex.p \u2264 0.05). Pearson\u2019s coefficients were calculated to test correlation between parameters. In the sensory triangle tests, the significance of differences was assessed using the standard references tables from Amerine et al. [Comparisons between least-square means were performed by Tukey\u2019s test when the effects were significant . In addition, the DWB contributed to double the content of total phenolic acids, consisting largely of ferulic acid, and therefore, the total polyphenols. Thus, the greater antioxidant capacity (TEAC) detected for the DWB20 concentrate can be certainly attributed to the higher presence of phenolic compounds in the bran, especially ferulic acid, which is recognized for its antioxidant power . With reThe growth performance and feed intake of lambs during the first phase, from 45 to 90 days of age, are shown in p = 0.052) due to the faster growth rate of males compared to females. These effects were responsible for lower final body weights in lambs fed restricted DWB20 diet and in females, although the differences were not significant.The considered factors significantly affected the weight gain of lambs, which strongly decreased when the DWB-based diet was offered at a restricted level; since the feed intake did not differ between the restricted DWB0 and the DWB20 lambs, this result is certainly attributable to the higher fiber content of the DWB20 concentrate which, reducing the digestive utilization, did not allow the restricted lambs to satisfy their nutritional needs for growth. In addition, in this more advanced phase, when the first signs of sexual dimorphism begin to emerge, the effect of sex on weight gain from 90 to 120 days was almost significant  than in DWB20 diet (70%), and the same (73%) for concentrates. Overall, the restriction in both DWB0 (69%) and DWB20 diets (72%) has a more pronounced result than that planned at 75% of the ad libitum intake. As expected, the ferulic acid and the total phenolic acids were ingested at highest levels with the diet containing DWB offered ad libitum, followed by the restricted DWB20 diet.Due to the lower weight gain, the levels of the concentrate and diet conversion ratios were particularly high in the lambs fed with the restricted DWB20 diet; indeed, the interaction FL \u00d7 diet, which expressed a different trend of diets within the feeding levels, tended towards statistical significance. Instead, the feed conversion ratios were significantly higher in the females, which, being characterized by a more precocious adipogenesis compared to males, require more energy to develop adipose tissue which, as known, increases the feed conversion ratio.The growth rate of lambs recorded from 90 to 120 days of age was comparable to that equal to 178 g/day detected in male lambs of Comisana breed fed ad libitum from 80 to 130 days of age with an analogous diet consisting of alfalfa pelleted hay and a concentrate composed of faba bean (76%) and barley (24%), while the feed conversion ratio was markedly higher than that recorded for the same Comisana lambs, which was equal to 4.82 . HoweverThe parameters related to slaughter performance and carcass traits are reported in The feeding plan, defined by different slaughter age of lambs and feeding level, affected significantly the weight of lambs at slaughter; indeed, both the slaughter body weight  and empty body weight  were obviously higher in 120 day-old lambs, especially when fed ad libitum, although the 120L and 120R plans did not have statistically dissimilar results. As a consequence, the weight of the entire  and half carcasses  were significantly lower for the younger lambs in comparison with the 120-day-old lambs. On average, the carcasses of 90L lambs weighed 12.5 kg, which is heavier than the carcasses produced from 130-day-old Comisana lambs fed with an analogous diet (11.3 kg) , and comCompared to the 120L lambs, the 90L lambs showed a higher incidence of head (8.11% vs. 7.57 %), a lower presence of perirenal and pelvic fat (2.15% vs. 2.68 %), and a tendency towards a lower incidence of the internal organs. These results can be attributed to the different growth rate of these body regions , which iA tendency towards significance of the feeding plan also emerged for the incidence of the empty gastrointestinal tract and its content, which slightly increased in 120R lambs, and can be explained by the slower gastrointestinal transit associated to restricted feeding that would favor a longer permanence of ingested feeds in the digestive tract and, as a consequence, a slight increase recorded in its volume and content.Regardless of the feeding plan, the carcasses of lambs fed DWB showed a greater incidence of head, which, also in this case, can be related to the greater development of the more precocious bone tissue , favoredThe sex of the lambs influenced the carcass yield expressed as a percentage of the slaughter body weight (SBW), which was higher in the females due to only their lower gastrointestinal content, since the same difference did not emerge for the carcass yield referred to the empty body weight (EBW). The carcasses of the females also showed a greater adiposity, as indicated by the higher content of perirenal and pelvic fat. In contrast, the males had a higher incidence of head, which denotes their greater skeletal development compared to the females. These expected results are in line with several findings on lambs from various breeds and weight ranges  and are With regard to the tissue composition  that resSimilarly to the carcass traits, the parameters of physical quality of LD meat were also more affected by the feeding plan rather than by the other factors considered ; neverthThe meat of the lighter carcasses from the 90L lambs showed an ultimate pH (6.01), measured at 24 h after slaughtering, higher than that of meat from 120-day lambs . As known, a higher meat pH is linked to a lower post mortem acidification, which, in turn, could depend on the mobilization of the muscle reserves of glycogen to sustain an increasing energy demand ; in thisDespite its higher pH, which should be linked to an increasing water holding capacity , the LD 2). This result is in line with D\u2019Alessandro et al. [The higher values of shear force recorded for the 90L cooked meat indicates a lower tenderness of meat from young lambs, especially in comparison with the 120R meat , and were both influenced by feeding plan, diet and their interaction. The significance of the interaction shows that a higher presence of polyphenols in the meat, and the consequent improvement of the meat antioxidant capacity, occurred only in the 120-day-old lambs fed with DWB, regardless of the feeding level. Presumably, this result can be linked to the higher DWB intake of 120-day lambs, but also to a longer accumulation in the tissues of the compounds with antioxidant activity contained in the ingested DWB, such as phenolic acids, especially ferulic acid [The content of total polyphenols and the antioxidant capacity of LD meat, detected exclusively for the male lambs, were strongly correlated  and 120R lambs , but not in the meat of 120L lambs . Instead, the effect of the feeding level was perceived significantly only in the meat from lambs fed the diet without the DWB , and not in the meat from lambs fed the DWB20 diet .In the triangle tests, the panelists were able to detect sensorial differences between samples of cooked meat due to the presence of DWB in the diet and the feeding level, restricted or ad libitum, although these differences were always perceived at a moderate level. In particular, the assessors discriminated significantly the diet in the meat of 90L , recognized for its hypolipidemic effect, which is important for human health to reduce plasma cholesterol and triglycerides [Oleic acid , and an increase of n-3 FA for the main contribution of the eicosapentaenoic acid (EPA); these results corresponded to the favourable strong reduction of the n-6/n-3 ratio to values strictly close to the threshold (\u22645) recommended by the FAO/WHO  in the hHowever, EPA and DHA are recognized for their multiple health benefits, mediated by their anti-inflammatory actions ,57 and etrans vaccenic acid (VA) only showed a reduction with the restricted DWB20 diet.On the contrary, the diets with DWB were responsible for the tendency for a lower level of rumenic acid (RA), the main isomer of the conjugated linoleic acids (CLA), known for its health benefits ,60; thisThe VA is an intermediate of the biohydrogenation of dietary polyunsaturated FA to stearic acid performed by the micro-organisms in the rumen, whereas the RA is produced by the endogenous desaturation of VA in the tissues due to the \u03949-desaturase . Thus, tSignificant interactions with the FL \u00d7 diet emerged for the total of saturated FA (SFA) and unsaturated FA (UFA), and then for their PUFA/SFA and UFA/SFA ratios. The effect of DWB on the level of saturated FA was opposite in relation to the feeding level, since the byproduct induced a decrease of saturated FA, especially palmitic (C16:0) and stearic (C18:0) acids, when offered ad libitum. This result could be a confirmation of the effect of restricted DWB20 diet in favouring the complete biohydrogenation process in the rumen. The levels of saturated FA with DWB20-120L and DWB0-120R diets (37\u201338 g/100 g FA) resulting from this study were lower than those recorded in meat from light lambs of the Leccese breed (from 50 to 55 g/100 g FA)  and the Compared to the DWB0-120L diet, the DWB20 diet offered ad libitum was responsible for the rise in unsaturated FA, which occurred as a consequence of the reduced saturated FA, and despite the reduction of \u03b1-linolenic acid (ALA). Since ALA represents the precursor of the long-chain n-3 FA, its reduction could be linked to the major biosynthesis of EPA, DPA and DHA that emerged with the DWB20-120L diet. Significant interactions also emerged for the health indexes used to assess the health value of meat fat; indeed, when the lambs were fed the DWB20 diet ad libitum, the trombogenic index and the h/H ratio improved by decreasing, whereas the health promoting did not decrease, as occurred with the DWB20 restricted diet.Moreover, the DWB20-120L diet led to improvements in the PUFA/SFA and UFA/SFA ratios, approaching the level recorded with the DWB0-120R. In particular, the ratio PUFA/SFA of both DWB0-120R and DWB20-120L diets reached closer values to that recommended for human health (0.45) .On the whole, these results show that the presence of DWB in the diets of lambs increased the level on n-3 FA in the meat, especially EPA and DHA, reducing the n-6/n-3 ratio; moreover, when the DWB was offered in a greater amount, as obtained when the DWB20 concentrate was fed ad libitum, the byproduct was able to further improve the health profile of meat FA by reducing the saturated FA, and increasing the more beneficial unsaturated FA and the PUFA/SFA and UFA/SFA ratios.The feeding plans proposed in this investigation, consisting of a feed restriction at 75% applied after 90 days of age (DWB0-120R), or based on the inclusion of DWB as a fibrous source in a diet offered constantly ad libitum (DWB20-120L), were both suitable to increase the slaughter age of dairy breed lambs to up to 120 days, and produce lean carcasses of about 15 kg, which are heavier than those of traditional milk-fed lambs. Regardless of diet, the quality of meat from 120-day lambs, compared to that from 90-day lambs, improved in terms of major water retention, tenderness and lightness. The use of DWB reduced the fat level of 120-day lamb meat and improved its health properties by increasing the polyphenols content, the antioxidant capacity and the level of n-3 polyunsaturated FA. However, the use of DWB associated to feed restriction is not recommended, since it resulted in a lowest growth performance and carcasses weight of 120-day lambs.Ultimately, the results obtained applying the DWB0-120R or DWB20-120L feeding plan were analogous in terms of lambs\u2019 growth rate, feed conversion and carcass weight. Thus, the choice between them depends on the preferred method to limit feeding costs, which can be obtained by reducing feed intake by 30% due to the feed restriction, or by using a less expensive feeding source, such as the DWB, but also taking into account the benefits linked to the use of the DWB in terms of environmental sustainability, the oxidative stability of meat and protection of consumers\u2019 health."}
+{"text": "A variety of single-stranded DNA-binding proteins (ssDBPs) and single-stranded RNA-binding proteins (ssRBPs) have evolved that bind ssDNA and ssRNA, respectively, with varying degree of affinities and specificities to form complexes. Structural studies of these complexes provide key insights into their recognition mechanism. However, computational modeling of the specific recognition process and to predict the structure of the complex is challenging, primarily due to the heterogeneity of their binding energy landscape and the greater flexibility of ssDNA or ssRNA compared with double-stranded nucleic acids. Consequently, considerably fewer computational studies have explored interactions between proteins and single-stranded nucleic acids compared with protein interactions with double-stranded nucleic acids. Here, we report a newly developed energy-based coarse-grained model to predict the structure of ssDNA\u2013ssDBP and ssRNA\u2013ssRBP complexes and to assess their sequence-specific interactions and stabilities. We tuned two factors that can modulate specific recognition: base\u2013aromatic stacking strength and the flexibility of the single-stranded nucleic acid. The model was successfully applied to predict the binding conformations of 12 distinct ssDBP and ssRBP structures with their cognate ssDNA and ssRNA partners having various sequences. Estimated binding energies agreed well with the corresponding experimental binding affinities. Bound conformations from the simulation showed a funnel-shaped binding energy distribution where the native-like conformations corresponded to the energy minima. The various ssDNA\u2013protein and ssRNA\u2013protein complexes differed in the balance of electrostatic and aromatic energies. The lower affinity of the ssRNA\u2013ssRBP complexes compared with the ssDNA\u2013ssDBP complexes stems from lower flexibility of ssRNA compared to ssDNA, which results in higher rate constants for the dissociation of the complex  is very challenging due to their high flexibility. Furthermore, the interface between proteins and single-stranded nucleic acids is often chemically more heterogeneous than the interface between proteins and double-stranded DNA. In this study, we developed a coarse-grained computational model to predict the structure of complexes between proteins and single-stranded nucleic acids. The model was applied to estimate binding affinities and the estimated binding energies agreed well with the corresponding experimental binding affinities. The kinetics of association as well as the specificity of the complexes between proteins and ssDNA are different than those with ssRNA, mostly due to differences in their conformational flexibility. Interactions between nucleic acids and proteins are essential and central to many biochemical processes. Protein\u2013nucleic acid complexes have very diverse structures and the interface may depend on both the shape of the protein and the structure of the nucleic acid. The diversity of DNA and RNA sequences dictates their structures, which in turn control their binding specificity to proteins. The structure of protein\u2013DNA complexes may vary and sometimes even small nuances in the geometrical parameters of the major or minor grooves are fundamental to achieving specificity ,2 and thCompared with dsDNA, ssDNA structures are highly flexible \u201312 and tThe situation is somewhat similar with respect to ssRNAs, which are an important component of RNA biology ,16. RNA Studying the conformational heterogeneity of ssDNA and ssRNA is challenging using common approaches because they can provide only limited information either on the global conformation or on the detailed molecular characteristics. Nevertheless, ssDNA and ssRNA were studied by atomic force microscopy AFM; ,18), flu, flu18])Interactions between ssDNA and ssDBP or between ssRNA and ssRBP are fundamentally different from the interactions of dsDNA or dsRNA with proteins. Predicting their structures is complicated by the much greater flexibility of ssDNA/ssRNA compared with their double-stranded analogs. In many cases, ssDNA/ssRNA molecules of variable sequences but of similar length are able to adopt different conformations to engage with the same protein binding site. It was reported that the binding mode adopted is affected by salt concentration. For example, an ssDBP interacts differently with ssDNA at low and high salt concentrations . In the Although electrostatics (in which the negatively charged backbone of the nucleic acid is attracted to the positively charged residues on the binding surface of the protein) plays a crucial role in the interactions of both single and double-stranded nucleic acids with proteins, ssDNAs and ssRNAs are highly flexible in solution and thus they do not possess a definite shape . By contS. pombe binds a hexanucleotide ssDNA sequence strongly with an equilibrium dissociation constant, KD, in the nano-molar range but does not bind when a single nucleotide at the center of the sequence is altered  calculated for the near-native structures  The Cold shock protein from ures see .A very low \u03bb (<<1)for the B protein of Bs-Csp (Bs-CspB) indicates the importance of aromatic interactions for this protein, with this also clear from the predicted structures in i.e., 0.2>\u03bb>0.4, see In the coarse-grained model, the contribution of the aromatic energy to the stability of the interface between ssDNA and the telomeric proteins was 2\u20134 times higher than the contribution of the electrostatic energy  of the simulated complexes were compared with the experimentally measured equilibrium dissociation constants (KD). bind and ln(KD) for different oligonucleotide sequences that bind six ssDBP and three ssRBPs. For Pot1pc, we calculated Ebind for seven different ssDNA sequences for which structures are available[KD values are in good agreement with Ebind (r = 0.66) indicating that the model captures the energetics of interaction between various proteins and ssDNA as well as ssRNA. In each case, Ebind was calculated by considering only near-native conformations  (2ES2.pdb) that binds with nM affinity, the other one with hexa-Uracil (dU6) (3PF5.pdb), whose binding is weaker than that of dT6 but nevertheless in the nM range. The Bs-CspB binding site can interact with six to seven nucleotides[6 for all ssDNA sequences. Starting from dT7, T was progressively replaced by C to investigate their preferences at each position, as was tested experimentally. The binding constant KD of the resulting sequences varied in the \u03bcM to nM range, showing a preference for poly-Thymine over poly-Cytosine. Likewise, for all nine ssRNA sequences, the bound conformations were considered to be the same as in the Bs-CspB.dU6 complex. The binding constant of ssRNAs also varied in the \u03bcM to nM range, however the values were lower than for ssDNAs.To compare Ecleotides. The nuccleotides. In the bind versus KD plot for 13 ssDNA (solid red circles) and nine ssRNA  that bind to Bs-CspB is shown in bind) of the polythymine and polycytosine sequences in the coarse-grained model indicate that the former is more stable. However, the Ebind was less sensitive in predicting the effect on KD of a single mutation at different positions. This is expected, as achieving such accuracy is beyond the scope of any coarse-grained model. Nonetheless, results from our simulations agreed well with the experimental binding affinities when nucleotide content was taken into account, and thus such simulations can be used in binding specificity predictions.The Ekon) was estimated by the elapsed time for binding (defined by Dconf <5\u00c5) when starting from an unbound state, and similarly the rate constant for dissociation (koff) was estimated by the elapsed time for dissociation (defined by Dconf>5\u00c5) when starting from the bound complex. The association constant ratio kon(ssDNA)/kon(ssRNA) from the coarse-grained simulations is ~1, in very good agreement with the experimental data. The dissociation constant ratio koff(ssDNA)/koff(ssRNA) from the simulations is ~0.2. The value of this ratio based on the experimental results is 0.1[D of Bs-CspB\u2013ssRNA compared with Bs-CspB\u2013ssDNA.To understand the origin of the higher affinities of Bs-CspB for ssDNA than for ssRNA, we used the simulated binding events to estimate the association and dissociation rates for the interactions of the GTCTTTA ssDNA sequence and GUCUUUA ssRNA sequence with the cold shock protein, for which experimental kinetic results are available. Computats is 0.1, yet botThe energy contribution from electrostatic and aromatic interactions plays a significant role in ssDNA/ssRNA binding with proteins. Nevertheless, it is not only the charged or aromatic side-chains that interact with the nucleic acid backbone or bases, respectively, to govern the protein\u2013ssDNA/ssRNA assembly. For example, some charged residues that do not interact directly with DNA or RNA can still have a strong electrostatic effect on binding ,81. UnboThe flexibility of ssDNA or ssRNA is often judged by their persistence length, where a lower persistence length value corresponds to greater flexibility. The persistence length of both ssDNA and ssRNA decreases with increasing salt concentration. HoweverKD values is much weaker  while their association rate constants (kon) are of similar values. The complexes of ssRNA are also found to be less specific than those of ssDNA, which might be linked to their greater stiffness.Our results suggest that the origin for the weaker stability of the complexes formed between proteins and ssRNA compared with ssDNA is the lower flexibility of ssRNA. The lower affinities of ssRNA\u2013ssRBP compared with ssDNA\u2013ssDBP are coupled with larger dissociation rate constants , to S1 FigThe population distribution of predicted conformations is shown for ssDBP\u2013ssDNA  and ssRBP\u2013ssRNA  complexes. The plots are similar to those presented in Fifure 2 but for six different ssDBP-ssDNA and ssRBP-ssRNA. Representative conformations from three regions marked 1\u20133 in the current figure are shown in (TIF)Click here for additional data file.S2 FigD1, D2 \u2264 5\u00c5).The heterogeneous model refers to the model presented in the current manuscript and the homogenous (polyT) model refers to the model presented in ref. # 47. The number in the right-bottom corner of each panel corresponds to the percentage of native-like conformations ((TIF)Click here for additional data file.S3 FigThe regions are labelled 1, 2, and 3 in (TIF)Click here for additional data file.S4 FigGroup A and B corresponds to the backbone and base beads, respectively. This analysis was performed for the native-like conformations.(TIF)Click here for additional data file.S5 Fig-1) is plotted versus DSite and DConf for each of the ssDBP\u2013ssDNA  and ssRBP\u2013ssRNA  complexes. The points encircled in green, red, and blue correspond to the respective ssDNA/ssRNA conformations shown in The binding energy Click here for additional data file.S6 FigThe complexes between Pot1pc (4HIO) and Cdc13 (1S40) telomeric proteins and seven different sequences of ssDNA were studied. The energy plots demonstrate that the specific positions of ssDNA bases with respect to the aromatic residues  dictate the binding specificity for heterogeneous sequences. The effect of sequence shuffling is larger for 4HIO with ssDNA comprise all four nucleotides than the more homogeneous ssDNA sequences for 1S40 in which the interface also does not have any Trp.(TIF)Click here for additional data file.S7 FigDsite. For most systems, the aromatic interactions govern the shape of the energy landscape for binding. The exceptional case is 3VKE that is stabilized by electrostatic interactions.The total binding energy (right column) is decomposed into aromatic energy (left column) and electrostatic energy (middle common) along (TIF)Click here for additional data file."}
+{"text": "The aim of this study was to analyze the safety and effectiveness of stenting using partially covered self-expandable stents in palliation of dysphagia in patients with unresectable esophageal cancer.Retrospective analysis of hospital records of all patients who underwent esophageal stenting in the period 2008\u20132015 was performed. The study included patients with unresectable esophageal and esophagogastric cancer.p\u2009=\u20090.00001). During the follow-up, 55 (12.4%) patients experienced recurrent dysphagia due to tumor or granulation tissue overgrowth, and in 18 (4.1%) patients, migration of the stent occurred, for which an independent risk factor was adjuvant chemo- and/or radiation therapy (p\u2009=\u20090.001). Minor complications included chest pain (54.5%), delayed complete stent expansion (12.0%), feeling of a foreign body (25.3%), hiccup (1.6%), gastroesophageal reflux (45.6%) and post-discharge pneumonia (2.5%). A feeling of a foreign body in the esophagus was significantly more common after stenting of the cervical esophagus (p\u2009=\u20090.0001), and hiccup was more common after stenting of the esophagogastric junction (p\u2009=\u20090.02). Major complications included bleeding (1.3%), respiratory insufficiency (0.7%), esophageal perforation (0.9%) and irregular heartburn (2.3%). Overall procedure-related mortality was 0.4%. The median survival time was 117.8\u00a0days (range 2\u2013732).There were 442 patients included. Mean age was 56\u00a0years (range 28\u201389), and 379 were males. In 40 (9.0%) patients, stenting was performed in the cervical, in 150 (39.3%)\u2014in the middle thoracic, in 141 (31.9%)\u2014in lower thoracic esophagus and in 111 (25.1%)\u2014in the esophagogastric junction. Stenting resulted in significant alleviation of dysphagia grade (3.0 vs. 1.0, Stenting is an effective procedure in relieving dysphagia in patients with unresectable malignant esophageal stenosis and is associated with low rate of postoperative and long-term complications. Squamous cell carcinoma of the esophagus is the eighth most frequent cancer in the world, whereas carcinoma of esophagogastric junction (OGJ) is the tumor with the highest dynamics of incidence in the last two decades \u20134.It is estimated that in North America there are 5\u201310 cases of esophageal cancer per 100,000 inhabitants; however, depending on the geographical area, the incidence may increase up to 100/100,000, such as in Iran .Multimodal treatment consisting of preoperative chemoradiation therapy followed by complete resection and lymphadenectomy is a standard in therapeutic management. However, curative-intent treatment is possible in only 20\u201340% of patients. In a majority of them, palliative management is the only option, due to local advancement and/or distant metastases . In suchIn this study, we present one of the largest retrospective analyses with prospective follow-up of patients with esophageal cancer, who underwent esophageal stenting due to unresectability of the tumor or medical inoperability. The aim of this study was to evaluate the safety and efficacy of stenting in patients with esophageal squamous cell carcinoma and carcinoma of the esophagogastric junction, complications, re-interventions and survival after the treatment.This retrospective analysis of hospital records included data of a consecutive group of patients with advanced esophageal carcinoma, treated between 2008 and 2015 in Department of Thoracic Surgery, Jagiellonian University Collegium Medicum. Demographic and clinical data including age, sex, weight, dysphagia, dyspnea, chemotherapy/chemoradiation, technical success rate, stent migration, complications and survival were evaluated.The study included all patients treated in the period 2008\u20132015 for unresectable or medically inoperable esophageal or (OGJ) cancer, regardless of histological type.Preterminal condition, Karnofsky score\u2009\u2264\u200940%;Patients with mediastinal infiltration causing dysphagia in the course of lung cancer, lymphomas and other malignancies.Exclusion criteria included:0\u2014no dysphagia;1\u2014swallowing of a semiliquid diet;2\u2014swallowing of a liquid diet;3\u2014dysphagia to the liquids and saliva.Unresectability was determined on the basis of chest radiography, abdominal ultrasound, computed tomography (CT) of the chest and the upper abdomen, positron-emission tomography (PET) and endoscopy, with the endoscopic ultrasound (EUS) and endobronchial ultrasound (EBUS). Disease staging was based on the UICC classification . DysphagType 1\u2014fistula to the mediastinum;Type 2\u2014fistula to the trachea;Type 3\u2014fistula to the bronchus;Type 4\u2014fistula after stenting.Patients diagnosed with fistula in the course of esophageal or bronchogenic cancer were classified into four groups according to fistula location :Type 1\u2014f0\u2014less than 30% of tracheal or/and bronchial stenosis, no dyspnea;1\u201430\u201350% stenosis, dyspnea upon exercise;2\u201450\u201370% stenosis, dyspnea during daily activities;3\u2014more than 70% stenosis, dyspnea while resting.Dyspnea severity was assessed with a four-grade scale :0\u2014less tPatient performance status was assessed according to Karnofsky score .Esophageal stenting was performed under general anesthesia. Location of the stenosis was endoscopically identified, and in case of narrow stenosis, dilatation was performed with Savary\u2013Gilliard dilators, up to the size of 10 Fr. After the dilatation, the neoplastic infiltration length was assessed using a small-diameter endoscope, then a guidewire was inserted and the esophageal stent was introduced over it. Deployment of the stent was performed under endoscopic control. Partially covered self-expandable metallic stents  were used.Airway compression or infiltration posing the risk of severe airway compromise after expansion of the esophageal stent.Double stenting was performed in patients with unresectable esophageal cancer involving the airway, with dysphagia and dyspnea;As a rule, airway stenting was performed before esophageal stenting. The double stenting procedure was performed under general anesthesia. The self-expandable Ultraflex stents  were used for stenting of fistulas to the trachea and silicone Y stents  in case of fistula located in the tracheal bifurcation and main bronchi. Stenting with the use of silicone Y stents was performed using the Freitag forceps according to the technique described elsewhere .Peri-, intra- and postoperative complications and any additional procedures were recorded. Complications after stenting were classified as minor or major. Minor complications were defined as those subsiding spontaneously or following pharmacological treatment only, potentially requiring endoscopy. All other, including life threatening or fatal, were defined as major complications.Postoperative complications were defined as early (occurring within\u2009\u2264\u200930\u00a0days following stenting) or late (occurring later than\u2009>\u2009\u200a30\u00a0days following stenting).Following the procedure, patients received a liquid diet the same day. Routinely, on the first day after the procedure a follow-up chest radiogram was obtained and dyspnea and dysphagia scores were assessed. Patients received detailed instructions regarding nutrition at discharge from the hospital. Patients were followed up every 30\u00a0days thereafter. If the follow-up visit in the clinic was not feasible, patients were interviewed by phone. During the follow-up visit, the patients\u2019 general condition, dysphagia and dyspnea were assessed.p\u2009<\u20090.05 was considered as statistically significant.Statistical analysis was performed using the STATISTICA 11 PL software package . The Mann\u2013Whitney test was used to compare two samples. Kruskal\u2013Wallis test was used to compare three or more attempts. In order to evaluate the changes over time , the Wilcoxon signed rank test was applied. To assess the significance of connections between data on nominal scale, the Fisher\u2019s Chi-square test or Fisher\u2019s exact test was used. The logistic regression model was used to find the risk factors for complications after stenting. If an important factor was found, odds ratio (OR) was calculated along with 95% confidence interval. Survival was calculated using Kaplan\u2013Meier method. Gehena\u2013Wilcoxon test was used to compare survival curves. 46 patients with lung cancer;2 patients with thyroid cancer;1 patient with colorectal cancer;1 patient with breast cancer;7 patients with lymphomas;45 due to the preterminal state or Karnofsky score\u2009\u2264\u200940%;14 patients who were lost from postoperative follow-up;48 patients stented before planned surgical resection.Between 2008 and 2015, 606 patients underwent esophageal stenting for malignant esophageal obstruction. The flowchart of the study is presented in Fig.\u00a0The final analysis included homogenous group of 442 patients with esophageal or OEJ cancer, who underwent esophageal stenting procedure.Patients presented with body weight loss from 4 to 40\u00a0kg, dysphagia, cough and cachexia. The mean length of neoplastic infiltration in the esophagus was 5.9\u00a0cm (range 4\u201312\u00a0cm).28 (6.3%) and 30 (6.34%) patients with the tumor located between 18 and 21\u00a0cm from the incisors;12 (2.7%) and 18 (4.16%) patients with the tumor located between 22 and 25\u00a0cm from the incisors.In 40 (9.0%) patients, stenting of the upper segment of the esophagus was performed, including:In 150 (39.3%) patients, stenting was performed in the middle part of the esophagus, in 141 (31.9%)\u2014in the lower thoracic part of the esophagus and in 111 (25.1%)\u2014in the OGJ.Nineteen (4.3%) patients had primary fistula to the mediastinum or the airway. Fifteen (3.04%) patients with fistula developed after the stenting procedure. Adjuvant chemo- and radiation therapy was administered to 201 (45.5%) patients (Table\u00a0Stenting procedure could not be performed in 3 (0.6%) patients due to complete obstruction of the esophagus. These patients underwent laparotomy and gastrostomy. Thus, the technical success rate was 99.4%.p\u2009=\u20090.00001).After stenting procedure, swallowing improvement was observed in all the patients. The mean dysphagia score improved from 3.0 (range 2\u20133) before stenting to 1 (range 1\u20132) after the stenting procedure (p\u2009=\u20090.004). In 209 (42.3%) patients, mild analgesia was required and the pain subsided within 2\u20134\u00a0days after the procedure, whereas in 33 (7.5%) patients long-term analgetic medication was needed.After esophageal stenting, 241 (54.5%) patients reported chest pain: in 28 (6.3%) patients with stent in the proximal esophagus, in 94 (21.2%)\u2014in the middle part, in 78 (17.6%)\u2014in the lower part and in 49 (11.0%)\u2014in the EGJ. Pain occurred more frequently in patients with stents the proximal and middle part of the esophagus (Incomplete immediate stent expansion occurred in 53 (12.0%) patients. In all of them, the stent expanded fully without any intervention within 48\u00a0h.p\u2009=\u20090.0001). These symptoms subsided completely or partially within 3\u20137\u00a0days.Difficulties in swallowing, associated with the feeling of a foreign body, were reported by 112 (25.3%) patients, including 29 (6.6%) with a stent in the proximal esophagus, 30 (6.8%)\u2014in the middle part, in 21 (4.8%)\u2014in the lower part and in 29 (5.7%) of them after the stenting of OGJ. The feeling of a foreign body was present only in patients with stents in the proximal part of the esophagus (p\u2009=\u20090.02), requiring stent removal in 1 (0.2%) patient. In 3 patients with squamous cell carcinoma and in 4 with OGJ, carcinoma early migration of the stent occurred. Two hundred and two (45.6%) patients reported reflux symptoms, and they required treatment with proton-pump inhibitors. After discharge from the hospital, in 11 (2.5%) patients pneumonia occurred and they received outpatient treatment.Hiccup occurred in 7 (1.6%) patients after esophageal stenting: In 4 of them, it happened after stenting of the EGJ and required stent removal in 3 cases, and in 3 (0.7%) patients after stenting of the lower part of the thoracic esophagus  patients, and in 3 (0.7%) of them, transfusion of 2\u20134 units of packed red blood cells was necessary.Irregular heartburn occurred in 10 2.3%) patients (Table\u00a0.3% patiep\u2009=\u20090.06) ]. There were no significant differences in the migration rates when SCC was compared with adenocarcinoma of the OGJ (p\u2009=\u20090.06).In 18 (4.1%) patients, migration of the stent occurred. It happened in the middle thoracic part of the esophagus in 3 (0.7%) the patients, in the lower part in 7 (1.6%) patients and in 8 (1.8%) patients in the OGJ (6) Table\u00a0. AdjuvanIn 7 patients with squamous cell carcinoma and in 4 with OGJ carcinoma, late migration of the stent occurred.p\u2009=\u20090.54). In patients with stent obliteration by tumor or granulation tissue ingrowth, restoration of patency was performed with the use of argon plasma coagulation followed by re-stenting.Dysphagia associated with stent obliteration by the ingrowing granulation tissue occurred in 55 (12.4%) patients: in 46 (10.4%) at the proximal end of stent and in 9 (2.0%) patients at the distal end. In 2 (0.4%) patients, ingrowing tumor was observed. Development of granulation tissue was observed in the period from 27 to 103\u00a0days (mean 72\u00a0days) since stent implantation. In 5 (1.1%) patients, ingrowing granulation tissue was observed in the proximal part of the esophagus, in 16 (3.6%) patients in the middle part, in 19 (4.3%) patients in the lower part and in 16 (3.6%) patients in the EGJ ].Stent removal and re-stenting procedure were required in 28 (6.3%) and 31 (6.3%) patients, respectively. Forty-six (10.4%) patients with stent obstruction received CTH and/or RTH, which was a risk factor for stent obstruction [In 4 (0.9%) patients, esophageal stenting resulted in compression of the airway. In 2 (0.4%) of them, symptoms of dyspnea required additional stenting of the bronchial tree with Y stent; in the remaining 2 (0.4%) patients, compression of the bronchial tree\u2009<\u200930% of lumen occurred, without symptoms of dyspnea and not requiring additional stenting. These patients were subjected to follow-up (Table\u00a0p\u2009=\u20090.06). Median survival time in patients with OAF was 74.5\u00a0days (range 41\u2013432).The follow-up period ranged between 1 and 732\u00a0days. Median survival time was 117.8\u00a0days (range 2\u2013732) , dyspnea score  and Karnofsky score  was achieved. There was no significant improvement in BMI . The median survival after stenting of the OAF was 74.5\u00a0days (range 41\u2013432) days. The survival did not correlate with the use of chemotherapy or chemoradiation (p\u2009=\u20090.54).Esophago-airway fistula (OAF) was found in 34 (7.7%) patients Table\u00a0. NineteeA primitive stent was used for the first time for intubation of the esophageal stenosis in 1885, and rapid development of stenting occurred together with the development of endoscopy . In the One of the most frequent complications of esophageal stenting is ingrowth or overgrowth of granulation tissue or tumor at the ends of the stent. In our study, the percentage of tissue overgrowth was 11.99%, with the applied stenting margin of 4\u00a0cm. These results are consistent with the literature data, showing its incidence of 4\u201347% (higher in cases of the use of non-covered stents) , 18. ResThe second most frequent complication is stent migration, which reportedly occurs in 0\u201320% cases \u201323. Our Although esophageal stenting is characterized by a very high technical success rate, it is associated with the risk of life-threatening complications. One of them is development of an OAF following the procedure. According to the literature, it occurs in up to 10% of patients after the stenting procedure. Ferreira et al. reported the occurrence of such fistulas in 7 out of 126 treated patients and Uitdehaag et al. in 2 out of 44 patients, after the application of SX Ella stents with anti-migration mechanism , 32. In Other most frequent severe complications include: hemorrhage, which occurs in 2\u201328%, perforations, perioperative mortality, which was estimated to be 0.5\u20137%, and a 30-day mortality that ranges from 7 to 18% , 32\u201337. Esophageal stenting may enable the introduction of chemotherapy or chemoradiation. Chemotherapy enables relief of dysphagia and full oral nutrition, so the European Society of Gastrointestinal Endoscopy recommends its administration , 39. TheGenerally, the use of partially covered self-expandable metallic stents in inoperable/unresectable esophageal cancer is safe and effective palliative procedure, with low rate of complications and perioperative mortality."}
+{"text": "We aimed to evaluate the safety and efficacy of combined airway and esophageal stents under fluoroscopy guidance and local anesthesia for patients with malignant tracheobronchial and esophageal disease. This retrospective analysis included 35 consecutive patients underwent combined stenting from March 2012 to August 2016. All patients underwent chest computed tomography scans before stenting and during follow-up. Thirty-nine airway stents and 43 esophageal covered stents were implanted. The indication of stenting, technical success and postinterventional complications were collected and analyzed. Thirty-nine airway stents and 43 esophageal covered stents were implanted. Stenting failed in 1 airway stent, and 2 esophageal stents, with technology success rates of 97.4% and 95.3%, respectively. No procedure-related death occurred, only 1 patient died from failure of respiration due to esophagotracheal fistula. The median interval between 2 stenting was 13.0 days. Both dyspnea and dysphasia were significantly relieved after stenting. Restenosis after stenting (7.7%) was the most common complication for airway stenting, all these cases required second stenting. Stent migration (7.0%) was the most common complication after esophageal stenting, 1 case had to receive airway stenting and 1 case received replacement of esophageal stent. During follow up, 23 patients were clinically cured, 2 patients were improved in symptoms, and 1 was invalid. Eight deaths were found in total. The 1-year, 3-year, and 5-year survival rates were 82.4%, 78.8%, and 78.8%, respectively. In conclusion, combined airway and esophageal stents implantation under fluoroscopy guidance and local anesthesia are safe and effective for malignant tracheobronchial and esophageal disease. Those patients are usually poor candidates for surgical treatment due to the advanced tumor stage.\u20136 The palliative treatment simply relieve patient's symptom to improve the patient's quality of life. The self-expandable metallic stent can effectively relieve airway and esophageal stricture, which is widely used clinically for patients with airway\u201312 or esophageal stenosis.\u201315 Patients with advanced lung or esophageal cancer often have respiratory or esophageal distress/fistula caused by tumor invasion, and stenting therapy not only for esophagus but also for airway. Although 80% of esophagotracheal fistula can be palliated by esophageal stenting, an additional seal can also be provided by airway stenting if esophageal stent failed. Small case series have reported combined stents for management of combined malignant airway and esophageal stenosis\u201319 or solely to allow safe placement of the esophageal stent. Combined stenting could be necessary for approximately 9% to 27.5% of patients presenting with tracheobronchial fistula.\u201323 However, majority of these small sample studies were performed under general anesthesia and tracheal intubation,19,20,22 by using rigid bronchoscopes and esophagoscopes,,19,22\u201324 less is known regarding the management of this complex condition under fluoroscopy guidance. In this study, we determined the safety and feasibility of combined stenting in the management of esophagostenosis, tracheostenosis, or esophagotracheal fistula.Airway and esophagus may be involved simultaneously or successively by esophageal cancer, lung cancer due to the close anatomic relationship.2This retrospective study was approved by the committee board of Zhengzhou University, all procedures were performed in accordance with the guidelines and regulations for clinical study. Informed consents were obtained from all participants enrolled in this study. This study included 35 consecutive patients , who underwent combined stenting from March 2012 to August 2016. Combined stents were needed for patients with esophagostenosis combined with tracheostenosis, and airway stent was placed before esophageal stent under this circumstance. Otherwise, the stents were placed according to the order of occurrence of the airway and esophageal stenosis. Esophageal stenting was the first choice for esophagotracheal fistula, and airway stent was used if necessary, such as failure of esophageal stent, recurrence of fistula, or airway stenosis compressed by esophageal stent, and so on. Airway stenting was performed for patients with airway stenosis and/or airway fistula, and esophageal stenting performed if repeated failure or recurrence of airway diseases. Airway stents were intended to remove at a time interval of 3 to 6 months to avoid the long-term complications, especially severe airway stenosis, or left in for the long-term when patients showed a survival time may less than 6 months. Esophageal stents were left in for the long-term, and removal performed only for patients with complications, such as recurrence caused by significant migration, airway compression, or severe restenosis.2.1All patients underwent chest computed tomography (CT) scans before stenting and during follow-up. Pre procedure bronchoscopy/endoscopy performed if necessary, such as confirm diagnosis, treatment for severe stenosis. The airway or esophageal stenosis was diagnosed according to the patient's symptoms, history, and results of chest CT with or without bronchoscopy/endoscopy. The locations, length, and grade of airway and esophageal stenosis was evaluated and measured before stenting. All measurements, proximal and distal landing zones and selection of stent diameter and length were based on chest CT scanning.2.2All airway covered stents were individually manufactured . The straight airway covered stent range 18 to 26\u200amm in diameter and 40 to 100\u200amm in length. Large Y-shaped stent was used for patients with stenosis or fistula in bilateral main bronchi. Diameter of the main body, left main bronchus, right main bronchus range 18 to 22\u200amm, 12 to 14\u200amm, and 12 to 16\u200amm, respectively, and the length of main body, left main bronchus, right main bronchus were 30 to 60\u200amm, 15 to 35\u200amm, and 10 to 25\u200amm, respectively  was introduced, a 0.035-inch stiff guide wire (Cook Corporation) was then advanced, and stent was implanted along the stiff wire . A 12 to 14 F long sheath was prepared to manage the airway and ventilation during airway stent placement or removal.2.4 . Clinical cure defined as successful management of disease without complications or symptom.Technical success of stenting was defined as exact stenting with no severe procedure-related complications. Peroperative stenting failure was defined as severe stent migration or repeated migration required stent removal; or stent release failure due to the technology factor. The Hugh\u2013Jones classification was used to assay dyspnea before and after airway stenting. Dysphagia was also evaluated before and after esophageal stenting according to previous report2.5All patients underwent chest CT within 1 week after stenting to confirm the location of stents. The chest CT, clinical examination, or bronchoscopy/endoscopy were performed 1 month after stenting and then about every 3 months thereafter during follow-up. Telephone follow-up was carried out for patients unable to visit hospital.2.6t test was performed to compare continuous variables. Fisher exact test was used for compare incidence of complications. A P\u200a<\u200a.05 was considered statistically significant.All statistical calculations were performed using Prism 5.0 software . Descriptive statistics were used to describe the conditions of patients. Continuous variables are summarized as mean\u200a\u00b1\u200aSE. The student 33.1One airway stent failed to release due to the stent and binding wire was wound together. After repeated attempts, the stent had to removed, and airway stent was successfully placed again 1 week later. A chest CT obtained within 1 week after stenting confirmed the correct location of all airway stents. The correct location of esophagus stents within 1 week after stenting was confirmed in 35 patients. Two esophageal stents failed to treat fistula due to migration 2 and 5 days after stenting, and airway stents were used. The technology success rates were 97.4% and 95.3% for airway and esophageal stent, respectively. The mean stenting time was 30.4\u200a\u00b1\u200a3.4\u200aminutes and 34.5\u200a\u00b1\u200a3.4\u200aminutes for airway and esophagus stents, respectively , airway stent restenosis n\u200a=\u200a3), esophagostenosis combined tracheostenosis due to esophageal carcinoma (n\u200a=\u200a3) were the main indications for airway stenting. Recurrence of esophagotracheal fistula after stenting (n\u200a=\u200a6), esophagotracheal fistula (n\u200a=\u200a3), malignant esophagostenosis (n\u200a=\u200a4) were the most common indications for esophageal stenting. Esophagostenosis combined tracheostenosis due to esophageal cancer or lung cancer (n\u200a=\u200a12) and esophagotracheal fistula with/without esophagotracheal stenosis (n\u200a=\u200a10) were indications for both airway and esophageal stenting  was the most common complication for airway stenting, all these cases required second stenting. Stent migration (7.0%) was the most common complication after esophageal stenting Fig. . All cas3.5Eleven esophagus stents and 2 airway stents were withdrawn in 11 patients; the mean indwelling duration of stent ranged 2 to 270 days. Two esophagus stents and 1 airway stent were regularly removed according to doctor's advice to avoid long-term complication. Two esophagus stents and 1 airway stent were removed due to repeated migration of the stent. Three esophagus stents were withdrawn due to airway compression, and 2 due to restenosis of esophageal stent. Four esophagus stents and 1 small y type airway stent were implanted immediately after esophagus stent removal owing to failure recovery of esophagotracheal fistula (n\u200a=\u200a1), restenosis of esophagus (n\u200a=\u200a3), or retained stent pieces (n\u200a=\u200a1).3.6P\u200a<\u200a.0001). The mean dysphagia grade decreased from 3.2\u200a\u00b1\u200a0.2 to 1.2\u200a\u00b1\u200a0.1 after esophageal stenting (P\u200a<\u200a.0001). During follow up, 23 patients were clinically cured, 2 patients were improved in symptoms, and 1 was invalid.The symptoms of esophagotracheal fistula improved after placement of covered stent. Both respiratory and dysphagia symptoms were immediately improved in all patients after stenting. The mean Hugh\u2013Jones grade decreased from 3.0\u200a\u00b1\u200a0.1 to 1.3\u200a\u00b1\u200a0.1 after airway stenting  was the main and severe complication after esophageal stenting in our study, which was lower than the previous report.Airway and/or esophageal stent insertion provided an effective approach to improve the quality of life in patients with malignant airway\u2013esophageal fistula.,22 however, less than 10 patients had fistulae before stent therapy.,18 Nomori et al reported a high risk of fistula occurring due to necrosis of airway/esophageal walls, and occurrence/recurrence of fistula due to growth of fistula after stenting. Besides, considering that there is a high risk of fistula occurring after combined stents placement in the future, the covered metallic stents should be used in esophageal cancer invading the airway, even for patients without esophagotracheal fistula. In this study, no fistulae were caused by combined stenting.Combined stenting has also been reported in the treatment of esophagotracheal fistulas using rigid bronchoscopes and esophagoscopes;,12,26 most of airway stents were often performed under general anesthesia and tracheal intubation.,19,20,22 However, patients with malignant stenosis and/or fistula are usually poor candidates for surgical treatment due to the advanced tumor stage.\u20136 The esophageal metallic stents can be placed under local anesthesia,,15 and inserted under fluoroscopic guidance. All stents were implanted under local anesthesia and fluoroscopic guidance in this study.Although airway stents can be inserted under fluoroscopic guidance,This study had some limitations. First, this is a retrospective study in a single institution. Second, the sample size is still small, making it difficult to make definitive conclusions regarding this technique.In conclusion, combined airway and esophageal stents implantation under fluoroscopy guidance and local anesthesia are safe and effective for malignant tracheobronchial and esophageal disease.Study design was done by HXW and WG; Data collection was done by BYH, RJZ, CHM, and BLL; Data analysis was done by BYH, RJZ, CHM, and BLL; Written by BYH, RJZ, and WG; Study was approved by HXW, and WG.Conceptualization: Xinwei Han, Gang Wu.Data curation: Yonghua Bi, Jianzhuang Ren, Hongmei Chen, Liangliang Bai.Formal analysis: Yonghua Bi, Jianzhuang Ren.Funding acquisition: Yonghua Bi.Investigation: Yonghua Bi, Jianzhuang Ren, Hongmei Chen, Liangliang Bai.Methodology: Hongmei Chen.Project administration: Xinwei Han, Gang Wu.Resources: Yonghua Bi.Software: Hongmei Chen, Liangliang Bai.Supervision: Xinwei Han, Gang Wu.Validation: Xinwei Han, Gang Wu.Visualization: Xinwei Han.Writing \u2013 original draft: Yonghua Bi, Jianzhuang Ren, Hongmei Chen.Writing \u2013 review and editing: Xinwei Han, Gang Wu."}
+{"text": "Tramadol has gained popularity among the drugs of the most active population especially the respondents in Ghana abuse especially farmers who nicknamed as \u201cfarm and buy cow.\u201d It has recently become a public health concern, and stakeholders are worried about tramadol abuse and its implications on health in the Upper West Region. The study sought to measure the prevalence of tramadol/related substance abuse and the associated factors.  A community-based analytic cross-sectional study involving 420 respondents was conducted. The participants were selected using a multistage sampling technique. Semistructured questionnaire was used to generate the data. p = 0.009], compared to respondents with no history of any substance abuse. Respondents who take tramadol to enhance sex were 4 times more likely to abuse tramadol . Formal sector employment was protective against tramadol abuse  compared to self-employment and the unemployed. In addition, use of nonopioid prescription drugs for posttraumatic/pain management reduced the risk of tramadol abuse  compared to the posttraumatic/pain management dependence on prescription of only opioid like tramadol.  About 77.6% of the respondents abuse tramadol while 83.9% of the participants take at least one other related substance or drug. Participants with history of any substance abuse were 5 times more likely to abuse tramadol [AOR = 5.15; 95% CI (1.501-17.656);  An infantile municipality like Jirapa is challenged with high level of tramadol and related substance which has serious repercussion on the health system in the Jirapa district. It is important that measures are taken by the stakeholders to stop tramadol and related substance and mitigate the impact of drug abuse in the district. The world is in opioid crisis; in fact, prescription drug abuse has monumentally increased; cocaine and opium absolutely hitting the highest records in the history of the world presenting multiple challenges on multiple fronts . The extThe current trend of substance abuse among women is a major national concern. It has detrimental effects on women's health and behavior and could lead to death .The extent of abuse of tramadol and codeine among pupils at both basic and junior high schools in the Upper West Region is alarming . It is tTramadol is sometimes abused alongside other drugs, which is called polydrug use. Typically, users combine tramadol with other substances to increase their high or self-medicate. The following drugs are commonly combined with tramadol: alcohol, other painkillers, sedatives, like benzodiazepines and sleeping pills, and cold medicine .The study was conducted specifically in the Jirapa Municipality of the Upper West Region with a territorial size of 1,188.6 square kilometers. The municipality is divided into submunicipals for effective health services delivery purpose. There are seven (7) submunicipals administratively managed by Submunicipal Health Teams (SMHTs). The study took place in Hain, Tizza, Duori, Tuggo, Yaga, Sabuli, and Jirapa urban submunicipals. In 2017, the entire population of the municipality is estimated to be 101,899. The LI 1902 established Jirapa District which was carved out of the then Jirapa-Lambussie District in light of Ghana's decentralization processes in 2007 and was upgraded into a municipal status in 2018.The study was an analytical community-based cross-sectional design involving the most active population within the ages 15-55 years. All persons below 15 years and above 55 years were excluded. All critical ill-patient was equally excluded. Persons who currently abuse/misuse tramadol were included in the study.Sample size was estimated using the Cochran's formula for quantitative continuous data. In addition to the 10% nonresponder rate, a sample size of 420 persons was used for the study. A prevalence of 50% was used for the estimation of the sample size with an acceptable margin of error of 5%. The respondents were selected across the seven submunicipals for the study.A multistage sampling procedure was used for the study. Stratified sampling was used to stratify the seven submunicipals into stratums. Simple random sampling procedure was used to select the communities within each stratum for the study. This was randomly done using the ENA sampling software. A comprehensive list of all households that constituted the sample frame was compiled from a chosen cluster, and systematic sampling technique was used to select the study households. The first household was selected using the table of random numbers. In the selection of the study participants, only one eligible respondent was selected using a simple random sampling technique in a household.A semistructured questionnaire made of both open and closed-ended questions was used to collect the data from the respondents. The questionnaires were personally administered in a face-to-face interview by research assistants. The tool was to enable us to collect both qualitative and quantitative data. In addition, it enables us to collect large range of different responses from respondents. Also, the choice was made for purposes of data triangulation of responses. The questionnaire was the main instrument used for the collection of data. There were some open-ended questions as well as closed or multichoice questions. The open-ended questions provided in-depth understanding into some of the reasons underpinning the use of tramadol and other abusive substances. Face to face interview was administered because other modes  would have been problematic because abusers of tramadol would have been suspicious as it is illegal to take it in Ghana.R = 0.8.Ample time were given to the respondents to study the pattern of the instruments and to answer appropriately without being rushed. Respondents' preferred choices were ticked. However, in instances where respondents cannot read and write, the data collector read out to the respondent in a language (Dagaare) that he/she understood. Piloting of the questionnaire was done a day after the practical training sessions of the enumerators. The team reconvened to discuss thoroughly about the entire exercise, and some misconceptions, flexibility issues of the data collection tool, and interpretations were further clarified. Also, the validity and reliability tests were ran to determine precision level of the tool. The determination was based on Cronbach's alpha. Some closed-ended questions were modified, and some were deleted until Data on sociodemographic and economic variables , peer pressure, weak enforcement of regulations, posttraumatic/management dependence, curiosity, psychological/mental health challenges, and ignorance were collected. Also, data on the knowledge of respondents on the effects of tramadol abuse, the perceptions of the study population on the associated benefits of tramadol use, and the abuse of other related substances/drug were taken. In addition, data on other variables like awareness of the availability of the drugs, taking the drug with or without medical officer approval, duration, and dosages of tramadol mostly patronized were collected. A rating scale was used to assess the level of perception on the benefits associated with tramadol use and the knowledge on the consequences of tramadol abuse. The rating scale sought to ascertain the degree of acceptance or rejection to some parameters associated or related with tramadol among the study group. A scale of agree, strongly agree, do not know, disagree, and strongly disagree was used for the assessment.Statistical Package for the Social Sciences (SPSS) Version 21 was used for the data entry, cleaning, and analysis. Missing data and wrong entries were checked, and all irregularities were corrected. The abuse/misuse of the tramadol by this study was defined as inappropriate use of the drug or use of the drug without physician's approval. The approved tramadol dosage strengths for use in Ghana by the FDA are 50\u2009mg and 100\u2009mg in tablets and capsules and 50\u2009mg/ml-2\u2009ml in injections, and therefore any intake above these strengths were considered as abuse.Results from the rating scale assessment were statistically transformed into dichotomous variables of yes/no. Agree and strongly agree were considered as accepted (yes) while disagree and strongly disagree were considered as rejection (no) to a particular parameter. Knowledge on the effects/consequences of tramadol was a composite indicator of the study participants who have the knowledge that its abuse/misuse has an effect on a person and the number of the study group who are knowledgeable that the abuse/misuse of the tramadol can lead to mental problems including depression, insomnia, and addiction, while the knowledge on the consequences of tramadol abuse was classified as low or high. In addition, the perception of the respondents on the benefits associated with the abuse/misuse of tramadol was categorized as having low or high perception on the benefits. It was also a composite indicator. Tramadol abuse was the main dependent variable.p < 0.5. Multivariate analysis was done to control for confounders and to exactly identify determinants/predictors of the tramadol abuse among the study population by using logistic regression analysis. All the independent variables that proved statistically significant  at the bivariate level were put for multivariate analysis. Socioeconomic and demographic variables , knowledge on the consequences of tramadol abuse , perceptions on benefits of tramadol , and other variables  with p values less than 0.05 were considered for the modeling.Descriptive statistics were generated from the data. Chi-square test was used for the bivariate analysis to establish the relationship between tramadol abuse and independent variables. A relationship was considered significant when The mean age of the study participants was 28.3 \u00b1 7 years. Most of the study participants (40.7%) were found in the age group 18-25years. About 81.4% of the respondents were males. Majority of the respondents (specify the figure) were self-employed. Most participants 191 (45.5%) had at least secondary education. About 46.7% of the participants were married whereas the majority (53.3) were single. Again, most participants (67.6%) were Christians .The prevalence of tramadol use is 36.2% among respondents in the municipality with 77.6% of the users inappropriately taking or misusing/abusing the drug. Averagely, the daily milligram intake of tramadol was 100\u2009mg \u00b1 42.6\u2009mg. About 32.9% of the participants misuse tramadol without knowing the various strength/dosages they take. Regardless of the strength, 17.1% of the study participants can take at least 4 tablets/capsules at once. The vast majority of the respondents patronize dosages \u2265 100\u2009mg .The first three commonest reasons why majority of respondents took tramadol were peer influence (38.8%), improve physical performance (37.5%), and improve physical strength/become more active (24.3%).Summary of the reasons indicate that majority (54.6%) took the drug to solve a single challenge while 29% use the drugs for multiple reasons. However, a significant number has no reason for the use of the drug . The infAlso, about 95.7% of the study participants were aware of the existence of tramadol in the municipality. Majority (81.5%) of the participants voted \u201cyes\u201d for licensed chemical shops as the main and reliable source of tramadol. Not surprising, almost 61.1% of the respondents took drug peddlers as the second most reliable source, whereas 36.7% of respondents voted \u201cyes\u201d for black markets as the third reliable source of tramadol for the respondents . The remAbout 64.5% abuse alcohol, and almost half of the respondents inhale substances popularly known as sera or Enye or snuff. Also, 17.9% and 6.4% were found abusing prohibited substances like cocaine/heroine and wee/marijuana, respectively. Interestingly, quite a number of respondents were engaged in abusing emerging substances such as \u201cWuole\u201d (17.1%), sniffing glue (6.2%), inhaling petrol/turpentine (1.9%), and drinking of soaked/boiled diapers/pads (1.4%) .p = 0.046) was observed between the educational status of the participants and abuse of tramadol. Compared to the unemployed (39.9%) and respondents engaged in formal employment (2.5%), majority (57.6%) of abusers were self-employed. However, the employment status of the respondents was significantly associated with tramadol abuse (p = 0.003). A statistically significant positive association also was observed between religion and tramadol abuse. In spite of this, about 59.4% of the abusers were Christian. Finally, it was gender/sex showed a strong positive association with tramadol abuse (p = 0.009).About 38% and 34% of the abusers attained at least secondary education and basic education, respectively. The remaining 28.8% had no formal education . A positp = 0.027). Also, taking tramadol for purposes of enhancing sexual performance/prolongation of the time of intercourse (p = 0.001) and taking tramadol to improve on euphoria/pleasurable effect (p = 0.046) were statistically significantly associated with tramadol abuse. It was observed that quite majority (65.1%) did not have adequate knowledge on the dangers/consequences associated with tramadol abuse. However, there was a positive association (p = 0.009) between tramadol abuse and the knowledge index on dangers.A higher proportion (63.2%) of the respondents believed that there are extraordinary benefits associated with abusing tramadol. Therefore, the perception of respondents on the benefits of tramadol use was significantly related to its abuse (p = 0.001). It was noted that almost 74.3% of the tramadol users have a friend or a relative abusing tramadol. Though a few (38.2%) assented to role of posttraumatic/pain management dependence in tramadol abuse, a positive association was observed between them (p = 0.044). A lot of (77.6%) of the respondents were involved in other related substance abuse. A positive significant association was also observed between history of other substance abuse and tramadol abuse (p = 0.001) which were relatively linked to tramadol abuse. Close to 78.9% have justified reasons for the abuse of tramadol . Compared to respondents with no history of any substance/drugs abuse, respondents with history of any substance abuse were 5 times more likely to be engaged in the misuse/abuse of tramadol . The odds of abusing tramadol for purposes of sexual enhancement was higher among respondents. Compared to respondents who rejected the perception that tramadol was a sex enhancer, the respondents who take tramadol to enhance sex were 4 times more likely to take tramadol without physician prescription/inappropriately engaged in taking tramadol . The odds of respondents abusing tramadol was significantly higher among unemployed respondents  compared to respondents who were engaged in government work or self-employed. Comparatively, there is an increased risk of 23.7% of the respondents likely to continuously depend on opioid prescription-only drugs like tramadol after been used to manage trauma or pain-related illness in the hospital .This set of variables accounted for 30.5% of the variability of abuse/misuse of tramadol (Nagelkerke p \u2265 0.05) and were removed by hideout from the model during the modeling process  and 280 (66.7%) of the respondents had heard and saw tramadol, respectively. Out of this number 152 (36.9%) and 118 (77.6) had individually used and abused/misused tramadol. Elhabiby in his systematic analysis found similar findings especially in many African countries . The WorThe abuse of substances and other drugs in the municipality is alarming as unearthed by the study. The emergence of new forms of abuses puts the municipality at risk of public health disasters and rising trends of social vices. The called by the District Director of Health Services of Sunyani West district in the Brong-Ahafo region of Ghana reaffirmed the findings of the current study about the looming danger of abuse of \u201cWuole\u201d (a mixture of akpeteshie or other jin and wee or cocaine with or without ginger) in Ghana . About 6On high prevalence of substance abuse, almost half the respondents were found abusing alcohol, Indian herm or wee, mahogany/bitter roots with alcohol/akpeteshie, and \u201csera\u201d or \u201cEnye.\u201d In this regard, the findings of the following studies are consistent with findings of the current study \u201320. The The present study established that gender, religion, education status, and employment status were significantly related with the abuse of tramadol. A case study of tramadol use in Lagos state, Nigeria, established the relationship between socioeconomics, demographic characteristics, and tramadol abuse which is in support of the findings of the current study . SimilarThe present study found that having low or high perception on the benefits of tramadol influences the abuse of the drug. The perception that the use of tramadol is a sexual enhancer and improves euphoria was significantly related with tramadol abuse. Ghana drugs report in 2019 noted that tramadol can induce a sense of euphoria and enhance sexual prowess . The resThe main findings indicated that the history of abuse of other substances, sexual enhancement, postdependence from trauma/pain management, and occupation were associated negatively with the respondents abusing tramadol. This is consistent with a study conducted in the Western Region of Ghana . A studyDespite the addiction potential of this drug coupled with the adverse effects, the veracity of its use in the management of trauma and patients in severest pains cannot be underestimated . The finIn view of the study outcomes, further larger and longitudinal studies are proposed. More biochemical analyses are needed especially the constituents of the emerging substances that attract the respondents. We will recommend more vigorous investigation on substance use patterns and the physical and psychiatric comorbidity among the respondents using these substances including prescription-only opioids like tramadol.The findings of the study revealed that, among the respondents in the municipality, 77.6% of the tramadol users are inappropriately taking or misusing/abusing the drug. In fact, history of abuse of other substances, sexual enhancement, postdependence from trauma/pain management, and occupation were the independent predictors of the tramadol abuse. Also, there is a looming danger since majority of the respondents are indulged in the abuse of other substances and drugs like codeine, alcohol, and snuff/sera. The trend of the insurgence of abuse of emerging substances like the smoking of dried faeces and drinking of soaked diapers/used pads is a worrisome situation and a wake-up call to stakeholders especially the law-enforcement authorities within the municipality."}
+{"text": "The field of synthetic microswimmers, micro-robots moving in aqueous environments, has evolved significantly in the last years. Micro-robots actuated and steered by external magnetic fields are of particular interest because of the biocompatibility of this energy source and the possibility of remote control, features suited for biomedical applications. While initial work has mostly focused on helical shapes, the design space under consideration has widened considerably with recent works, opening up new possibilities for optimization of propellers to meet specific requirements. Understanding the relation between shape on the one hand and targeted actuation and steerability on the other hand requires an understanding of their propulsion behavior. Here we propose hydrodynamic simulations for the characterization of rigid micropropellers of any shape, actuated by rotating external magnetic fields. The method consists of approximating the propellers by rigid clusters of spheres. We characterize the influence of model parameters on the swimming behavior to identify optimal simulation parameters using helical propellers as a test system. We then explore the behavior of randomly shaped propellers that were recently characterized experimentally. The simulations show that the orientation of the magnetic moment with respect to the propeller's internal coordinate system has a strong impact on the propulsion behavior and has to be known with a precision of \u2264 5\u00b0 to predict the propeller's velocity-frequency curve. This result emphasizes the importance of the magnetic properties of the micropropellers for the design of desired functionalities for potential biomedical applications, and in particular the importance of their orientation within the propeller's structure. Rotation of a magnetic field results in propulsion if the micropropeller has a rotation-translation coupling, which usually requires chirality of the structure. Therefore, the first magnetic micropropellers were magnetic helices, inspired by the propulsion of bacteria due to the rotation of their flagella  on the swimming behavior to identify optimal simulation parameters. For the validation and parametrization of our simulations, we focus on the helical geometry, as this is the best described behavior so far. We observe velocity reversals upon a change of the rotation frequency of the magnetic field, a feature that was recently predicted theoretically for helices , rotation ('rr') or the coupling of translation and rotation ('rt' and 'tr'). The mobility matrix is symmetric, thus Mrt = (Mtr)T.Here, TCM = m \u00d7 B, with the magnetic moment m of the propeller and the magnetic field B, which is taken to rotate counterclockwise in the yz-plane in the lab frame of reference, B = , Bsin(2\u03c0fBt)). fB is the frequency of the field and B its absolute value. For the rotation in the yz-plane, a net movement of the cluster along the x-axis is expected. Since only a torque and no external forces act on the propeller, the geometry of the propeller must have a non-zero rotation-translation coupling, Mtr \u2260 0 in order to lead to a non-zero translational velocity vCM \u2260 0.In the case of magnetic micropropellers that are rotated by a  magnetic field, the force is usually zero and the external torque is given by N \u00d7 6N mobility matrix for N beads appears in the equations of motion for the beads,v and \u03c9 are the 3N-dimensional translational and angular velocity vectors of the beads. F, T are the corresponding  vectors of the forces and torques, respectively, that are applied on the individual beads. The \u03bcij are 3N \u00d7 3N mobility matrices . The elements of the \u03bc matrices can be calculated through the Rotne-Prager approximation . In the present paper, rotational-translational couplings \u03bctr, \u03bcrt are neglected, which are known to result in artifacts for elastic structures \u22121, \u03bcr = (8\u03c0 \u03b7a3)\u22121 are the mobility coefficients of a sphere, \u03b7 is the kinematic water viscosity, a is the radius of the bead, rij is the distance between the i-th and j-th bead, and i-th and j-th bead. where M for the center of mass is obtained from the mobility matrix \u03bc for the individual beads by projecting onto the center of mass M=. The position of the center of mass xCM is then obtained by integration ofHere \u03b2. \u03b3 is obtained from the other two vectors through orthogonality.The orientation of the object is described by a triad of orthogonal unit vectors ai, bi, ci) in the body system defined by t \u2243 10\u22125s. Simulations were stopped if the error accumulated to The simulation was implemented in Fortran 90  The continuous surface is approximated with beads; (ii) The Rotne-Prager equations are an approximation themselves, valid for spheres at sufficiently large distances, but used here also for rather small distances (spheres touching each other); (iii) Some terms in the equations are neglected, in particular the rotational-translational terms and the off-diagonal rotational-rotational terms; (iv) Rigid bonds correction were not used. We will go through these points in the following.The approximation with beads of a continuous surface was previously used in the literature  effects . These correspond to two different orientations of the axis of rotation with respect to the long axis of the propeller. In the simulation though, thermal noise is not included, so to reproduce and to study branching, simulations are run with different initial configurations of the propellers, i.e., different orientations of the propeller in the lab system and respect to the rotating magnetic field. To determine branches systematically, we run simulation that walk along the branches as in a hysteresis curve: the frequency of the magnetic field increases in steps using the final configuration of one simulation as initial configuration for the next simulation, thus very likely staying on the same branch. When the highest frequency to be simulated is reached, the procedure is repeated decreasing the frequency, typically exploring the other branch.fso in the simulations, we check if the frequency of the propeller fCM is synchronized with the frequency of the magnetic field fB. If oscillations of the propeller frequency fCM(t) can be seen in time instead of the constant value given by fB, then the asynchronous regime has started. The frequency at which the change happens is the step-out frequency. See To determine the step-out frequency Inspired by the rotating bacterial flagella, helical propellers are the best-studied type of micropropellers, both from theoretical and computational points of view  is achieved). Here the wobbling angle is not constant, but oscillates. For more general propeller shapes and for helices with other orientations of the magnetic moment, the synchronous regime is divided further, as will be discussed below (see sections 3.3 and 4). Figure mag = 0 from our simulations, which indeed exhibit the synchronous and asynchronous regimes. Thus, the behavior shown in Figure vm, the step-out frequency fso, and the linear coupling cv \u2243 vm/fso. When two of these are read out from the simulation data, the velocity-frequency relation is fully determined and given by . The coupling coefficient becomes tr and \u03bcrr are the rotation-translation coupling and the rotation rotation coupling, respectively. For more complex cases, it is not easy to obtain a simple form for the coupling coefficient.For the helix shown in Figure rr Vach, , where tn = 4, r = 2.5\u03bcm, p = 4r and a gap size of 0 between beads (our standard parameters). The two bead sizes are a = 0.1 \u03bcm and a\u2032 = 1.5a. To maintain the gap sizes of zero , we need to adjust the number of beads per turn . Changing the bead size (while keeping the gap size between beads the same) has two effects: on the one hand, it changes the discretization of the helix, which ideally should not affect the simulation results, as different discretizations describe the same geometry. On the other hand, it also changes the thickness of the helix and thereby the area of the surface in contact with the fluid, increasing its friction from where a true physical effect is expected. To quantify this difference, we performed simulations for two helices discretized by different bead sizes, but with all other geometric parameters the same. This results in the two frequency-velocity relations that we show in Figure This can be explained by the higher friction arising from the increased surface area and should be considered as a result of the change in helix geometry rather than a discretization effect. Thus, to match experimental data, these effects should be taken into account. For example, beads with a diameter corresponding to the thickness of the helix can be used. Alternatively, an approximation of the real thickness of the helix can be implemented with multiple parallel chains of smaller beads instead of the one chain of large beads.npt = 4 \u2212 9 beads per turn of the helix). For all different discretizations, we determined frequency-velocity curves and extracted the three characteristic parameters fso, vm, and cv. These are plotted in Figure npt. In particular, a discretization with beads touching each other (npt = 9) gives essentially the same results as the case npt = 6, where the gap is big enough to use the Rotne-Prager approximation. For large gaps however, the velocity is noticeably reduced, indicating that the continuous structure of the helix is poorly represented. Thus, a sufficient density of beads is needed for a good representation of the continuous geometry, but the precise choice of the discretization is unimportant if the beads are sufficiently dense. For the further study of helices we therefore used beads touching each other.The choice of the number of beads per turn (for constant bead size) does not change the geometry of the helix, but only how the helix is represented in the discretized model: fewer beads should result in a poorer representation of the continuous surface of the helical propeller, while for more beads and thus smaller gaps, the Rotne-Prager approximation becomes invalid. To test the influence of the number of beads per turn, we approximate the same helix with different numbers of beads that are equally distributed along the curve  or from below (angles \u226583\u00b0), as shown in Figure The parameters a Figure . In partThe method proposed here allows us, in principle, to simulate any shape. An example of a shape that was assembled in a random fashion is shown in section 2 of the As an example, we study a propeller geometry reported by Bachmann et al. . The proTo test the influence of the discretization, we generate bead representations with different numbers and sizes of beads, from a very coarse grained representation with 55 beads to ten times more (518 beads), and finally to ~100 times more, equivalent to one bead each voxel 4,276 beads). To that end, voxels in the three-dimensional reconstruction of the propeller shape are coarse-grained as described in section 2.4. The resulting bead representations are shown as insets in the Figures  beads. TfB/fso and v/vmax and compared to the rescaled experimental data. The rescaling is necessary to correct for possible shifts of the velocity-frequency curve due to the approximations and to take the unknown absolute value of the magnetic moment into account. The closest match between computational and experimental curves was found for the magnetization direction shown as the red vector in Figure To determine the direction of the magnetic moment, which is unknown for the experimental propeller and which could impact the propeller behavior, we run simulations for 100 random orientations of the magnetic moment using the ~500 beads-representation. The shape of the curve varies considerably within this dataset see . The 100Since the direction of the magnetization emerges as a crucial parameter needed to describe the experimental behavior, we tested how sensitive the results are to changes in this direction, by making small perturbations to it Figure . For peretc. (Peyer et al., The aim of this study was to develop a method for simulating magnetic micropropellers with complex shapes in order to predict their behavior, specifically their propulsion velocity and its dependence on the strength and frequency of the magnetic field actuating these propellers. Such a simulation method is desirable for optimizing the design of magnetic microswimmers for specific purposes or applications, as magnetically actuated microswimmers are envisioned to perform tasks such as drug delivery, fertilization, artery cleaning, biopsies We first used the method to simulate the well-studied helical geometry, for which our simulations successfully recovered the known behavior. Small shifts in the velocity-frequency curve arise from the choice of the mobility matrix and approximations involved in it. This could be improved by re-designing the matrix elements. The systematic study on the influence of the simulation parameters such as bead dimension and bead separation suggests that beads touching each other can be used despite the use of the Rotne-Prager approximation, but this must be done cautiously, depending on the way a shape is represented. Changing the dimension and number of beads could lead to different results, so the discretization of the original shape must be done such as not to change the main features of the propeller geometry.We have then used the model to explore aspects of helical propellers that have not received much attention before, in particular, the influence of the orientation of the magnetic moment, which can result in velocity reversals both below and above the step-out frequency. Velocity reversals are accompanied by a change of propeller orientation relative to its direction of motion. Velocity reversals for helices at small frequencies were not seen experimentally yet, but have been seen in recent theoretical work and simulations (Morozov et al., While the results we obtain for helices are rather robust with respect to the representation of the propeller shape, this is less so for the random-aggregate propeller we studied next. These simulations show that it is crucial to reconstruct the propeller shape accurately as well as to know the orientation of the magnetic moment, extending previous results (Ghosh et al., All these results highlight the importance of accurately determining the orientation of the magnetic moment, on which most of the dynamics depend. Not only is this needed to understand and predict the behavior of existing geometries, but it can also be used to design new propellers with the required characteristics: not only shape must be taken into account, but magnetization too. However, designing the magnetization direction constitutes another challenge, as a design that works well in simulations may be difficult or even impossible to synthesize in the lab. The most promising technique to that end is 3D printing, with which in principle any shape can be implemented. However, the magnetization represents another problem. Existing techniques such as different coating approaches (sputtering etc. Fischer and Ghosh, AC, DF, and SK designed research, AC wrote the code and performed simulations. All authors analyzed data and wrote 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": "In all cases, methyl docking dominates by at least a factor of two, whereas DFT-optimized structures suggest a very close balance for the larger alcohols, once corrected by CCSD(T) relative electronic energies. Together with inconsistencies when switching from a C4 to a C5 alcohol, this points at deficiencies of the investigated B3LYP and in particular TPSS functionals even after dispersion correction, which cannot be blamed on zero point energy effects. The search for density functionals which describe the harmonic frequency shift, the structural change and the energy difference between the docking isomers of larger alcohols to unsymmetric ketones in a satisfactory way is open.The influence of distant London dispersion forces on the docking preference of alcohols of different size between the two lone electron pairs of the carbonyl group in pinacolone was explored by infrared spectroscopy of the OH stretching fundamental in supersonic jet expansions of 1:1 solvate complexes. Experimentally, no pronounced tendency of the alcohol to switch from the methyl to the bulkier  In nature, directional hydrogen bonds to carbonyl groups ,2 are frThis has led to the concept of ketone solvation balances, which were introduced for acetophenone and its derivatives in combination with alcohols as hydrogen bond donors ,11 and tbarriers ,14,15) aThe results of such studies can be used to benchmark the ability of different density functionals to predict the interplay of hydrogen bonding with distant London dispersion and Pauli repulsion, by simply comparing the predictions to experiment. This can be done strictly at the level of observables, without consulting any energy decomposition models ,17,18, atert-butyl (tBu) group. This removes aromatic\u2013aliphatic dispersive interactions and brings in more bulky donor-acceptor constellations. Cyclopentanol (CpOH) is introduced as a further, more disk-like and flexible alcohol, in addition to methanol (MeOH) and tert-butyl alcohol (tBuOH), which have been previously explored with acetophenone [The hypothesis that B3LYP-D3 and TPSS-D3 show an  acceptable performance for ketone dispersion balances obviously calls for further falsification attempts and this is the task of the present study which involves the purely aliphatic pinacolone see , where tophenone . Pinacolophenone , but in le \u03c4 see  with alcIn this work, we show that, in alcohol\u2013pinacolone balances, the methyl docking side is consistently preferred. According to exploratory calculations, this may extend to many alcohols beyond the experimentally investigated ones. Further, we show that the predictive quality of the two density functionals which were successful for acetophenone (B3LYP and TPSS) decreases with the size of the alcohol, including significant failures for the largest (CpOH). The proposed assignments and observed trends are discussed and an analysis of dispersion interactions on the docking side preference is presented. We provide initial evidence that some of the superficially satisfactory DFT performance for ketone balances must be fortuitous.We start with the theoretical description of alcohol\u2013pinacolone 1:1 complexes at the level of DFT before comparing to the experimental findings and finally consulting wave-function theory.tBuOH  and CpOH (cyclopentanol) for the solvating alcohols are consistently used. In From now on, the abbreviations Pin for the studied ketone pinacolone and MeOH (methanol), tBuOH to CpOH. On the tBu docking side of Pin, even MeOH is already displaced by 35\u201337As detailed in The structural trends are reflected in the calculated OH stretching wavenumbers see , which atBu docking sides fall between 0 and 3 The energy differences between Me and quencies ,20.tBu side of Pin together with the flexibility of alcohols provides possible explanations. The latter allows the alcohol to dock on the sterically more accessible Me side and at the same time to exploit London dispersion interaction with the tBu side. A good example is benzyl alcohol, where the Me sided structure is almost 2 tBu group of the Pin while the OH group is docking to the Me side of Pin.The predicted spread in docking energy difference of about tBu docking structure. The interconversion path is distinctly out-of-plane, relaxing the hydrogen bond angle Another important feature of carbonyl balances is the feasibility of the isomerization under supersonic jet expansion conditions. A transition state search between the two competing structures for MeOH\u2013Pin yielded an interconversion barrier height of about 3 ophenone  and its Before switching to experiment, two important observable predictions need to be explored. One is a sufficiently robust infrared cross section ratio for the docking isomers, which is a precondition for reliable experimental abundance determinations from spectral intensities. As shown in tBu differences involve systematic cancellation of anharmonic contributions for similar docking environments. Furthermore, the structural effect of increasing alcohol size is qualitatively similar on both docking sides, as pointed out above, and should translate into relatively uniform wavenumber splittings as a function of the number of alcoholic C atoms. This is illustrated in The most important theoretical assignment aid concerns the predicted positions and differences or splittings of the OH stretching fundamental vibrations. While the harmonic approximation is too crude for absolute predictions, the harmonic Me-nd width  by more With a single exception (TPSS for CpOH for the larger basis set), all predicted harmonic splittings are within \u00b1 12  \u03c4 angle . They ar \u03c4 angle , the lartBuOH (orange) and CpOH (blue) are shown. They feature the rovibrationally broadened alcohol monomer OH stretching bands , the downshifted hydrogen-bonded homodimer signals  level  were calFirst, they allow judging which of the DFT methods is likely closer to the true minimum by looking at the absolute CCSD(T) energies . In all tBu docking is expected but Me docking is predominantly observed  difference and keep the structural and ZPVE contributions from the DFT level. This generates a variant of rved see b. Only Mrved see  promote tBu docking, by 1.5 to 3.1 A third application of DLPNO-CCSD(T) is to provide dispersion contributions to the interaction energy in the LED scheme ,17 Tabl. This istBuOH docking, B3LYP predicts borderline Returning to the conformational freezing temperature analysis, now with DLPNO-CCSD(T)-corrected values , only MetBuOH docking). Compared to acetophenone [In summary, the DLPNO analysis shows that dispersion-corrected TPSS docking structures are imbalanced, more so than B3LYP structures. It confirms that beyond MeOH, the best isomer energy predictions are inconsistent with experiment or at best borderline  or cyclopentanol ). The gas mixture is filled into a 67 L reservoir at a pressure of 0.75 bar and pulsed through six magnetic valves into a pre-expansion chamber which is terminated by a 600 mm long and 0.2 mm wide slit nozzle. During about 0.2 s, the gas flows through this slit into a vacuum chamber connected to a buffer volume  is led through a temperature-controlled gas-flow system, where it passes separate gas saturators filled with the analytes pinacolone (Alfa Aesar > 97%) and alcohol (methanol (Roth \u2265 99.9%), lsewhere . No evidTo determine the band integral ratios .5) cm\u22121). The proDFT calculations were used for assignment purposes and to trigger future benchmarking of their ability to describe the combination of hydrogen bonding and distant London dispersion interactions. Therefore, they were limited to two functionals and two basis sets, but others are invited to find more powerful density functionals for this challenge. The initial structural search  was carain text . A transain text ,42) and ain text .tert-butyl-facing lone electron pair of the keto group. As generally predicted for almost two dozen alcohols by dispersion-corrected B3LYP calculations, the methyl side is preferred for methanol, tert-butyl alcohol and cyclopentanol. This was qualitatively confirmed by infrared spectroscopy of supersonic jet expansions in combination with approximate IR absorption cross sections. Quantitatively, the DFT predictive power in terms of the spectral splitting decreases with increasing alcohol size. In addition, the observed spectral abundance does not correlate systematically with the predicted energy difference. DLPNO-CCSD(T) energy calculations indicate that B3LYP provides a somewhat better description of the combined hydrogen bond and London dispersion interaction than TPSS. However, in combination with the experiment, they suggest that docking on the methyl side is systematically underrated by both density functionals on the 1 Three alcohols of increasing size were combined with pinacolone to determine the hydrogen bonding preference to either the methyl- or the tert-butyl alcohol. The qualitative failure of theory to describe the experimentally observed cyclopentanol docking invites studies of related complexes, such as cyclohexanol\u2013pinacolone and cyclopentanol\u2013acetophenone. Larger modifications involve the use of phenol [Intermolecular energy balances are thus shown to be powerful benchmarking tools to assess the ability of DFT methods to describe hydrogen bonding in competition with London dispersion. The ketone balance variety is particularly useful, as it involves systematically compensating zero-point-energy contributions and therefore allows judging electronic structure predictions in a rather direct way. For acetophenone, only a slight deficiency of the B3LYP functional could be identified . For pinf phenol  and the The goal is to find a density functional which systematically reproduces the harmonic wavenumber splitting between docking isomers within better than about 10"}
+{"text": "The growing aging population are increasingly suffering from the negative health consequences of the age-related decline in their senses, especially their chemical senses. Unfortunately, however, unlike for the higher senses of vision and hearing, there is currently nothing that can be done to bring back the chemical senses once they are lost (or have started their inevitable decline). The evidence suggests that such chemosensory changes can result in a range of maladaptive food behaviours, including the addition of more salt and sugar to food and drink in order to experience the same taste intensity while, at the same time, reducing their overall consumption because food has lost its savour. Here, though, it is also important to stress the importance of the more social aspects of eating and drinking, given the evidence suggesting that a growing number of older individuals are consuming more of their meals alone than ever before. Various solutions have been put forward in order to try to enhance the food experience amongst the elderly, including everything from optimising the product-intrinsic food inputs provided to the remaining functional senses through to a variety of digital interventions. Ultimately, however, the aim has to be to encourage healthier patterns of food consumption amongst this rapidly-growing section of the population by optimising the sensory, nutritional, social, and emotional aspects of eating and drinking. An experimental dinner with the residents of one such home where nostalgic-flavoured healthy ice-creams were served is described. The chemical senses, namely smell (olfaction), taste (gustation), and the trigeminal sense, just like the higher spatial senses of vision, audition, and touch e.g., ,2,3,4) s,3,4 s2,3Our sensory and perceptual functions start their inevitable decline at different ages and at very different rates see ,21,22). ,22. 21,2The decline in the sense of smell with aging ,31,32 isOne of the key problems for those hoping to optimise the design of food and drink products for the elderly is that the variability in sensitivity to olfactory stimuli across the population tends to increase as we age . What thIntriguingly, those who have lost the ability to taste, for example, in the case of herpes zoster oticus in the case of the psychophysicist Pfaffmann (see ), reportMeanwhile, in a study of nearly 2000 people ranging from 5 to 99 years in age, Doty et al.  reportedResearch directly comparing age-related declines in olfactory and gustatory sensitivity suggests that olfactory losses tend to start earlier and to be more severe than those seen for taste . MoreovAlthough it is not often mentioned in the literature, it is important to note that saliva also plays a key role in helping us to masticate and swallow food , and, on occasion, sound  and touIt has long been suggested that meals should be made more colourful and sonically interesting for elderly and hospitalized individuals e.g., ,96). Fur. Fur96])Many older individuals, including those who have lost their teeth, are often fed pureed meals as they can find it hard to deal with solid foods . UnfortuJapanese researchers have developed a headset that older people in this situation can wear that presents mastication-like sounds elicited by jaw movements ,108. It Ice-cream is often noted as being a popular food amongst many older individuals e.g., ,110). In. In110])Working with this idea, and against the common conception of ice-cream as an unhealthy (and possibly childish/necessarily indulgent) food, chef Jozef Youssef of Kitchen Theory  created While the concept of savoury ice-creams is currently unfamiliar to many Western consumers, they are nevertheless popular in Japan, as well as in the context of many modernist restaurants around the world ,119,120)Chef Jozef Youssef and his team created a range of savoury ice-creams with various flavours chosen to elicit positive nostalgia amongst older individuals. Given the UK base for this particular intervention, the meal incorporated Heinz cream of tomato soup, prawn cocktail, and bone marrow ice cream flavours see . MeanwhiAnother relatively-simple low-cost intervention to enhance food behaviour at mealtimes is to use music, or ambient soundscapes, to help relax those individuals who might otherwise be too agitated to eat. This is apparently a common problem amongst many psychiatric patients as well as a growing number of those older individuals who are suffering from Alzheimers/dementia e.g., ,139,140),140139,1Research conducted with individuals suffering from amnesia suggests that it is external cues that often trigger the initiation of meal consumption in the absence of awareness/memory of the meals that may just have been consumed . At the In order to try and address the latter problem, Prof. Spence was involved as a consultant in a project a few years ago designed to try and help older individuals, specifically early-stage Alzheimers/dementia patients who might otherwise need to be hospitalized due to undernutrition , to retaThe six food aromas developed for the launch included fresh orange juice, cherry Bakewell tart, homemade curry, pink grapefruit, beef casserole, and Black Forest gateau. They were specifically chosen to be representative of food aromas that were likely to be familiar to those in the target age group . The reFlavour is undoubtedly one of the most multisensory of our everyday experiences , potentiFor example, Laurienti, Burdette, Maldjian, and Wallace  have argThe authors are not, however, aware of any research that has specifically addressed the question of whether there is any central impairment affecting the multisensory integration of the flavour senses  with advancing years. Here, it is worth stressing that it is not only the neural sites of multisensory integration that differ between the chemosensory and the spatial senses, but also the very nature of the rules governing that integration  cues, in particular, exert a significant modulatory effect over the sensory-discriminative and hedonic aspects of tasting (see ,161, forAttention plays a key role in both the phenomenon of oral referral ,167,168 However, over and above any perceptual decline, a large part of the poor food consumption behaviours that have been evidenced in older populations are likely to result, at least in part, from the increasingly isolated living that many older people face, and the consequent lack of social interaction that have been documented amongst older age groups in many countries . To giveRecognizing this growing social issue, there is an emerging interest in the field of digital commensality, with a number of researchers trying to bring back the enjoyment of eating by means of digital technologies . While aA wide body of evidence now points toward the conclusion that the rapidly-growing aging population are engaging in a variety of unhealthy eating behaviours. On the one hand, this takes the form of adding excessive amounts of sugar and salt to taste , while, As stressed in this review, one promising vehicle for the delivery of nutritional requirements in at least some elderly individuals may be nutritionally-enhanced ice-creams (see ; see alsFurther, given the many reports of chemosensory loss constituting one of the most common symptoms of COVID-19 ,192, a l"}
+{"text": "Understanding the relationship between natural selection and phenotypic variation has been a long-standing challenge in human population genetics. With the emergence of biobank-scale datasets, along with new statistical metrics to approximate strength of purifying selection at the variant level, it is now possible to correlate a proxy of individual relative fitness with a range of medical phenotypes. We calculated a per-individual deleterious load score by summing the total number of derived alleles per individual after incorporating a weight that approximates strength of purifying selection. We assessed four methods for the weight, including GERP, phyloP, CADD, and fitcons. By quantitatively tracking each of these scores with the site frequency spectrum, we identified phyloP as the most appropriate weight. The phyloP-weighted load score was then calculated across 15,129,142 variants in 335,161 individuals from the UK Biobank and tested for association on 1,380 medical phenotypes. After accounting for multiple test correction, we observed a strong association of the load score amongst coding sites only on 27 traits including body mass, adiposity and metabolic rate. We further observed that the association signals were driven by common variants  with high phyloP score (phyloP > 2). Finally, through permutation analyses, we showed that the load score amongst coding sites had an excess of nominally significant associations on many medical phenotypes. These results suggest a broad impact of deleterious load on medical phenotypes and highlight the deleterious load score as a tool to disentangle the complex relationship between natural selection and medical phenotypes. This study aims to augment our understanding of the complex relation between natural selection and human phenotypic variation. We developed a load score to approximate the relative fitness of an individual and correlate it with a set of medical phenotypes. Association tests between the load score amongst coding sites and 1,380 phenotypes in a sample of 335,161 individuals from the UK Biobank showed a strong association with 27 traits including body mass, adiposity and metabolic rate. Furthermore, an excess of nominal associations at suggestive levels was observed between the load score amongst coding sites and medical phenotypes than would be expected under a null model. These results suggest that the aggregate effect of deleterious mutations as measured by the load score has a broad effect on human phenotypes. One of the primary questions of interest in the study of human population genetics is the relation between natural selection and the evolution of human phenotypes, from quantitative traits to complex disease. With the emergence of biobank-scale datasets, along with new statistical metrics to approximate strength of purifying selection at the variant level, it is now possible to both estimate the net impact of deleterious mutations for each individual in a large population sample and correlate it to a range of medical phenotypes exhibited by that individual. This provides an opportunity to simultaneously study the genetics of individuals within a relatively homogenous population and the potential impact of natural selection on annotated phenotypes.A large body of literature exists on evolution and estimation of the deleterious mutation load from human population samples, with particular emphasis on cross-ancestry comparisons \u20136. RatheIn this study, we aim to augment our understanding of the relation between natural selection and human phenotypes by focusing on the net impact of purifying selection on the fitness of each individual, and correlating this quantity to the set of phenotypes acting on that individual. Previously, this has been difficult for two reasons: first, we do not have a direct measure of the fitness of individual humans that can be estimated from genetic information, and second, there were no large databases available to quantify the wide range of phenotypes possessed by each individual. Biobank-scale datasets that contain both individual genotypes and phenotypes, such as the UK Biobank ,8, finalWe ventured to apply computational tools that predict aspects of purifying selection for individual alleles to published genotypes of 335,161 white British individuals from the UK Biobank to estimate the fitness impact of derived variation present in each imputed genome in this sample. Most of the variation in the sample exists at appreciable frequencies, and is likely under relatively small selective disadvantage, but in aggregate the fitness impact can be substantial. Using this representation of each individual\u2019s relative fitness, we probed correlations between the impact of common deleterious variation in an individual\u2019s genome and their personal phenotypic makeup. This provides a different lens into questions about the relation between fitness vis-\u00e0-vis mutation load and human traits by looking at a per-individual measure correlated to fitness, rather than focusing on the distribution of selective effects in the population as a whole. This allows us to ask which phenotypes, if any, are highly correlated to the aggregation of deleterious variation, and probe the relation between the ensemble of phenotypes and fitness loss due to common variation in individuals.The additive effects of deleterious variation can be quantified in aggregate by a genome-wide score representing the net action of purifying selection on an individual under the assumption that effects of individual variants can be summed additively. Multiple methods have been developed to characterize purifying selection, including methods that predict deleterious selection acting on the level of a single allele  towards rare alleles, with a steeper slope of the AFS indicating stronger purifying selection. We evaluated the extent to which each scoring method captures the deleteriousness of an allele by grouping alleles by the scores provided by each method and measuring the slope of the resulting AFS. The more strongly a score is related to the strength of selection, the more marked the increase in slope will be for high-scoring alleles relative to lower scoring alleles.We evaluated this correlation using whole genome sequencing data from a non-Finnish European population in the Genome Aggregation Database (gnomAD) . For eacTo further compare between CADD and phyloP, we examined the DAF distribution for protein coding variants and noncoding variants separately. Both scores performed similarly for coding variants, but phyloP showed better separation in noncoding variants . This isWe calculated a per-individual deleterious load score by summing the total number of derived alleles per individual, weighting each derived allele by its phyloP score to account for the strength of purifying selection. We considered three load scores: a genome-wide load score, a coding-specific load score, and a non-coding-specific load score. Each score was computed for 335,161 unrelated, white-British ancestry individuals in the UK Biobank using 6,774,062 variants from imputed genotypes  with positive phyloP scores . The observed population distribution across all sampled individuals appear very close to normal for each of our three scores  with 1,380 traits, after adjusting for age, sex, genotyping chip, and assessment center. To account for potential confounders, we further included a set of geographical and socioeconomic variables available in the UK Biobank data as additional covariates . We note-5). However, 27 traits were significantly associated with the load score calculated from coding SNPs; these included body mass, metabolic rate, and several adiposity traits such as body mass index and waist circumference  but not close to fixation (DAF < 70%), while stratification by phyloP score shows that they are more pronounced when limiting to variants with higher phyloP scores . However, rather than the load score having a strong effect on a single disease, we hypothesized that the load score may have subtle effects on many diseases, leading to an excess of weak associations that do not individually reach statistical significance. To test this hypothesis, we compared the number of phenotypes nominally associated with the load score  to a null distribution generated by random permutation of individual load score values . For this analysis, we restricted to associations with clinical phenotypes defined by phecodes. Out of 539 phecodes, 46, 24, and 27 phecodes  were fouIn this study, we have described a polygenic load score that estimates the deleterious load carried by an individual, and applied this score to 335,161 white British individuals from the UK Biobank. Our analysis produced two major results: First, while we found no significant associations between individual medical phenotypes and the genome-wide load score, we found that more phenotypes are nominally associated with the coding load score than would be expected under a null model . This suThere are several limitations to our method. We computed the load score from imputed genotypes rather than sequenced whole genomes, which gives us little information about extremely rare variants in the population, masking potentially large contributions to the load from variants under the strongest selection. As a future topic of research, the same methodology can be applied to include rare variants, which would shed light on the relative contribution of common and rare variation to the phenotypic associations of load. Previous studies have shown that rare variation contributes substantially to differences in deleterious load between human populations, so we may expect it to have a significant impact on individual load in this context as well ,2. FurthOne potentially exciting application for this approach is applying it to different populations to discover population-specific insights into phenotypic associations with deleterious load. Since PhyloP scores can be calculated without any reference to specific human populations , there iThe deleterious load score presented here provides a new approach to investigate the complex relationship between natural selection acting on individuals, individual medical phenotypes, and the human phenome at large. We expect that as the available biobank data continues to grow in size and scope, this method can be applied to larger and more diverse populations to gain additional insights into how load varies between different populations, possibly empowering population-specific medical discoveries with deleterious load.This research has been conducted using the UK Biobank Resource under Application number \u201816218\u2019. UK Biobank has received ethics approval from the North West Multi-centre Research Ethics Committee, the National Information Governance Board for Health & Social Care, and the Community Health Index Advisory Group.We evaluated the dependence of derived allele frequency of single nucleotide polymorphisms (SNPs) discovered in the whole genome sequences of 7,509 non-Finnish European individuals in the GnomAD data set  on each For each deleteriousness score, we divided the SNPs into multiple groups with arbitrarily defined intervals based on the range of each score. The intervals used were:  for fitcons,  for GERP,  for CADD, and  for phyloP.The load score of each individual was calculated by adding up the number (dosage in case of imputed SNPs) of derived alleles at each SNP, weighted by the phyloP score at that site, across the entire genome. Derived alleles were determined based on the six-way EPO alignment, as described above. Since we are focusing on the effect of purifying selection, only SNPs with positive phyloP score  were included. In this paper, we computed three load scores using three different SNP sets: the coding load score summed only over coding variants, the non-coding load score summed only over non-coding variants, and the genome-wide load score computed from both coding and non-coding variants. All load scores were computed using PRSice-2 software  under anThe UK Biobank consists of genotype, phenotype, and demographic data of more than 500,000 individuals recruited across the United Kingdom. Individual genotypes were generated from either the Affymetrix Axiom UK Biobank array  or the UK BiLEVE array , each contains ~0.9 million markers. Additional variants were then imputed using the Haplotype Reference Consortium (HRC) combined with the UK10K haplotype resource, with a total of ~96 million variants available in the latest released imputed data (version 3). To compute per-individual load scores, we restricted to variants with imputation quality INFO score > = 0.9. We excluded samples that were outliers in heterozygosity or missing rates, samples with putative sex chromosome aneuploidy, and samples with self-reported non-white British ancestry. We also excluded one individual from each pair of samples with relatedness up to the third degree. This produced a subsample of 335,161 individuals. All information used to exclude samples is included in the UK Biobank resource page.http://www.nealelab.is/blog/2017/9/15/heritability-of-2000-traits-and-disorders-in-the-uk-biobank). This subgroup covers phenotypes in most of the core categories, including early life and reproductive factors, family history, cognitive function, physical measures, lifestyle and health outcomes.UK Biobank provides a wide range of medical phenotypes from base line assessment, biochemical assays, dietary questionnaire, and health records. In the present study, we focused on 2,419 phenotypes which had been selected for heritability estimation by the Neale group  codes from electric health records. We converted ICD codes (including ICD-9 and ICD-10 codes) into phecodes using Phecode Maps 1.2 ,38. ThisThe remaining 1,800 phenotypes  were pre-processed using PHESANT , a packaThe association between load score and each phenotype was tested using a regression test in PHESANT: linear regression / lm R function for continuous, ordered logistic regression / polr R function for ordered categorical, multinomial logistic regression / multinom R function for unordered categorical, and binomial regression / glm R function with family = binomial for binary. Besides the commonly used covariates of age, sex, genotyping chip, assessment center and 40 principal components, we added five variables as covariates in all association tests that might denote population structure: birth location, home area population density, Townsend deprivation index, and UK deprivation index.To explore which variants drive the association between the 27 adiposity traits and the coding load score, we stratified variants by derived allele frequency  and phyloP score . Simultaneous stratification was performed with four groups of SNPs: DAF<0.05 and phyloP\u22642, DAF<0.05 and phyloP>2, DAF\u22650.05 and phyloP\u22642, DAF\u22650.05 and phyloP>2.A null distribution of the number of clinical phenotypes weakly associated with load score was created by repeatedly running the association test between load scores and phenotypes after randomly shuffling the load scores of individuals within the tested sample. The phenotypes included in this permutation analysis were all 539 phecodes. The same set of covariates used in phenome-wide association study (PHEWAS) tests above was applied. For each permutation, the number of phenotypes nominally associated with the load score  was then computed. The permutation p-value was calculated as the fraction of permutations for which the number of nominally associated traits was at least as large as the observed number of nominally associated traits.S1 FigTop row: Derived allele frequency spectrum of coding variants. Bottom row: Derived allele frequency spectrum of non-coding variants. Each solid line represents derived allele frequency spectrum of polymorphic sites belonging to one score category and three dashed lines represent derived allele frequency spectra of three control categories: synonymous (syn), missense (mis), and loss of function (LOF) variants.(TIF)Click here for additional data file.S2 FigQuantile-quantile plot of -log10 p-values for the phenotypic association of A) load scores (weighted by phyloP); B) burden scores (unweighted); C) burden scores restricted to rare variants (DAF<5%); and D) burden scores restricted to common variants (5%< = DAF<70%).(TIF)Click here for additional data file.S3 FigNull distribution of the number of clinical phenotypes weakly associated with genome-wide load score (left) and non-coding load score (right) was obtained from 2,000 permutations each. For each permutation, the load score was shuffled randomly among 335,161 samples and the number of associations on the x-axis was the count of phenotypes which yielded p-value < 0.05 in the association tests between the permuted load score and 539 phecodes. The red dashed lines indicates the observed number of clinical phenotypes nominally associated with genome-wide load score  and non-coding load score .(TIF)Click here for additional data file.S4 FigNull distributions of the number of clinical phenotypes weakly associated with burden scores were obtained using the same procedure to obtain the null distributions for load scores Figs  and S3. (TIF)Click here for additional data file.S1 TableThis table shows the result of linear regression tests between the slopes of DAF spectra and score category ranks for each scoring method. The slope of a DAF spectrum is the slope of the best-fit linear regression line. Score categories from low to high are coded as an integer starting from 1.(DOCX)Click here for additional data file.S2 TableThis table shows the results of association tests between a set of potential confounders and each load score. Logistic regression was used for each category of population density and linear regression was used for the others.(XLSX)Click here for additional data file.S3 TableAssociations between the 27 phenotypes  and four(XLSX)Click here for additional data file.S4 TableAssociations between the 27 phenotypes  and five(XLSX)Click here for additional data file.S5 TableAssociations between the 27 phenotypes  and four(XLSX)Click here for additional data file.S6 TableWe used the number of non-reference variants counted for each individual as an additional covariate in our linear regression model to test the association between load score and the 27 phenotypes .(XLSX)Click here for additional data file.S7 TableThis table shows the associations between the 27 phenotypes  and codi(XLSX)Click here for additional data file.S8 TableThis table shows the associations between the 27 phenotypes  and codi(XLSX)Click here for additional data file.S9 TableThis table lists the clinical phenotypes (defined by phecodes) which nominally associate  to genome-wide, non-coding, and coding load scores.(XLSX)Click here for additional data file."}
+{"text": "The formation of the central nervous system (CNS) involves multiple cellular and molecular interactions between neural progenitor cells (NPCs) and blood vessels to establish extensive and complex neural networks and attract a vascular supply that support their function. In this review, we discuss studies that have performed genetic manipulations of chick, fish and mouse embryos to define the spatiotemporal roles of molecules that mediate the reciprocal regulation of NPCs and blood vessels. These experiments have highlighted core functions of NPC-expressed ligands in initiating vascular growth into and within the neural tube as well as establishing the blood\u2013brain barrier. More recent findings have also revealed indispensable roles of blood vessels in regulating NPC expansion and eventual differentiation, and specific regional differences in the effect of angiocrine signals. Accordingly, NPCs initially stimulate blood vessel growth and maturation to nourish the brain, but blood vessels subsequently also regulate NPC behaviour to promote the formation of a sufficient number and diversity of neural cells. A greater understanding of the molecular cross-talk between NPCs and blood vessels will improve our knowledge of how the vertebrate nervous system forms and likely help in the design of novel therapies aimed at regenerating neurons and neural vasculature following CNS disease or injury. The formation of the central nervous system (CNS) begins early in development following the specification of the ectodermal germ layer . The neuHere, we will review signals produced by the neuroepithelium that direct the growth and maturation of blood vessels in the CNS, as well as reciprocal roles of CNS vasculature in regulating NPC behaviour. We will particularly focus on studies that have explored relevant molecular mechanisms through the analysis of conditional mouse mutants, because this allows us to distinguish the relative contribution of specific factors in endothelial versus neural cells during neurogenesis. We will further highlight gaps in our current knowledge about the molecular interplay between both developing systems and provide an outlook on potential directions for future research in this field.To ensure an adequate supply of oxygen and nutrients, the neuroepithelium has to produce chemoattractive guidance cues that ensure the formation of patent blood vessels in positions where neurons form and function.de novo ).Itgb8 gene that encodes \u03b28 in nestin-expressing NPCs disrupts vessel morphology, in addition to compromising the adhesion of NPCs to each other [In the developing CNS, integrins also promote normal vascular development by modulating transforming growth factor beta (TGF-\u03b2) signalling B. In parch other . In contch other . The NPCch other . These fch other ,21. Intech other ,21.Unc5b-null mouse cortex and spinal cord and the morpholino-treated zebrafish neural tube have significantly more tip cell filopodia [netrin mutants have not been analysed to date to investigate these possibilities, and additional work is therefore required to understand the complexity of mechanisms by which netrin signalling affects ECs.Netrins are laminin-related secreted molecules that bind to UNC5 and DCC receptors to exert repulsive or attractive actions in axons, respectively. Netrin signalling can also have pro- or anti-angiogenic effects during developmental angiogenesis, but it is not known whether this is due to the use of alternative receptors . It has ilopodia . Howeverilopodia . In suppilopodia ,77. As tDuring neurogenesis in vertebrate embryos, NPCs expand in number and specialise to give rise to partially committed progenitors that then differentiate into diverse subtypes of neurons and glia. Initially, the neuroepithelium contains primitive NPCs , which dThe symmetry of NPC cell division controls whether progenitors self-renew or differentiate into more committed NPCs and post-mitotic neurons . The divVegfa expression in the mouse cortex, achieved through in utero electroporation, promotes angiogenesis in the targeted areas and is accompanied by the supernumerary generation of TBR2+ BPs [+ APs and an increase of TBR2+ BPs, suggesting increased progenitor commitment [Myc [Two studies have shown that blood vessels promote NPC differentiation into more committed subtypes. Firstly, ectopic BR2+ BPs . Newly-fBR2+ BPs . Even thBR2+ BPs   project endfeet onto periventricular blood vessels and receive endothelial-derived notch and ephrin signals to remain in quiescence ,92. In ael areas . This adrneurons  (Figure rneurons . Howeverrneurons . Vessel The studies described above demonstrate the importance of extensive molecular crosstalk between NPCs and ECs in the developing CNS of several vertebrate species. We have discussed how NPCs direct vascularisation of the neuraxis through a number of complementary signalling mechanisms that regulate the initial invasion of the parenchyma by blood vessels, as well as their branching and eventual maturation within the neural tube. Yet, many outstanding questions remain to be answered before we will fully understand the process of CNS vascularisation. For example, we know now that VEGF-A has a key role in promoting mammalian CNS vascularisation, but we still need to define how it cooperates with other signalling pathways discussed here or described elsewhere to fine-tune the formation and maturation of neural vasculature. Moreover, it remains to be investigated which of these pro-angiogenic factors are of NPC or neuronal origin. Answering these questions may help devise effective therapies aimed at regenerating defective or regressed cerebrovasculature in ischaemic or degenerative CNS diseases.+ NPC subtypes [We have also discussed recent studies that highlight the importance of blood vessels in regulating neurogenesis. However, the specific mechanisms involved appear to differ, at least in part, across different regions of the developing CNS. For example, we have described above that a subset of forebrain NPC processes terminate with endfeet on periventricular vessels , whereassubtypes , whose gsubtypes . Indeed,in utero, as demonstrated, for example, by the divergent roles of NTF3 during adult and embryonic neurogenesis [Extending the idea that blood vessels regulate embryonic neurogenesis via oxygen provision, both forebrain and hindbrain studies have shown that blood vessels additionally provide angiocrine signals that regulate NPC behaviour, independently of oxygenation ,24. Thisogenesis ,98. It wUltimately, the knowledge gained on the vascular regulation of embryonic neurogenesis will increase our understanding of CNS formation and hopefully lay the foundation for innovative repair strategies in adult brain disease by exploiting developmental principles. For example, activating vasculature in the adult brain may be an ideal means to stimulate existing NSC niches and also create new niches that can support the survival and function of transplanted neural stem and progenitor cells for adult brain repair ."}
+{"text": "Our results were consistent regardless of age, sex, CKD stage of the participants (G2 or G3), daily dosage of astragalus root, or duration of astragalus-containing preparations. No severe adverse reactions were recorded in the charts of the study participants. Our results suggest that there is eGFR improvement after taking astragalus-containing preparations in mild to moderate CKD cases as reported previously. The findings should be considered with caution due to major limitations such as small sample size without optimum control, short follow-up period, and incomplete data. Further adequately powered and designed studies are needed to confirm the efficacy and safety of the long-term use of astragalus root in patients with mild to moderate CKD.In this self-controlled case series, we aimed to investigate the variation in estimated glomerular filtration rate (eGFR) after taking astragalus-containing preparations in patients with mild to moderate chronic kidney disease (CKD) by retrospectively reviewing their charts in our clinic. We set the inclusion criteria as first-visit patients aged 20 years or older presenting to our clinic between 1 October 2014, and 31 June 2019, and who were prescribed astragalus-containing herbal preparations for any reason. We calculated the mean eGFR from the readings taken 6\u00a0months before (pre) and after (post) the intake of astragalus-containing preparations for each participant. Among the 37 patients included in our final analysis, we found a statistically significant improvement in the eGFR after prescribing astragalus-containing preparations (pre, 66 \u00b1 12\u00a0ml/min/1.73\u00a0m Chronic kidney disease (CKD) is a long-term and globally widespread health condition. Moreover, this condition causes a huge economic burden due to end-stage renal disease requiring dialysis .Causes of CKD include high blood pressure, diabetes, glomerulonephritis, polycystic kidney disease, and long-term use of certain medications, such as non-steroidal anti-inflammatory drugs . UnfortuAstragalus membranaceus Bunge or Astragalus mongolicus Bunge (Leguminosae)  , which iminosae) . In addiminosae) . These Rminosae) . HoweverIn this study, we aimed to investigate the variation in estimated glomerular filtration rate (eGFR) after taking astragalus-containing preparations in patients with mild to moderate CKD by retrospectively reviewing their charts in our clinic. Our study focused on patients whose chief complaints were not CKD treatment, successfully including patients with mild to moderate CKD who took astragalus root unintentionally.This self-controlled case series was conducted at Keio University Hospital in Tokyo, Japan. The study was approved by the Keio University School of Medicine Institutional Review Board , and the protocol was registered at the UMIN Clinical Trials Registry (unique ID: UMIN000020478).2, and had an acute kidney injury (AKI) episode during the observational period. Written informed consent was obtained from all the participants prior to the study.We set the inclusion criteria as first-visit patients aged 20\u00a0years or older presenting to our clinic at Keio University Hospital between 1 October 2014, and 31 June 2019, and who were prescribed astragalus-containing herbal preparations  for any reason. We checked the adherence of these patients by following their medical records with prescriptions each time. We also allowed the continuous prescription of anti-hypertensive drugs, including renin-angiotensin-aldosterone system inhibitors. The exclusion criteria included patients who had no blood test before and/or after 6\u00a0months from the prescription of astragalus-containing preparation, had a baseline eGFR >90\u00a0ml/min/1.73\u00a0mt-test for these comparisons, and statistical significance was set at p < 0.05.All statistical calculations and analyses were performed using the R software  with Rstudio (version 1.4.1717 2021-05-24 for macOS). Descriptive statistics were used to assess demographic characteristics. The eGFR values were extracted from the chart; however, they contained completely random timing observations on eGFR. Therefore, we calculated the mean eGFR value from the readings taken 6\u00a0months before (pre) and after (post) the intake of astragalus-containing preparations for each participant to compare the eGFR values between these two periods. Additionally, we compared the baseline eGFR value with the first observation before taking the astragalus-containing preparations or the last observation after taking the astragalus-containing preparations. We employed a paired 2; and one patient was excluded due to an AKI episode during the observation period. Thus, 37 patients were included in the final analysis . Contrarily, the comparison between the baseline eGFR value and the last observation after taking the astragalus-containing preparations showed a statistically significant difference . Plasma concentration of hemoglobin or albumin did not differ significantly, and we hesitated to compare the degree of proteinuria due to lacking urinalysis data. Our results were consistent regardless of age, sex, CKD stage of the participants (G2 or G3), daily dosage of astragalus root, or duration of astragalus-containing preparations.We confirmed a statistically significant improvement in the mean eGFR after prescription of astragalus-containing preparations (pre, 66 \u00b1 12\u00a0ml/min/1.73\u00a0mFurthermore, we performed subgroup analysis by dividing the participants based on the presence of eGFR improvement in the observational period, but we did not find any difference in age, sex, CKD stage, daily dosage of astragalus root, or duration of astragalus-containing preparations.There was no severe adverse reaction recorded in the study participants\u2019 charts.Our results suggest that there is improvement in the eGFR after taking astragalus-containing preparations for mild to moderate CKD. Our study successfully included patients with mild to moderate CKD who unintentionally took astragalus root.Astragalus root is also effective in reducing fasting blood glucose and albuminuria levels, in reversing the glomerular hyperfiltration state, and in ameliorating the pathological changes of early diabetic nephropathy in animal models (Previous studies have reported on the various bioactive chemicals in astragalus root  and relal models . Notablyl models . Despitel models  that preAstragalus membranaceus, possibly suggesting higher efficacy if a higher dosage was used in our study population. Third, our study was observational, including small sample size, obtaining incomplete renal data, and the daily dosage and dosing duration varied significantly. Fourth, we could not conclude the efficacy and safety of long-term interventions in our short follow-up study. Fifth, we did not compare patients treated with Kampo formulas without astragalus root with the present self-controlled study. Therefore, we additionally performed regression analysis and confirmed the negative eGFR trend over time before astragalus-containing preparations (This study had several limitations. First, the CKD-G stages of our participants were G2 and G3, and it is unclear whether our results can be applied to patients with CKD stage G4 or G5. Although, previous studies have already reported favorable effects of astragalus roots in this population. Second, previous studies employed a higher daily dosage of arations . Lastly,In conclusion, Our results suggest that there is eGFR improvement after taking astragalus-containing preparations in mild to moderate CKD cases as reported previously. The findings should be considered with caution due to major limitations such as small sample size without optimum control, short follow-up period, and incomplete data. Further adequately powered and designed studies are needed to confirm the efficacy and safety of the long-term use of astragalus root in patients with mild to moderate CKD."}
+{"text": "Hepatocellular carcinoma (HCC) is one of the leading causes of tumor-associated deaths worldwide. Despite great progress in early diagnosis and multidisciplinary tumor management, the long-term prognosis of HCC remains poor. Currently, metabolic reprogramming during tumor development is widely observed to support rapid growth and proliferation of cancer cells, and several metabolic targets that could be used as cancer biomarkers have been identified. The liver and mitochondria are the two centers of human metabolism at the whole organism and cellular levels, respectively. Thus, identification of prognostic biomarkers based on mitochondrial-related genes (Mito-RGs)\u2014the coding-genes of proteins located in the mitochondria\u2014that reflect metabolic changes associated with HCC could lead to better interventions for HCC patients. In the present study, we used HCC data from The Cancer Genome Atlas (TCGA) database to construct a classifier containing 10 Mito-RGs  for predicting the prognosis of HCC by using 10-fold Least Absolute Shrinkage and Selection Operation (LASSO) cross-validation Cox regression. Based on the risk score calculated by the classifier, the samples were divided into high- and low-risk groups. Gene set enrichment analysis (GSEA), gene set variation analysis (GSVA), t-distributed stochastic neighbor embedding (t-SNE), and consensus clusterPlus algorithms were used to identify metabolic pathways that were significantly different between the high- and low-risk groups. We further investigated the relationship between metabolic status and infiltration of immune cells into HCC tumor samples by using the Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm combined with the Tumor Immune Estimation Resource (TIMER) database. Our results showed that the classifier based on Mito-RGs could act as an independent biomarker for predicting survival of HCC patients. Repression of primary bile acid biosynthesis plays a vital role in the development and poor prognosis of HCC, which provides a potential approach to treatment. Our study revealed cross-talk between bile acid and infiltration of tumors by immune cells, which may provide novel insight into immunotherapy of HCC. Furthermore, our research may provide a novel method for HCC metabolic therapy based on modulation of mitochondrial function. Hepatocellular carcinoma (HCC) is one of the most common malignant cancers and is currently the fifth and seventh leading cause of cancer-related deaths worldwide in females and males, respectively . DespiteMitochondria are centers of cellular metabolism that regulate metabolite and energy flow essential for cell growth, proliferation, differentiation, and death . TherefoThe liver is the key regulator of whole-body metabolism and maintains metabolic homeostasis. Recent studies have demonstrated that substantial metabolic changes are associated with various types of cancers, including HCC . Thus thIn the present study, we constructed a classifier containing 10 Mito-RGs for HCC cell survival by utilizing Least Absolute Shrinkage and Selection Operation (LASSO) Cox regression. Based on the risk score calculated by the classifier, the samples were divided into low- and high-risk groups. We further investigated changes in metabolism and metabolic subgroups of HCC samples by Gene Set Variation Analysis (GSVA), t-distributed Stochastic Neighbor Embedding (t-SNE), and consensus clusterPlus. Additionally, we used the Cell-type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT) algorithm and the Tumor Immune Estimation Resource (TIMER) database to investigate the relationship between metabolic status and infiltration of HCC samples by immune cells. Our results demonstrate that the Mito-RGs-based classifier can be used as a reliable predictor of HCC patient survival. The suppression of metabolic processes governing bile acid biosynthesis may play a vital role in the development and poor prognosis of HCC, providing a potential approach to treatment. Moreover, our research reveals cross-talk between bile acid and infiltration of tumors by immune cells, which may provide novel insight into immunotherapy of HCC. Therefore, our research may provide a novel method for HCC metabolic therapy based on modulation of mitochondrial function.Bioinformatics analyses were performed using the procedure shown in https://xenabrowser.net/datapages/) based on information in the TCGA database. For validation, RNA-seq data and clinical information of an additional 232 HCC tumor samples were obtained from the ICGC portal (https://dcc.icgc.org/projects/LIRI-JP). HCC patients with complete survival data and RNA-seq data were included in the subsequent analysis. HCC data were annotated by the Homo_sapiens.GRCh38.84.chr.gtf (ftp.ensembl.org) file in this study.An RNA-seq dataset and the corresponding clinical parameters of HCC tissues (n = 374) and normal liver tissues (n = 50) were downloaded from UCSC-Xena (http://software.broadinstitute.org/gsea/msigdb). A total of 23 cellular component gene sets related to mitochondria and 1571 unique genes were ultimately screened as Mito-RGs  cellular component gene sets in the molecular signatures database (MSigDB) database |) was used to relate every pairwise gene-gene relationship. An adjacency matrix was then constructed using a \u201csoft\u201d power adjacency function ija = Power = |ij|\u03b2s where ijs is the co-expression similarity, and ija represents the resulting adjacency that measures connection strengths. The adjacency matrix was then used to define a network distance measure, or more precisely, a measure of node dissimilarity based on a topological overlap matrix. Specifically, the topological overlap matrix is given byWeighted gene co-expression network analysis (WGCNA) for all Mito-RGs in the HCC dataset was performed according to the protocols of WGCNA , 8, as di and j are connected, and u indexes the nodes of the network. The topological overlap matrix (TOM) is given by \u03a9 = [\u03c9ij], where \u03c9ij is a number between 0 and 1 and is symmetric . The rationale for considering this similarity measure is that nodes that are part of highly integrated modules are expected to have high topological overlap with their neighbors. Clusters of genes with high topological overlap were identified as \u201cgene modules\u201d, utilizing a measure of dissimilarity (=1\u2212TOM).where, Correlations between modules and clinical characteristics were calculated by Pearson\u2019s correlation tests to identify modules with significant clinical meanings. The modules that exhibited high correlations with HCC clinical characteristics were selected as modules of interest for further study.A univariate Cox regression was performed for all Mito-RGs in modules of interest and the genes with P < 0.05 were identified as prognostic Mito-RGs.Since only the TCGA cohort was enrolled in the present study, the 10-fold LASSO cross-validation Cox regression analysis was applied to all prognostic Mito-RGs for selection of the most useful prognostic biomarkers and to construct a survival-predicting classifier. LASSO is a popular method of regression with multiple dimensional parameters . LASSO iWe then used the cutoff of the median risk score to divide the HCC patients into low- and high-risk groups. The predictive ability of the model for the training and validation cohorts, which was randomly split in a 1:1 ratio, as well as for the total cohort, was evaluated using the Kaplan-Meier log-rank test. Furthermore, the application value of the model was tested by Receiver Operating Characteristic (ROC) curve analysis, and by univariate and multivariate Cox regression analysis.In order to investigate any changes in mitochondrial function and metabolic pathways between high- and low-risk groups, we performed Gene Set Enrichment Analysis (GSEA) and Gene Set Variation Analysis (GSVA).GSEA is a method for determining whether a given gene set is significantly enriched in a list of gene markers ranked by their correlation with a phenotype of interest. The first step of GSEA is to sort genes according to the degree of differential expression in the two sample phenotypes . Then, the GSEA method calculates an Enrichment Score (ES) by proceeding through the list, increasing a cumulative sum when a gene is in the gene set and decreasing it if a gene is not. According to the ES, we can estimate the degree of enrichment of a gene set for the phenotype. Furthermore, GSEA normalizes the ES for each gene set to account for the variation in gene set sizes, yielding a normalized enrichment score (NES) . The cluGSVA is a gene set enrichment method that estimates variation of pathway activity over a sample population in an unsupervised manner . GSVA trT-SNE is one of the most effective methods to reduce dimensionality while maintaining the similarity between low-dimensional descriptors and high-dimensional data. In the t-SNE method, the low-dimensional space maintains the pair-wise similarity to the high-dimensional space, leading to a clustering in the embedding space close to the clustering in the high-dimensional space without losing significant structural information , 16. ConTo deduce the metabolic status of a sample, we used the t-SNE and Consensus ClusterPlus R packages  to cluster HCC samples into different metabolic subgroups.In order to further explore the relationship between metabolic status and immune cell infiltration, the CIBERSORT algorithm  was usedhttp://www.R-project.org) and GraphPad Prism 8.0 statistical software . The correlation between risk score and clinicopathological characteristics was analyzed by the chi-square test. The statistical significance of normally distributed variables of the two sample groups was estimated by the two-tailed unpaired t-test. P<0.05 were considered statistically significant.All statistical analyses were conducted by R version 3.6.1 (\u22126), pathologic T , tumor stage , vital status , and days to death   \u2009+\u2009(0.767 * expression level of ADPRHL2)\u2009+\u2009(0.211 * expression level of ATAD3A)\u2009+\u2009(1.226 * expression level of BSG)\u2009+\u2009(0.369 * expression level of FAM72A)\u2009+\u2009(0.202 * expression level of PDK3)\u2009+\u2009(0.419 * expression level of PDSS1)\u2009+\u2009(0.128 * expression level of RAD51C)\u2009+\u2009(0.028 * expression level of TOMM34)\u2009+\u2009(1.033 * expression level of TRMU). Patients in every cohort were further divided into high- and low-risk groups at the cutoff value of the median risk score. Expression levels of every biomarker in different groups were analyzed. The results showed that the levels of all 10 biomarkers were much higher in the high-risk group than in the low-risk group  and total  cohorts, histologic grade in the training , validation , total  cohorts, pathologic T (pT) in the training  and total  cohorts, pathologic N (pN) in the total  cohort, and tumor stage in the training  and total  cohorts showed significant differences between the two groups.As shown in As shown in To further assess the prognostic value of the classifier, a Kaplan-Meier test was conducted. As shown in In addition, in the time-dependent ROC curve analysis, the Area Under The Curve (AUC) for overall survival rates for 1, 3, and 5 years were, respectively, 0.838, 0.771, and 0.834 in the training cohort, 0.716, 0.627, and 0.608 in the validation cohort, and 0.787, 0.696, and 0.705 in the total cohort Figure 5Furthermore, the results of univariate Cox regression analysis in the training, validation, and total cohorts further validated the prognostic value of the classifier Table 3.In addition, we compared the predictive capability of our classifier with other previously published classifiers. As shown in Furthermore, to test the robustness of the classifier, the HCC patients from the ICGC cohort were also categorized into high- or low-risk groups using the median value calculated from the formula described above. As shown in These results indicate that the Mito-RGs-based classifier provides a useful prognostic tool with clinical value for appropriately categorizing patients with HCC.Since bile acid is a liver-specific metabolic substance, we investigated a possible role for measurements of primary bile acid biosynthesis in prognosis of HCC. As shown in Finally, we conducted Kaplan-Meier analyses to further verify the value of these processes to the prognosis of HCC. As shown in In order to further investigate the role of bile acid metabolism in HCC, we conducted cluster analysis based on gene expression of the primary bile acid biosynthesis pathway. As shown in These results further demonstrate that metabolic processes governing bile acid biosynthesis affect the prognosis of HCC.The bar plots in The Wilcoxon rank-sum test revealed that tumor-infiltrating CD8 T cells (P = 0.023), activated CD4 memory T cells (P = 0.01), Tregs (P<0.001), M0 macrophages (P = 0.005), and neutrophils (P = 0.004) were significantly higher in cluster 1. However, resting NK cells (P = 0.031), M1 macrophages (P<0.001), monocytes (P<0.001), and resting mast cells (P<0.001) were significantly higher in cluster 2 , while patients with a high proportion of Tregs (P = 0.019), M0 macrophages (P\u00a0= 0.013), and neutrophils (P = 0.024) exhibited lower overall survival  between clusters 1 and 2 from the HCC samples. As shown in HCC is one of the leading causes of cancer-related deaths because it is highly malignant, recurrent, metastatic, drug-resistant, and usually diagnosed late in its progression . Thus, iIn the present study, a 10 Mito-RGs-based prognostic classifier for HCC was constructed and validated for prognosis HCC patients for the first time. The classifier performed well in predicting the progression of HCC patients in the TCGA training and ICGC external validation cohorts, supporting the repeatability and utility of the classifier for prognosis of HCC overall survival. Furthermore, the prediction efficacy of the classifier was superior to those of histologic grade and tumor stage (TNM stage), which are two previously reported major risk factors for tumor progression , 30. AddAll 10 Mito-RGs of the classifier, ACOT7, ADPRHL2, ATAD3A, BSG, FAM72A, PDK3, PDSS1, RAD51C, TOMM34, and TRMU, were risk-associated, and more highly expressed in the high-risk group. Among them, ACOT7, ADPRHL2, ATAD3A, BSG, FAM72A, PDSS1, RAD51C, and TOMM34 were overexpressed in HCC compared with normal liver tissues, indicating potential roles of these genes in the initiation and development of HCC.via modulating fibroblasts and tumor cells themselves to disrupt the HCC microenvironment (ACOT7 is an isoform of the acyl-CoA thioesterase (ACOT) family, which is responsible for cleaving fatty acyl-CoAs to free fatty acids . High exironment . FAM72A,ironment . Previouironment . In addiironment , 54. RADironment . Accuratironment , 57. TOMironment . A previironment , ovarianironment , and breironment  and servMetabolic changes are a well-founded hallmark of cancers, including HCC . The widEarly in the 1970s, it was shown that plasma bile acid concentrations are elevated in HCC patients compared with healthy individuals , indicatA recent study found that bile acids can serve as messengers in the gut microbiome to control accumulation of hepatic NKT cells by upregulation of CXCL16 and anti-tumor immunity against both primary and metastatic liver tumors in the liver , suggestThe immune-modulatory effects of bile acids have been widely researched in the gastrointestinal tract  and liveThe mechanism of bile acid-mediated immune cell infiltration of tumors remains unclear. In the present study, we found that the genes controlling primary bile acid biosynthesis, CYP7A1, CYP8B1, SLC27A5, and CYP27A1 negatively correlated with infiltration of CD8+ T cells, macrophages, and neutrophils. These results provide clues for further investigation. However, the mechanism by which these genes mediate infiltration of tumors by immune cells requires further exploration.Inevitably, the present study has some limitations. Firstly, it was a retrospective study based on public online databases. Second, only two cohorts consisting of 606 samples were included. Therefore, large-scale, multi-center studies are needed to verify our results before the Mito-RGs-based classifier can be used in the clinic.In conclusion, we first identified and validated a classifier containing 10 Mito-RGs with independent prognostic significance for patients with HCC. Based on the classifier, we showed that the primary bile acid biosynthesis pathway was correlated with the prognosis of HCC, indicating that this pathway and related metabolites provide potential targets for anti-tumor treatments. Moreover, our research reveals cross-talk between bile acid and immune cell infiltration of tumors, which may provide novel insight into immunotherapy of HCC. Finally, our research may provide a novel method for HCC metabolic therapy based on modulation of mitochondrial function.https://portal.gdc.cancer.gov/) (TCGA-LIHC); the NCBI Gene Expression Omnibus (GSE76427 and GSE10143).Publicly available datasets were analyzed in this study. This data can be found here: The Cancer Genome Atlas  and the National Key Research and Development Project (No. 2018YFC2001802).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": "Lycium barbarum, a highly nutritious and medicinal crop. However, the influence of environmental factors on AMF communities remains largely elusive. Based on MiSeq sequencing, we analyzed AMF communities in rhizosphere soils of L. barbarum with growth synchronization in three typical L. barbarum cultivation sites in China. The Zhongning region has poor soils with a high richness of AMF communities. Geographical environmental variances lead to differences in AMF communities which in turn affects the active ingredients of L. barbarum fruit. Furthermore, different genera of AMF showed significant correlations with environmental factors and fruit ingredients. The three genera, Claroideoglomus, Dominikia, and Funneliformis correlated to environmental factors and fruits ingredients in a similar manner affecting the whole sugar (TS) and flavonoids (FLA) contents in the fruits of L. barbarum. Also, these showed a significantly positive correlation with soil pH. This fact was unknown so far due to different soil acidity/alkalinity in different studies.The symbiotic relationship of arbuscular mycorrhizal fungi (AMF) is important for IMPORTANCE The climatic and ecological environment is a complex phenomenon, involving various environmental factors that regulate the diversity and population distribution structure of AMF communities affecting plant growth, crop composition, and yield. Current studies on the effects of environmental factors on AMF communities have mainly focused on soil conditions and host plants. Fewer studies have been conducted on the correlation between temperature, enzyme activity, plant fruiting, and AMF communities. The present study investigated the diversity of AMF communities and the influence of environmental factors on their distribution patterns, which showed similar effects on some AMF species. The results suggest that screening AMF fungicides that meet the target may significantly help soil restoration reducing the use of chemical fertilizers and a large amount of human and material resources. Lycium barbarum L , originally cultivated in East Asia (\u2013Lycium barbarum polysaccharides)  . This suharides) , 10. AlsArbuscular mycorrhizal fungi  are the most widely distributed endophytic mycorrhizae, which constitute a group of root obligate biotrophs that exchange mutual benefits with most plants , 12. The13\u2013Change in soil characteristics due to different locations, both in latitude and longitude directions , 27, can31\u201334\u2013The traditional AMF taxonomic classification is mainly based on the morphological features of soil asexual spores, which has certain limitations , 40. RecL. barbarum from three areas and confirmed the symbiosis of mycorrhizal fungi , and FLA being significantly higher in the ZN region, betaine (BET) and FLA being highest in the DL region . P <0.05 was used to assess the significance of the data.All the nine soil physicochemical indicators showed significant differences among the three regions in the various months, with all soils being alkalinesee . From May to September, the soil pH rose, then fell, and finally ended up being slightly lower than the initial value. Notably, Zhongning (ZN) had the highest pH  and the remaining eight indicators were lower than in the other two regions. In all three regions, total soil nitrogen (TN), total phosphorus (TP), and total potassium (TK) tended to decrease from May to September, while available nitrogen (AN) and available potassium (AK) increased and then decreased; the increase in available phosphorus (AP) was significantly higher in the ZN region. In May, cellulase (CEL) was significantly higher in the ZN region compared with other regions, while urease (URE) was the lowest. In contrast, sucrase (SUR) was significantly higher in the Dulan (DL) region than in the other two regions . Importantly, temperature data were less significantly distinct , with TS, Rhizophagus, Dominikia, and Glomus each had seven OTUs; Funneliformis and Claroideoglomus had three and two OTUs, respectively.The dilution curves for all samples from the three regions flattened out, and a further increase in sequencing depth only added a small number of additional species. This indicated that sequencing depth was adequate . The optimized sequence number of 1959297 was obtained from 27 soil samples and clustered into 1,762 microbial operational taxonomic units (OTUs), dominated by Ascomycota with 1126 OTUs, followed by 152 Basidiomycota, 66 Chytridiomycota, 55 Mortierellomycota, and 51 Glomeromycota OTUs, while other fungal phyla were relatively low. The AMF OTUs belonged to two classes, three orders, four families, nine genera, and 15 species. Fifty-one Glomeromycota OTUs belonged to five genera; The community richness and diversity analyses revealed higher AMF abundance in ZN rhizosphere soil samples than in the Jinghe (JH) and DL samples . Among tobs, ACE, and Chao) revealed significantly higher AMF community richness in the ZN region than in JH and DL; among ZN samples, richness was higher in July and highest in September  between the sample source and pH, electrical conductivity (EC), soil organic matter (SOM), TN, TS, FLA, monthly average atmospheric humidity (MAAH), TP, and BET; and some correlations (P\u2009<\u20090.05) were also found with SUR, amylase (AMY), AK, LBP, monthly average soil temperature (MAST), monthly minimum temperature (MMinT), AP, URE, monthly average temperature (MAT), protease (PRO), monthly maximum temperature (MMT), and TK.Permutational multivariate analysis of variance (PERMANOVA) was performed to examine the relationship and significance analysis among variation in AMF communities, environmental factors, and fruit ingredients for different samples. The results showed significant correlations  and significantly correlated with TN, TP, TS, LBP, monthly average daily difference in temperature (MADTD), and MAST .A db-RDA analysis was performed for correlation analysis among AMF communities, environmental factors, and fruits ingredients in samples from different regions and months; 36%, 57%, and 35.97% of the variations in AMF communities are explained by each of the two ranking axes  and B. SThe correlations among environmental factors, fruit ingredients, and AMF communities are reflected at the genus level , with thClaroideoglomus, Dominikia, and Funneliformis, were significantly positively correlated with pH. Two genera, Dominikia and Funneliformis, were significantly positively correlated with TP, BET, AK, SOM, URE, TK, MAAH, and TN. Rhizophagus, Glomus, and unclassified_p__Glomeromycota were significantly positively correlated with MAST, MAT, and MMinT, and negatively correlated with TP and BET.Three genera, AMF symbiosis provides a range of benefits to the host plant in exchange for plant-produced carbohydrates such as improved nutrition, resistance to soilborne pests and diseases, drought tolerance, heavy metals tolerance, and soil structure \u201345. Howe\u2013https://unite.ut.ee).There is no consensus barcoding region for the determination of AMF taxa . InternaL. barbarum planting areas, with three sampling sites located in northwestern China, at an altitude of 1,123 m, 290 m, and 2,783 m in the Zhongning, Jinghe, and Dulan regions, respectively. More OTUs were detected in the ZN samples showing higher richness and diversity. In Brazil, variation in AMF communities was found along an altitudinal gradient of 700 m ; AMF density and richness were higher at intermediate altitudes, and species composition showed statistical differences at different altitudes , while ZN soil samples were highly enriched in Rhizophagus (23%), Dominikia (21%), and Glomus (18%). In general, Rhizophagus and Glomus are abundant in rhizosphere soil samples in both farming and restoration areas  between the soil samples from three regions; cellulase showed a significant positive correlation with AMF communities. Cellulase degrades cellulose into glucose for microbial use. This suggests that cellulase provides a conducive environment for the growth of AMF communities. Urease content indicates the nitrogen status of the soil. Spearman correlation analysis revealed that three genera, Claroideoglomus, Dominikia, and Funneliformis, were significantly negatively correlated with urease, showing a negative effect of urease and total nitrogen. AMF inoculation is known to increase the soil urease, convertase, and cellulase activities  of Northwest China has the highest pH, compared with the other two regions. Especially, the soil of the Zhongning region is poor with lower soil physicochemical indicators, but it has a higher abundance of AMF species. Many studies suggest that soil pH significantly affects the AMF communities , 63\u201366. 63\u2013L. barbarum producing regions in northwest China and found the highest AMF abundance in the Zhongning region despite poor soils. The results show that rhizosphere soil physiochemical properties, enzyme activity, climatic factors , and fruit ingredients correlate with AMF communities. Furthermore, different AMF genera  correlated to environmental factors and fruits ingredients in a similar manner affecting the TS and FLA contents in the fruits of L. barbarum.We analyzed AMF communities in rhizosphere soil from three high-quality L. barbarum single plant were raised using shoot cuttings breeding technology in Zhongning (Ningxia Province), Jinghe (Xinjiang Province), and Dulan (Qinghai Province) counties . This confirmed that rhizosphere AMF from all three regions could infest L. barbarum and produce symbiotic structures.The pure seed and healthy tree of \u201cNing Qi 1\u201d Chinese counties . TT-QXZ L. barbarum produces summer fruits with indefinite inflorescences at full flowering and fruiting stages. The summer fruits were harvested for five to seven crops, and autumn fruits were collected after summer dormancy was over. The crop with the most fruits was selected to represent the year\u2019s yield and the analysis of fruit's active ingredients.Three sites were randomly selected for the experimental survey, and the soil samples were collected at three different time points in May (berry budding stage), July (berry blooming stage), and September (berry fruiting stage). A clean shovel was used to remove surface foliage, stones, and debris to collect soil samples at 20 cm to 40\u2009cm depth, which were well marked and stored in self-sealing bags. Three random soil samples were collected each month at one sampling site and then divided into two parts. Those were placed into two separate sterilized 50-mL centrifuge tubes in an ultra-low temperature (\u221280\u00b0C) refrigerator for fungal 18S rDNA high-throughput sequencing and soil physicochemical parameters analysis, respectively. Rhizosphere soil samples were collected from three areas in 3 months with three replicates, making a total of 27 samples. CTTGGTCATTTAGAGGAAGTAA-3\u2032) and ITS2R (5\u2032-GCTGCGTTCTTCATCGATGC-3\u2032) primers corresponding to the region ITS1 (length 350\u2009bp). PCR was carried out in a 20-\u03bcL reaction mixture consisting of a 10\u2009ng DNA template, 0.4 \u03bcL FastPfu Polymerase , 0.8 \u03bcL (5\u2009\u03bcM) Forward Primer, 0.8 \u03bcL (5\u2009\u03bcM) Reverse Primer, 4 \u03bcL 5\u00d7FastPfu Buffer, 2 \u03bcL 2.5\u2009mM dNTPs, 0.2 \u03bcL BSA, and ddH2O to complete the remaining volume. PCR conditions were as follows: 3\u2009min of denaturation at 95\u00b0C; 30 seconds at 95\u00b0C for 34 cycles; 30 seconds primer annealing at 55\u00b0C; 45 seconds extension at 72\u00b0C, 10\u2009min final extension at 72\u00b0C and lastly 10\u00b0C until removed for storage. Three PCR product replicates were produced for each sample and then mixed. The products were analyzed using 2% agarose gel electrophoresis and then gel purified using the AxyPrep DNA Gel Extraction Kit . Finally, the PCR products were quantified by a Quantus Fluorometer . The samples were diluted based on the amount required for sequencing.Soil DNA was extracted from all 27 samples using 0.5-g soil samples and FastDNA Spin Kit for Soil following the manufacturer\u2019s instructions . DNA purity, concentration, and integrity were determined by NanoDrop 2000 UV-vis spectrophotometer , and 1% agarose gel electrophoresis, respectively. The PCR was performed to amplify 18S rDNA fungal microbial fragments with ITS1F  following the standard procedures of Majorbio Bio-Pharm Technology Co. Ltd. .https://unite.ut.ee) was screened with RDP classifier version 2.2  were measured by double-beam UV-Vis spectrophotometry.Nine major soil physicochemical factors affecting microbial communities were analyzed , 76. TheL. barbarum were used for data analysis. The abbreviations of the indicators are summarized in , MAAH, and MAST during the growth and development period of rized in .obs , ACE (index for richness and evenness of species composition), and Chao (index for number of OTUs) indices using mothur (version v.1.30.1) (http://www.mothur.org/wiki/Schloss_SOP#Alpha_diversity) software. Shannon and Simpson indices, which reflect the community and spectral diversity, were also calculated. PD was used to measure the biodiversity of the microbial communities.The clumped mycorrhizae belonging to the phylum Glomeromycota were screened from respective samples. To study the diversity index, each sample was sampled flat for community diversity analysis, and dilution curves were constructed with a minimum number of sample sequences using the R language tool. To analyze the species richness of the samples, we calculated Spost hoc test for multiple test correction were applied. The Tukey-Kramer method (which requires the same number of samples) was used to analyze the variability between groups with a confidence interval of 0.95. To further assess the similarity of community composition between different samples, the Bray-Curtis distance algorithm was used at the OTU level considering the abundance and presence of species. ANOSIM test was used to find the difference between groups, and the number of Monte Carlo random permutations was set to 999 as default for PCoA analysis. PERMANOVA was used to decompose the total variance using the semi-metric Bray-Curtis algorithm to analyze the effect of different grouped environmental factors on sample differences. The statistical significance of the divisions was analyzed using Monte Carlo random permutations (999 times) in statistical analysis.The samples from three regions were analyzed for comparative analysis of species composition by selecting OTUs with 97% similarity. Venn diagrams were constructed to compare divergent and shared species, and community histograms were plotted at the genus level based on OTUs classification. The classification was statistically and graphically presented using R language tools. Given that the sample species abundance did not conform to a normal distribution, the Kruskal-Wallis rank-sum test without overall restrictions and the https://www.megasoftware.net/).To understand the history and sequence of biological evolutionary processes in AMF communities of the three regions, evolutionary trees were systematically constructed at the OTU level using maximum parsimony (MP) method in Mega software (version 10.0 L. barbarum fruits in different months using Tukey test or one-way ANOVA (P <0.05). To analyze the relationship between AMF communities and environmental factors, db-RDA  analysis was chosen for its ability to address the limitation of data type . Temporary Submission ID: SUB10211634.The raw reads have been stored in the NCBI Sequence Read Archive (SRA) database ("}
+{"text": "Pseudomonas aeruginosa is a major pathogen in humans and other animals, frequently harboring mechanisms of resistance to commonly used antimicrobials. Here, we describe the isolation of Pseudomonas bacteriophage Zikora. The full 65,837-bp genome was annotated and demonstrates similarity to Pbunavirus phages, making Zikora a new member of this genus of the Myoviridae family. Pseudomonas aeruginosa infections is becoming a global concern and has not spared Nigeria (P. aeruginosa is a Gram-negative opportunistic organism found in soils and aquatic environments (The emergence of multidrug-resistant (MDR)  Nigeria . P. aeruronments . It has ronments .P. aeruginosa strain ACE015 as a host and the classic double-layer agar (DLA) method, as described previously (https://www.patricbrc.org) and Unicycler (https://blast.ncbi.nlm.nih.gov/Blast.cgi). VIRIDIC , in May 2020. It has been propagated using nicycler . ARAGORNnicycler . Phage tnicycler . A Web-b VIRIDIC  was used VIRIDIC .Pseudomonas phage DRL-P1 (GenBank accession number MN564818) (97.77% identity with 100% coverage), phage DL52 (GenBank accession number KR054028) (97.38% identity with 100% coverage), and phage misfit (GenBank accession number MT119367) (95.63% identity with 100% coverage). Regarding its close identity with other pbunaviruses, i.e., a genus of the Myoviridae family of phages, Zikora was classified as a Myoviridae member by VIRIDIC. PhageTerm determined Zikora to package DNA by the classic headful mechanism  .The complete genome of Zikora was obtained as a single contig of 65,837 bp with a G+C content of 54.88%. A total of 92 open reading frames (ORFs) and no tRNAs were predicted. At the nucleotide level, the closest neighbors of Zikora were found to be the pbunavirus echanism . Zikora MW557846, BioProject number PRJNA693824, BioSample number SAMN17478038, and SRA number SRX11023225.The genome sequence and associated data for phage Zikora were deposited under GenBank accession number"}
+{"text": "Blumeria graminis f. sp. tritici (Bgt), is a widely occurring foliar disease of wheat worldwide. Wild emmer wheat  , the progenitor of the cultivated tetraploid and hexaploid wheat, is highly resistant to powdery mildew, and many resistance alleles were identified in this wild species. However, only Pm41 that encodes a nucleotide\u2010binding leucine\u2010rich repeat (NLR) protein has been cloned using a map\u2010based cloning approach  technology  was performed for three histone marks H3K27me3, H3K4me3, and H3K36me3 that are closely associated with gene activity. After quality control, 423\u00a0924\u00a0608 and 358\u00a0879\u00a0346 reads for the resistant and susceptible DNA bulks, respectively, were obtained for subsequent analysis.Powdery mildew resistance gene 9 Figure\u00a0, an intrd Figure\u00a0. We seleMlWE18 was located. Following stringent mapping and filtering steps, 8,461 high confidence single nucleotide polymorphisms (SNPs) were detected  reference genome. Since CS was susceptible to powdery mildew, we focussed on sequences that were partially mapped to CS . A total of 479 CGT\u2010Seq\u2010derived contigs were identified in the resistant pool and sequence annotation revealed that five contigs contained NB\u2010ARC domain, with lengths ranging from 595 to 2128\u00a0bp and sequence identities ranging from 15% to 56% between each other. Significantly, one 2128\u00a0bp contig (resk36_3047604) was highly homologous (99% identity) to the diploid T. urartu wheat Pm60 sequence  linked to MlWE18. Since MlIW172 was mapped to the same chromosomal region as MLWE18  derived from the Xuezao \u00d7 3D249 cross, three CGT\u2010Seq contig\u2010derived markers CGT1, CGT2, and CGT3 were found to co\u2010segregated with MlWE18, whereas CGT5 was closely linked (~0.05\u00a0cM)  allele and Pm60\u2010related contig resk36_3047604  were expressed in 3D249 before and after Bgt isolate E09 inoculation whereas the others were not  Figure\u00a0. No poly3 Figure\u00a0, corresp3 Figure\u00a0. RT\u2010PCR t Figure\u00a0. Since c9 Figure\u00a0, the NLRWE18NLR were cloned from the resistant 3D249, and no alternative splicing variant was found in the gene body. The WE18NLR contained only one exon and encoded a typical CC\u2010NBS\u2010LRR protein with 1454 amino acids that was allelic to Pm60 with six synonymous and three nonsynonymous SNPs  transformation. The construct contained a 12\u00a0230\u00a0bp genomic fragment with 2103\u00a0bp of presumed native promoter, 4365\u00a0bp exon region, and 5762\u00a0bp terminator .The genomic DNA and full\u2010length cDNA sequences of s Figure\u00a0. To furtr Figure\u00a0. Four po9 Figure\u00a0. These ret al., et al., MlWE18 (WE18NLR) whose locus is not present in the wheat reference genomes using a combined BSA and CGT\u2010seq strategies. The major advantage of this approach is that it is mostly reference genome free and can identify genes which are not present in the reference genomes but in a specific donor line. The proposed bulked segregant CGT\u2010Seq approach and the computational strategy can be used in further genetic mapping of genes controlling important agronomic traits in wheat and other crops with large genomes.Most of the cloned wheat disease resistance genes are NLRs that tend to be PAVs between the resistance and susceptible genotypes, especially the genes derived from wild relatives (Li The authors declare no conflict of interest.Z.L. and Y.Z. conceived the project. Q.W., Y.C., P.Z., H.Z., G.G., P.L., and M.L. performed fine mapping and map\u2010based cloning. F.Z. performed the CGT\u2010Seq experiment and data analysis. F.Z., J.X., L.D., and S.M. performed bioinformatic and micro\u2010collinearity analysis. T.F. and E.N. provided wild emmer wheat materials. Q.W., F.Z., H.L., T.F, Y.Z., and Z.L. wrote the manuscript."}
+{"text": "Short-track speed skating (STSS) is an extreme sport in pursuit of extreme speed and explosive force. In such a sport, once athletes fall down, they are susceptible to serious cervical spine injury (CSI) under the inertia of high-velocity movement. Nanohydroxyapatite/polyamide 66 (NHP66) bioactive cage is a high-tech product of nanotechnology in the medical field in recent years. With a structure similar to that of human cortical bone, NHP66 bioactive cage has extremely high toughness and strength, which tailors to the needs of STSS.  This study mainly analyzed the therapeutic effect of NHP66 on patients with CSI in STSS, aiming to provide new opportunities for the treatment of this patient population.  A total of 51 patients with CSI treated in our hospital were enrolled, including 19 cases of short-track speed skaters (observation group) and 32 cases of car accidents, falls from heights, or collision injuries (control group). The relevant surgical indicators , the incidence of adverse reactions, the Cobb angle of cervical lordosis before and after surgery, and the fusion segment height of the cage were observed and compared between the two groups. Postoperative pain was evaluated by the visual analog scale (VAS), improvement of spinal cord injury was assessed by the American Spinal Cord Injury Association (ASIA) Impairment Scale, and bone fusion, bone subsidence, and other motor functions were assessed by the Japanese Orthopaedic Association (JOA) score rating system.  The operation time, intraoperative blood loss, and incidence of adverse reactions in the observation group were significantly lower than those in the control group. The Cobb angle of cervical lordosis and the fusion segment height of cage increased significantly higher in both groups after surgery. In addition, the VAS scores of the observation group 2\u2009h and 3\u2009d after operation were significantly lower than those of the control group. In terms of improvement of spinal cord injury, ASIA and JOA scores in the observation group were significantly higher than those before treatment and in the control group. There was no significant difference in bone fusion activity between the two groups.  In this study, it is found through experiments that NHP66 has higher safety and application value than autogenous iliac bone, confirming that NHP66 can achieve significant results as a cage for anterior cervical decompression and iliac bone graft fusion and internal fixation in short-track speed skaters after CSI. In the fierce and fast movements of competitive sports, athletes are susceptible to injuries due to accidents . Short-tCurrently, conservative treatment is reserved for patients with mild CSI, while surgery is indicated for those with severe CSI combined with spinal cord injury (SCI) . AnterioNanohydroxyapatite/polyamide 66 (NHP66) bioactive cage, as a high-tech product with increasingly mature applications of nanotechnology in the medical field in recent years, has achieved remarkable achievements in decompression and fusion for cervical spondylosis . NHP66 hA total of 51 patients with CSI admitted to our hospital between May 2018 and April 2020 were enrolled. Among them, 19 STSS athletes were assigned to an observation group, and the other 32 patients injured due to traffic accident, falls from heights, or collision injuries were included in a control group. This study, approved by the Ethics Committee, was conducted in strict accordance with the Declaration of Helsinki. The subjects were fully informed of the purpose of the study and submitted an informed consent.Inclusion criteria: patients (20-40 years old) who were confirmed with cervical vertebra fracture by X-ray, CT, and MRI, with the presence of intervertebral height decrease, segmental kyphosis, cervical cord compression by fracture or ruptured cervical tissue, and vertebral subluxation, were enrolled. Exclusion criteria: patients with other cardiocerebrovascular diseases, congenital abnormality of cervical spine, cervical locked facet, or allergies to drugs used in this study were excluded, as well as referrals.The patient was placed in a supine position with the neck naturally tilted back under routine intratracheal intubation anesthesia. An anterior transverse incision was made based on the level of the injured vertebra, and blunt dissection was performed along the gap of loose connective tissue between the cervical visceral sheath and the carotid sheath. Then, the injured vertebra was located by X-ray. Subsequently, corpectomy of the injured vertebra was performed. The spinal canal was decompressed thoroughly, and the intervertebral space was distracted to restore the physiological curvature of cervical lordosis, so as to measure the height of intervertebral groove. For patients in the observation group, NHP66 bioactive cage was used as the vertebral cage, and the bone fragments dropping during spinal canal decompression were filled into the hollow part of the cage and implanted into the intervertebral bone groove. For patients in the control group, AIB was used as a cage. An oblique incision of about 4\u2009cm long was made on the patient's right iliac crest, and a full plate iliac bone strip of about 1.5 \u00d7 3.5\u2009cm in size was cut with an osteotome and implanted into the intervertebral bone groove. After implantation, the distractor was released, followed by appropriate compression, anterior cervical titanium plate internal fixation, drainage tube placement, and incision suture. After surgery, patients in both groups were given hormones, dehydrants, antibiotics, and neurotrophic drugs. In addition, they wore a neck brace for 3 months, and the stitches were removed on the 7th day after surgery.Clinical baseline data of patients in the two groups: surgical indicators: intraoperative blood loss and operation time were recorded in both groups. Safety: the incidence of adverse reactions after surgery was calculated in both groups. The cage: lateral cervical spine X-ray examination and CT scan were performed before surgery and 7 days after surgery, and the fusion segment height of the cage and the Cobb angle of cervical lordosis were measured. Pain: the visual analog scale (VAS), with a score ranging from 0 to 10, was used to evaluate the pain of patients at 2\u2009h and 3\u2009d after surgery; the score is proportional to the degree of pain. Alleviation of SCI: before surgery and 7 days after surgery, each patient was assessed for the degree of SCI according to SCI grading criteria developed by the American Spinal Injury Association (ASIA); on a 100-point scale, the score was in inverse proportion to the degree of injury. Bone fusion: the fusion of the implanted bone in the two groups was reexamined at 6 months after surgery. Once the implanted bone was fused, all the interfaces between the implant and the adjacent vertebral endplates became blurred, and new continuous bone trabeculae could be seen passing through the cage and the upper and lower endplates. The change of fusion segment height \u2265 3\u2009mm was judged as subsidence. Motor function: the Japanese Orthopaedic Association (JOA) score rating system, with a score ranging from 0 to 17 points, was used to assess the improvement of cervical function of patients before surgery and at 6 months after surgery; the lower the score, the more obvious the spinal dysfunction. Characterization and observation of NHP66: NHP66 tissues were ground into powder and added into a proper amount of normal saline. The powder sample was then observed under a microscope, and the particle size and potential of nanoparticles were evaluated with a potentiometer.n (%)) and analyzed using the chi-square test. Measurement data, such as age and body mass index (BMI), were expressed as , and the intraoperative blood loss (mL) was significantly less in the observation group compared with the control group  (The intraoperative blood loss and operation time were compared between the two groups. It was found that the operation time (min) of the observation group was significantly shorter than that of the control group  .P < 0.05, The observation group showed a total incidence of postoperative adverse reactions of 15.79%, including 0 cases of infection, 1 case of pain, and 2 cases of numbness, while the control group showed a total incidence of 43.75%, including 4 cases of infection, 5 cases of pain, and 5 cases of numbness. The incidence of postoperative adverse reactions in the observation group was significantly lower than that in the control group (P < 0.05), while increased greatly in both groups after surgery ; after surgery, the ASIA and JOA scores of both groups increased and the scores of the observation group were higher than those of the control group (both P < 0.05, The alleviation of SCI and improvement of motor function of the two groups were evaluated by the SCI grading criteria developed by the ASIA and the JOA score rating system before surgery and at 6 months after surgery, respectively. The results showed that there were no statistically significant differences in ASIA and JOA scores before surgery (P > 0.05, In the observation group, all patients had successful bone fusion, while in the control group, 93.75% of patients had bone fusion and 6.25% of them had bone subsidence. Nonetheless, there was no statistical difference in bone fusion between the two groups (Microscopically, NHP66 had an extremely dense and evenly distributed molecular structure, with a very small internanoparticle gap and particle size of about 150\u2009nm. As determined by a potentiometer, the zeta potential and PDI of NHP66 were approximately 3.26 and 0.36, respectively .In addition to the serious trauma of bone tissue, the fracture fragments and broken cervical intervertebral disc tissue generated in CSI may compress and stimulate the spinal cord, leading to neurological dysfunction . TherefoIn our study, we compared NHP66 with AIB in various aspects. First, we found that patients in the observation group treated by NHP66 experienced shorter operation time and less intraoperative blood loss than those in the control group treated by the ilium, which is consistent with our expectations. Patients treated with iliac bone as cage required not only cervical vertebra surgery but also additional removal of the iliac bone, so they experienced longer operation time and more blood loss than those in the observation group requiring only cervical vertebra surgery. Then, we calculated the incidence of postoperative adverse reactions in the two groups and found a significantly lower incidence in the observation group compared with the control group. The results suggest that NHP66 may be safer than AIB, and the reason behind it, we speculate, may be related to the shorter operation time and less blood loss in this procedure. In a long-term operation, the patients' internal environment and tissues are exposed for a longer time, which increases the possibility of oxidative stress and inflammatory reaction and consequently elevated risk of postoperative infection and pain. Subsequently, we compared the condition of the cage in the two groups. The results showed that the intervertebral height and Cobb angle of the cervical fusion segment of both groups were improved after surgery, with no significant difference between the two groups, suggesting the excellent improvement effects of both NHP66 and ilium. However, the observation group experienced better alleviation of postoperative pain and injury than the control group. In addition, all patients in the observation group had bone fusion, but some in the control group had bone subsidence. The results might be caused by the excessive curettage of adjacent vertebral bone endplate during surgery. Moreover, with a small and dense molecular structure, NHP66 can closely contact intervertebral nerve and muscle tissue and act as an annular cage, thus reducing the friction against endplate. After implantation, the molecular structure of NHP66 can slowly change to a height and curvature suitable for the cervical vertebra, and the nanomolecular particles can effectively adsorb osteoblasts, further improving the stability.The novelty of this study lies in the evaluation of clinical advantages of NHP66 bioactive cage in short-track speed skaters from multiple dimensions including surgical indicators, safety, cage, pain, alleviation of spinal cord injury, and improvement of motor function, providing a new effective treatment strategy for short-track speed skaters with CSI. However, due to the short experimental period, the prognosis of athletes implanted with NHP66, one crucial index for the evaluation of NHP66, has not been followed up. We will follow up all participants of this study for a longer time to improve our results.With excellent mechanical strength and toughness, NHP66 can deliver remarkable results as a cage for ACF combined with iliac bone graft fusion and internal fixation for athletes with CSI in STSS."}
+{"text": "Nelumbo nucifera Gaertn. is an important perennial aquatic herb that has high ornamental, edible, medicinal, and economic value, being widely distributed and used in China. The NAC superfamily  plays critical roles in plant growth, development, and response to abiotic and biotic stresses. Though there have been a few reports about NAC genes in lotus, systematic analysis is still relatively lacking. The present study aimed to characterize all the NAC genes in the lotus and obtain better insights on the NnNACs in response to salt stress by depending on ABA signaling. Here, 97 NAC genes were identified by searching the whole lotus genome based on the raw HMM models of the conserved NAM domain and NAC domain. They were characterized by bioinformatics analysis and divided into 18 subgroups based on the phylogenetic tree. Cis-element analysis demonstrated that NAC genes are responsive to biotic and abiotic stresses, light, low temperature, and plant hormones. Meanwhile, NAC genes had tissue expression specificity. qRT-PCR analysis indicated that NAC genes could be upregulated or downregulated by NaCl treatment, ABA, and fluoridone. In addition, NAC016, NAC025, and NAC070, whose encoding genes were significantly induced by NaCl and ABA, were located in the nucleus. Further analysis showed the three NAC proteins had transcriptional activation capabilities. The co-expression network analysis reflected that NAC proteins may form complexes with other proteins to play a role together. Our study provides a theoretical basis for further research to be conducted on the regulatory mechanisms of salinity resistance in the lotus. Considepression ,5. The sArabidopsis, 151 members in rice, 150 members in soybean, 261 members in M. sinensis, 132 members in peanut, 151 members in sunflower, 87 members in maize, 111 members in celery, 104 members in pepper, and 110 members in potato [Gene family analysis as a basic method plays an important role in investigating the functions of plant transcription factors. The members of the NAC family have been identified and reported in many species by gene family analysis. For instance, 108 members have been identified in n potato ,10,11,12n potato ,17,18. ONAC066, ONAC095, OsNAC2, OsNAC006, OsNAC67, OsNAC016, and OsNAP function as positive or negative regulators of the drought stress response [JUB1, SlVOZ1, SlNAC35, StNAC053, and CaNAC46, also play an important role in plant responses to drought stresses [StNAC053, grapevine VvNAC17, pepper CaNAC46, rice ONAC022, and wheat TaNAC29 enhance not only plant drought tolerance but also salt tolerance [Considering that abiotic stresses could adversely affect crop growth and productivity, it is very important to study the functions of NAC TFs in improving crop resistance to abiotic stresses, which have been well elucidated in many species in last decade. In grain crop rice, response ,22,23,24stresses ,27,28,29olerance ,30,31,32OsNAC3, OsNAC45, and VvNAC17 improved transgenic plant ABA sensitivity and salt tolerance [CrNCED5 expression to hinder ABA biosynthesis in citrus [NAC genes. In other words, NAC genes were regulated by plant miRNAs. Peu-miR164 regulated its target genes PeNAC070 and PeNAC081, which are involved in the poplar response to abiotic stress [OsCUC1 and OsCUC3, which are involved in rice meristem/organ boundary specification [NAC700 transcription to trigger phasiRNA formation in response to water deficit in legumes [NAC TFs can regulate several plant signal networks and be regulated by environmental stimuli, plant hormones, and miRNAs. Environmental stimuli, such as light, temperature, drought, salinity, water, etc., could induce the expression of NAC TFs ,34,35. Folerance ,37,38. Fn citrus . Interesc stress . osa-miRfication . In addi legumes . These rNelumbo nucifera Gaertn.), popular ornamental, edible, and medicinal plant which belongs to the small family Nelumbonaceae, will be an important model plant in horticulture [NnNAC genes have been found using the PlantTFDB 4.0, the NAC gene family still needs to be identified by searching the whole lotus genome. Here, all NAC members of lotus were identified, and their distribution, phylogenetic relationship, gene structure, and synteny were analyzed. In addition, certain candidate NAC TFs which had a higher expression in lotus leaves were further studied though subcellular localization and expression level analysis by qRT-PCR. These results will further deepen the understanding of NAC TFs and provide several candidate genes related to salinity and abscisic acid (ABA) response in lotus.Lotus  and NAC domain (PF01849) were used to seek the respective raw HMM models. Based on these two HMM models, 107 and 106 candidate members were identified, and the redundant forms of the same genes were removed. In total, 97 NAC TF members were identified and named NAC001\u2013NAC097 according to their chromosome location  was obtained from the Nelumbo Genome Database [https://pfam.xfam.org/ (accessed on 27 May 2022)). the raw HMM models were then used to search the Nelumbo nucifera Geartn. genome with the software TBtools [The Database . The raw TBtools  to obtaihttps://www.expasy.org/ (accessed on 5 June 2022)). The amino acid sequences of Arabidopsis and Nelumbo nucifera Geartn. were download from TAIR (https://www.arabidopsis.org/ (accessed on 5 June 2022)) and the Nelumbo Genome Database [The NAC genes were visualized on the chromosomes using the software TBtools . Their CDatabase , respectDatabase . The annNelumbo nucifera Geartn. and Arabidopsis was visualized using the applet Dual Synteny Plotter in TBtools [The applet MCScanX (default parameters) in TBtools was used to examine duplicate genes. Then, the homology of the NAC gene between  TBtools . The con TBtools . The motThe tissue expression data of NAC genes was downloaded from the Nelumbo Genome Database. The analysis was carried out, which included the 97 NAC genes in the different tissues and development stages, and then it was visualized by TBtools.The selected proteins were fused with the expression vector pCAMBIA1300, containing a GFP tag for subcellular localization analysis, as described previously . A dilutLotus seeds were germinated in soil in a greenhouse. Two-week-old seedlings were subjected to exogenous ABA and salinity treatments. For these treatments, seedlings were transferred to solutions containing 200 mM NaCl or 100 \u03bcM ABA. Seedlings were sampled at 0, 1, 2, 4, 8, 12, and 24 h after treatment. The samples were then dropped immediately into liquid nitrogen and stored at \u221280 \u00b0C.The samples were ground in liquid nitrogen, and the powder was used to extract the RNA. The cDNA synthesis was conducted following the manufacturer\u2019s instructions using a PrimeScript\u2122 1st Strand cDNA Synthesis Kit . Then, the expression patterns were analyzed with a BIO-RAD CFX MaestroTM system. In this assay, three biological and technical replicates were made. The histogram was made with the GraphPad Prism 8.0 software, and the standard error was calculated with the SPSS software. The primers used in this study can be found in The transactivation ability of NAC TFs was tested in yeast cells. The fusion plasmids NnNACs:pGBKT7 and the control plasmid pGBKT7 were transformed into Y2H Gold yeast cells. Transformed yeast competent cells were spread on plates containing SD/-Trp or SD/-Trp/-His/-Ade."}
+{"text": "To address the difficulty in assessing the implication of regulatory variants in diseases, a scoring scheme previously published allows the calculation of the Regulatory Variant Evidence score (RVE-score). The score represents the accumulated evidence for a causative role of a regulatory variant in a disease. Regulatory Evidence for Variants Underlying Phenotypes was built to calculate the RVE-score of regulatory variants, based on the 24 criteria, with a hybrid approach combining information retrieved from public databases and user input.http://www.revup-classifier.ca. The source code is available at https://github.com/wassermanlab/revup.RevUP is freely available at Bioinformatics online. To date, several DNA variant classifiers are available and most are aligned to the American College of Medical Genetics and Genomics (ACMG) consensus recommendations .With the growing use of clinical whole-genome sequencing, there is a need for extension of the classification systems suitable for regulatory sequence variants. Diverse regulatory variant prediction methods have been developed, such as PRVCS (http://www.revup-classifier.ca), a web-based classifier, was built to calculate the RVE-score of regulatory variants, based on the 24 criteria presented in Regulatory Evidence for Variants Underlying Phenotypes  instance with Nginx being used as the web server to route traffic. All code for RevUP is available on GitHub The scoring scheme presented in For RevUP to query external databases, the user must input the variant details and the suspected target gene. Six external resources are then queried: PhyloP . Therefore, the user will have the ability to answer \u2018Yes\u2019 or \u2018No\u2019 to these questions in order for the tool to calculate the score.Users may have additional evidence that was not available in the queried public databases, therefore, the user can modify the score for each evidence level as well as add comments. Comments were created to allow citations to relevant publications, or specific figures within publications (i.e. free text). It is not advised to change the scores unless the user has strong evidence to justify it.Based on the user input and on the values retrieved from the external databases, RevUP calculates the C-score, the F-score and the RVE-score for the submitted variant. The result page is composed of two parts. The top portion  presentsNOTCH1 and implicated in the tetralogy of Fallot (An example is shown in RevUP is a strong addition to the available variant classifier tools, as it will allow users to assess properties specific to regulatory variants. This scoring system does not conflict with the ACMG classification guidelines; rather it can be used as additional information when studying regulatory variants. The tool will save time for the user as it is able to query databases rapidly.btac157_Supplementary_DataClick here for additional data file."}
+{"text": "In Saudi clinical settings, cultural influences can give a patient\u2019s family authority to override the patient\u2019s autonomous right to make informed health-related decisions. Cultural values should not prevent patients from exercising their genuine preferences when making medical decisions in their own best interests.This article discusses the moral implications of family-centred medical decisions for autonomous patients who are competent and capable of making decisions. The author argues that socio-cultural values do not justify the decision to override patient autonomy when patients express a preference for making their own choices.The author recommends the use of a model of shared decision-making that accounts for both individual and relational conceptions of autonomy, approaching patients\u2019 preferences in all medical encounters with the aim of minimising the potential for socio-cultural values to undermine patient autonomy. Although this approach is a safeguard against both family and medical paternalism, allowance is made for clinicians to act in weakly paternalistic ways when patients at high risk of exacerbating existing medical conditions are likely to benefit from delaying or limiting the disclosure of potentially distressing but non-fatal diagnoses and prognoses. Thus, the author argues that even in a culture that supports family involvement in management decisions, physicians should respect patient autonomy by asking patients for their preferences in the disclosure of their medical diagnoses, prognoses and management options and verifying patients\u2019 preferences about the roles they wish their families to play (if any) in health-related decisions. Example (4): A 9-month pregnant lady came with embryonic fluid leakage; all vital signs of the embryo were stable permitting normal delivery. After examination, the gynecologist suggests to the husband doing a cesarean section giving that it is easier than normal delivery. The wife refused but the medical team tried again by telling the husband of possible harm to the baby and leading him to force his wife to do the unnecessary cesarean section even without taking the approval of the mother. , p.\u00a015.This footnote does not explicitly state whether the pregnant woman in the example retains or has lost decisional capacity. If she retains decisional capacity, why is the medical team seeking approval for a cesarean section from her husband? If she has lost decisional capacity, why does it state that \u201cthe wife refused\u201d? Further, the footnote does not state whether the husband is her representative in decision-making by default or if she nominated him.In 2019, the Saudi Ministry of Health (MOH) hoever kills a soul unless for a soul or for corruption [done] in the land\u2014it is as if he had slain mankind entirely. And whoever saves one\u2014it is as if he had saved mankind entirely .The Holy Quran describes the virtuous act of saving a single human life as being comparable to saving all human lives:According to a report from the Saudi Centre for Organ Transplantation, families refuse to allow organ donation from a deceased donor relative in 62% of cases . This reAccording to Al Sayyari et al. , p. 66, Darnell et al.  conducteCignarella et al.  remarks Islamic (shariah) law allows do not resuscitate orders (DNRs) for patients suffering from terminal illnesses. Under these orders, patients can receive all necessary treatment, hydration, nutrition, and pain control to die peacefully without receiving artificial resuscitation, as resuscitation to restore cardiac and/or respiratory function to arrested patients would only prolong their suffering in the absence of foreseeable benefits for prolonging their lives .Such decisions should be made through discussions with competent patients who retain the right to refuse life-sustaining measures. However, Chamsi-Pasha and Albar  state thWhen the patient\u2019s family refuse the DNR order which was issued by the patient themselves or by the medical team and the family requests efforts for resuscitation, they are allowed to do so by transferring the patient to another hospital where the patient could be resuscitated following cardiopulmonary arrest, as the signed DNR is only kept in the patient\u2019s records and is not valid outside the hospital .To prevent the prolongation of the patient\u2019s suffering and prevent family dominance through demanding unnecessary resuscitation, the Saudi MOH should provide a clear policy to put an end to unnecessary resuscitation that is applicable in all healthcare settings . PracticIn light of the abovementioned considerations, physicians must avoid extremes when considering what to disclose to their patients or when seeking the consent of competent patients for health-related decisions. These extremes can involve enabling strong family paternalism by allowing families to prevent the disclosure of diagnoses and prognoses to patients or overriding patients\u2019 consent for management options due to benevolent and protective cultural reasons. The other extreme is when physicians leave patients to make decisions without offering guidance or sharing in the decision-making process. Thus, the model of shared decision-making introduced in the following section, \u201cThis section presents a model of shared decision-making that accounts for both individual and relational concepts of autonomy, with the aim of minimising the potential for socio-cultural values to undermine patient autonomy.G\u00f3mez-V\u00edrseda et al.  argue thI.Compromised autonomy and paternalism: This occurs when a healthcare provider  or the family  takes full control of the decision-making process and ignores or fails to check the preferences of a competent patient.II.Delegating responsibilities: This occurs when the family and/or healthcare provider takes full control of the decision-making process because the patient prefers to let their caring and supportive family as well as the experienced physician take the lead.III.Shared decision-making: In this scenario, there is more emphasis on individual autonomy and the relationship between the individual patient and the healthcare provider, which does not include the family unless the patient wants to include them. Here the emphasis is on identifying and respecting the patient\u2019s values, preferences and beliefs, not just the disclosure of relevant clinical information. This process can also include elements of relational autonomy if the welfare of the patient is believed and decided (by the patient) to be inseparable from the welfare of his/her family.IV.Prioritising individual autonomy: This occurs when patients make their own decisions without any contribution from the family or healthcare provider apart from what is required as part of the consent process.According to G\u00f3mez-V\u00edrseda et al. , the proG\u00f3mez-V\u00edrseda et al.  hold thaVarious conceptions of relational autonomyFrom the perspective of the author\u2019s interests in this article, respecting patients\u2019 relational autonomy does not necessarily oppose the act of respecting their individual autonomy if both are incorporated into a model of shared decision-making that includes scenarios II and III from G\u00f3mez-V\u00edrseda et al.\u2019s  researchThis model of shared decision-making would be supported by continually checking the patient\u2019s preferences for medical disclosures and management decisions to clarify whether they are more in line with individual or relational understandings of autonomy. In this way, patients are able to provide clear guidance to healthcare professionals for when it is appropriate to contact people they trust, and to authorise them to make the best decisions on their behalf when their condition requires it.8Dove et al.  support The suggested preference approach involves the adoption of a model of shared decision-making that protects patients\u2019 preferences from being overridden by both family paternalism and medical paternalism. This approach involves checking patients\u2019 preferences for decision-making to ensure that patients who prefer to maintain control over their medical decisions are not disenfranchised by their families, regardless of their gender, age or socio-cultural background. The preference approach should be more detailed and autonomy-focused than what is already practised in Saudi medical settings, where patient consent is obtained but their preferences for the disclosure and the extent of family involvement in decisions are not adequately checked.The author recommends that in every medical encounter, all patients\u2019 preferences for knowing about their diagnoses and prognoses in addition to the roles (if any) they would like their families to play in their management decisions should be routinely checked.Care should be taken when checking elderly and female patients\u2019 preferences for medical decisions, as they may want to be informed of their diagnoses and prognoses personally, but may fear disappointing or angering their family for this decision. In such cases, their preferences for shared or delegated decisions to their family need to be verified without their family\u2019s presence to ensure they are not being coerced or manipulated .Nonetheless, even if patients decide to keep their family out of the decision-making process, there are beneficial reasons for them to choose otherwise. For example, because disclosing bad diagnoses and prognoses to cancer patients is typically stressful, it is possible that such disclosures could play a major role in worsening patients\u2019 physical and emotional conditions. Consider a patient with a medical history of cardiovascular disease (CVD),The preference approach must be grounded in a biopsychosocial approach to caring for patients in Saudi medical settings. This involves providing a comprehensive assessment of a patient\u2019s psychological and social wellbeing in addition to their physical wellbeing, since all these domains contribute to the patients\u2019 overall welfare.Under this approach, the team directly responsible for the patient\u2019s management is required to assess the patient\u2019s capacity to handle the disclosure of bad news and to obtain informed consent for subsequent medical interventions. Other healthcare service providers, such as social workers, oncologists and the other medical specialists the patient needs to treat their medical condition, should also play a role in supporting the patient\u2019s physical, psychological and social wellbeing. This support could help prepare the patient to accept and understand their diagnosis and prognosis, which is essential in providing care to a patient during a difficult diagnostic and therapeutic process, a factor which is all too often neglected.The extent of the family\u2019s contributions to the decision-making process should be determined by competent patients. This means that whether the family shares in the patient\u2019s health decisions, takes full responsibility for management decisions, or are kept out of the process entirely, should depend on the patient\u2019s preferences. Patients with co-existing medical diagnoses who give authority to their family to make medical decisions on their behalf must understand that the family may insist on delaying or limiting medical information from being disclosed to them if their medical condition justifies the family\u2019s decision to do so. However, even if a patient prefers to keep their family out of the decision-making process, physicians can justifiably delay or limit the disclosure of medical information to prevent significant harm to a patient if that patient is not imminently dying but has pre-existing conditions that can be worsened by full disclosures.In this article, the author identified a range of harms that arise in Saudi medical settings due to the influence of cultural values that support a role for families in medical decision-making that often overrides patient autonomy. Whether it involves medical disclosures, protecting patient privacy and confidentiality or consent, the priority given to family decision-making leaves little (if any) room for a genuine consideration of patient preferences, especially if the patients are female or elderly. This is reflected in the lack of national policies that are specifically intended to protect patient autonomy from family dominance over decisions in relation to medical disclosures, confidentiality and consent. In the absence of such policies, healthcare providers too often acquiesce to the cultural norm of family dominance in medical decision-making. Against this background, the author argued for the implementation of a model of shared decision-making that accounts for both individual and relational conceptions of autonomy in placing an obligation on physicians to maintain safeguards against strong medical and family paternalism.The continual assessment of patient preferences for medical disclosures and consent should be used in all medical encounters with patients in Saudi medical settings to verify the extent to which patients\u2019 families are authorised to participate in medical decision-making. Physicians have an obligation to ensure that their patients fully understand that while medical reasons to authorise the family to make decisions on their behalf are acceptable, socio-cultural expectations are not. The author also argued that if a patient has pre-existing conditions that could worsen from full disclosures, risk assessments of their existing condition may justify physicians delaying or limiting the disclosure of medical information to avoid exacerbating their condition."}
+{"text": "It is well known that hippocampal place cells have spatiotemporal properties, namely, that they generally respond to a single spatial location of a small environment, and they also display the temporal response property of theta phase precession, namely, that the phase of spiking relative to the theta wave shifts from the late phase to early phase as the animal crosses the place field. Grid cells in Layer II of the medial entorhinal cortex (MEC) also have spatiotemporal properties similar to hippocampal place cells, except that grid cells respond to multiple spatial locations that form a hexagonal pattern. Because the EC is the upstream area that projects strongly to the hippocampus, a number of EC-hippocampus learning models have been proposed to explain how the spatial receptive field properties of place cells emerge via synaptic plasticity. However, the question of how the phase precession properties of place cells and grid cells are related has remained unclear. This study shows how theta phase precession in hippocampal place cells can emerge from MEC input as a result of synaptic plasticity, demonstrating that a learning model based on non-negative sparse coding can account for both the spatial and temporal properties of hippocampal place cells. Although both MEC grid cells and other EC spatial cells contribute to the spatial properties of hippocampal place cells, it is the MEC grid cells that predominantly determine the temporal response properties of hippocampal place cells displayed here. In the navigational system of the brain, place cells in the hippocampus and grid cells in the medial entorhinal cortex (MEC) have functionally important spatiotemporal properties. However, little is known about the link between the temporal properties of grid cells and place cells. Recent experimental studies have suggested that temporal properties of hippocampal place cells may be inherited from the MEC. However, a learning model explaining how their relationship can be learnt via synaptic plasticity is still lacking. Here, we build a learning model based on the principle of sparse coding and demonstrate how input from the EC leads to the spatiotemporal properties of hippocampal place cells via synaptic learning.In early electrophysiological experiments involving freely behaving rats , neuroscThree decades after hippocampal place cells were discovered, Apart from grid cells in MEC, there are also many nongrid spatial cells in the MEC . In the Hippocampal place cells and MEC grid cells are fundamental units of the navigational system of the brain and there is a close relationship between these two types of cells. Experimental studies indicate that input from the EC is the principal input to the hippocampus . ConsequSeparate from the link between the spatial properties of place cells and MEC grid cells, some experimental studies also infer a link between the temporal properties of place cells and MEC grid cells. The learning model presented here is built on our previous work that shows that the model based on non-negative sparse coding can learn an efficient hippocampal place map using EC input such as MEC grid cells and EC weakly spatial cells . In thistv is the speed sampled from an Ornstein\u2013Uhlenbeck process with long-term mean t\u03b8 is the direction of movement, t\u03c9 is a standard Wiener process, and \u03b8\u03c3 is the parameter that controls the tortuosity of the running trajectory.The spatial environment used in this study is a 1m \u00d7 1m square box where a virtual rat runs freely. Similar to the study by t\u03b8) is set to the direction parallel to the wall. If the rat location generated by When the virtual rat is running toward the wall and very close to the wall (within 2\u2009cm), the running direction of the rat f of one example MEC grid cell is displayed in The spatial receptive field, ous work , the spaous work , these frk \u03b1c in  of each rk \u03b1c in . The rad\u03d5k is the parameter that controls the level of phase modulation and hence the extent of the resulting phase precession, F is the frequency of theta rhythm and set to 10\u2009Hz throughout the paper. The choice of F\u2009=\u200910\u2009Hz help with visualizing the results as every 0.1 s is a full period  as a linear function of the location, r:\u03d50 is the entry phase at location cr \u2013 R, \u0394\u03d5 is the phase change across the receptive field, and thus the exit phase at location cr + R is \u03d50 \u2013 \u0394\u03d5. Note that in 1D r and cr become scalars instead of vectors. An example of the linear relationship between \u03d5(r) and r described in In a 1D linear track, \u03d5(r) in R\u2032 is the projected radius onto the running direction. In any location within the receptive field, the firing phase However, determining Above all, the spatiotemporal responses of a MEC grid cells in a 2D environment are simply modelled as multiple grid fields at all the vertices of the hexagonal grid in which each grid field is characterized by the spatiotemporal model described above \u20134, 6.F when the virtual rat stays at a fixed location within the receptive field. Therefore, supposing the virtual rat remains stationary at r for 1 s, the spatiotemporal response over this 1 s will oscillate at frequency F with phase determined by From  animals , the spaR\u2009=\u20090.16 m. The response of one example MEC spatiotemporal grid cell is illustrated in Although MEC grid cells have a clear spatial structure, they account for only \u223c20% of the MEC cell population . FurtherSimilar to our previous work , we builI is the input, s represents the response (firing rate) of the model units, u can be interpreted as the membrane potential, A is the matrix containing basis vectors and can be interpreted as the connection weights between the input and model units, \u03c4 is the time constant, \u03b2 is the positive sparsity constant that controls the threshold of firing, and \u03b7 is the learning rate. Each column of A is normalized to have length 1. The non-negative constraints are incorporated into the system, as seen from the non-negativity of both s and A in and\u0394A=\u03b7. The EC-hippocampus connection undergoes a learning process while the virtual rat is exploring the environment. This scenario is designed to investigate the contribution of MEC spatiotemporal grid cells to the spatial and temporal properties of learnt hippocampal place cells.Modelled hippocampal cells receive input from MEC spatiotemporal grid cells together with EC weakly spatial cells. Similar to scenario 1, the EC-hippocampus connection is learnt. This scenario aims to validate and extend the results of scenario 1 to the situation in which other spatial cells in the EC also contribute to the firing of learnt hippocampal place cells.After the learning process of scenario 2, MEC spatiotemporal grid cells are inactivated and there is no learning in this scenario. This scenario is used to replicate the experimental setup of the study by r and time t according to A, between EC cells and modelled hippocampal cells is updated according to the learning rule described in The values of training parameters and definition of symbols can be found in A is a 900\u2009\u00d7\u2009100 matrix. A running trajectory of 3600 s is used to train the model and the connection weight matrix, A, is learnt during this training process.Only MEC spatiotemporal grid cells provide input to the modelled hippocampal cells, there are 900 MEC spatiotemporal grid cells, i.e., A is a 1300\u2009\u00d7\u2009100 matrix where the top 400 rows of A represent the connection weights from EC weakly spatial cells to modelled hippocampal cells and the bottom 900 rows represent the connection weights from MEC grid cells to modelled hippocampal cells. The same running trajectory of 3600 s, as used in scenario 1, is used to train the model.Both MEC spatiotemporal grid cells and EC weakly spatial cells provide input to the hippocampus. In addition to the 900 MEC spatiotemporal grid cells, there are also 400 EC weakly spatial cells, i.e., A  to zero and then normalizing each column of A to have length 1. In this scenario, there is no learning.After the learning process in scenario 2, MEC spatiotemporal grid cells were inactivated to investigate how this affects the spatial firing of learnt hippocampal place cells after spatiotemporal input from MEC grid cells is lost. MEC grid cells are inactivated by setting the bottom 900 rows of A is initialized according to a uniform distribution between 0 and 1 and then normalized to have length 1 for each column. For both scenarios 1 and 2, a running trajectory of 3600 s is used to train the model and the connection weight matrix, A, is learnt during this training process. After learning, another running trajectory of 1200 s is used to recover the spatial receptive fields of these 100 modelled hippocampal cells for all three scenarios.The connection weight matrix \u03d5k is chosen randomly from a uniform distribution between 0.8 and 1.2; \u03d50 and \u0394\u03d5 are both chosen randomly from a uniform distribution between 300\u00b0 and 340\u00b0; and F is 10\u2009Hz.The parameters in \u03c4 is 10\u2009ms, \u03b2 is 0.3, and the time step for simulating the modelled hippocampal cells by A, is updated using \u03b7 is 0.01.For the parameters in the model dynamics and learning rule , 8, \u03c4 isAfter training, another running trajectory of the virtual rat with a duration of 1200 s was used to recover the spatial receptive fields (rate maps) of modelled hippocampal cells. The 1m \u00d7 1m environment is discretized into a 40\u2009\u00d7\u200940 lattice and the receptive field is recovered as the averaged response across all locations along the running trajectory of 1200 s.lsqcurvefit, and the fitting error is defined as the ratio between the summed square of the fitting residual and the summed square of the recovered receptive field.In order to quantitatively characterize the spatial receptive fields of learnt hippocampal cells, the recovered receptive fields using the approach above is fitted by the following function:The criteria for categorizing a modelled hippocampal cell as a place cell are: (1) the center, A is learnt during the virtual rat navigation. To investigate the temporal properties of learnt hippocampal place cells after learning, the response of learnt hippocampal place cells over 1 s at a given location is fitted to the following function:lsqcurvefit. The fitting error is defined as the square of the ratio between the fitting residual and original response.The MEC spatiotemporal grid cells in the model have built-in spatial and temporal properties as described in In order to quantitatively investigate the temporal properties of theta phase precession of learnt hippocampal place cells after learning, the following approach was used. First, for the learnt hippocampal place cells that meet the criteria of a place cell described earlier , only cells whose entire place fields lie in the spatial environment are considered. Second, for each learnt hippocampal place cell, a virtual rat starts from the left side of the place field with an initial running rightwards direction and then a curved trajectory is generated according to https://github.com/yanbolian/Learning-Spatiotemporal-Properties-of-Hippocampal-Place-Cells.The code of implementing the model and analyzing results was written in MATLAB (R2020a), and is available at The results presented here are those from scenario 1, namely, the simulation in which only MEC spatiotemporal grid cells provide input for the modelled hippocampal cells. The MEC spatiotemporal grid cell to hippocampal cell connectivity was learnt using non-negative sparse coding over 3600 s of virtual rat navigation over the 1m \u00d7 1m environment, as described in Materials and Methods, Training. Through this learning, the learnt hippocampal place cells learn to pool different MEC spatiotemporal grid cells in such a way that they become selective to specific locations in the spatial environment. The receptive fields of the 94 out of 100 modelled hippocampal cells that meet the criteria for place cells described in Materials and Methods, Selecting place cells, are plotted in Furthermore, these learnt hippocampal place cells also display the temporal property of theta phase precession. This is illustrated in the response of a learnt hippocampal place cell over 0.5 s shown in The response of the same example place cells over 0.5 s as the animals runs from left to right of the place field (through the field center) along the straight trajectory 1 in x and y values of the position change, we plot the firing phase relative to the normalized pdcd  learnt hippocampal place cells, 58 place cells have their entire place fields inside the spatial environment. For each place cell, a random curved trajectory is generated that crosses the place field and the neuronal response is computed at different locations along the trajectory . The population statistics of the temporal properties are shown in In this section, we show the results of training with scenario 2, namely, where both MEC spatiotemporal grid cells and EC weakly spatial cells provide input to the modelled hippocampal cells. The results show that both MEC grid cells and EC weakly spatial cells provide spatial information for the hippocampus, so that a spatial hippocampal map can be retrieved from upstream input, while MEC grid cells provide temporal information that results in theta phase precession also being inherited from the upstream neural population.A demonstration of how EC weakly spatial cells and MEC grid cells contribute to the spatial and temporal properties of hippocampal place cells is given in After the learning process in scenario 2, MEC spatiotemporal grid cells in the model were inactivated, i.e., only the remaining EC weakly spatial cells provide spatial information for hippocampal place cells and there is no input  from MEC grid cells. Recall that there is no learning in scenario 3, so the spatial tuning of hippocampal place cells depends on the connectivity between EC weakly spatial cells and hippocampal place cells that was learnt in scenario 2.However, the learnt connection between EC weakly spatial cells and place cells is sufficient to maintain the place field of hippocampal place cells after MEC inactivation. After recovering the receptive fields of modelled hippocampal cells , 87 out of 100 modelled hippocampal cells were found to meet the criteria of a place cell. The place fields, together with their field centers, are plotted in There are 94 place cells before inactivating MEC grid cells . FurtherThis result is consistent with the experimental study by Combining results in this paper and our previous work , the conA, is learnt during this exploration period. After learning, A is kept fixed and another running trajectory of 1200 s is used to recover the place fields of learnt place cells. In addition, responses over 1 s at different positions across the place field are used to investigate the firing phases when the virtual rat traverses the place field. Similar to the study of In this study, a model based on sparse coding is built that demonstrates that the spatial and temporal properties of hippocampal place cells can be learnt simultaneously via plasticity as the virtual rat freely explores an open environment. In the training phase, a virtual rat runs for 3600 s and the connectivity weight matrix, Our model demonstrates that the temporal properties of hippocampal place cells can be inherited from MEC grid cells via synaptic learning. However, there are also other models that describe the temporal properties of place cells from other perspectives.The intrinsic oscillator mechanism explains how phase precession operates on the basis of single neurons, but hippocampal phase precession can also be explained by a network mechanism in which the interaction between neurons in the network plays a crucial role. Both the intrinsic oscillator mechanism and network mechanism require a baseline theta rhythm as the input, but the two mechanisms differ in how the firing phase shifts relative to the baseline oscillation: one is caused by the faster oscillation of single place cell and the other is caused by the interaction between place cells in the network. However, this does not imply that these two mechanisms are completely contradictory. Instead, they can be unified within a perspective that hippocampal place cells require both external input (extrinsic) and local interaction (intrinsic) to achieve spatial-temporal properties, as supported by a recent experimental study .Experimental studies indicate that phase precession may not originate in the hippocampus, but rather it is inherited from the upstream processing . In addiThe work presented in this paper models temporal properties of hippocampal place cells as being inherited from upstream processing, as proposed by earlier studies . AlthougOur model is also related to the intrinsic oscillator mechanism and the extrinsic oscillator mechanism because it requires oscillatory input and local interactions between cells in the network: the oscillatory input comes from the MEC grid cells that have temporal response properties and the local interactions between cells introduce competition (sparsity) into the network such that cells learn different representations. Experimental studies suggest that the projections from medial septal areas also control hippocampal oscillations , but ourEssentially, our model provides a learning framework in which spatial-temporal properties of hippocampal place cells can be inherited from MEC via synaptic learning. This framework may interact with other mechanisms to help us better understand the hippocampus.Early computational models of place cells were mostly based on a feedforward structure, where cells in the EC provide spatial input to the hippocampus . Some moRecent research has provided experimental evidence that place fields emerge earlier in development than MEC grid cells . There iWhile this study offers a picture of how the EC contributes to the spatial and temporal properties of hippocampal place cell, there remain a number of interesting outstanding questions to be addressed. First, the question of how the feedback from the hippocampus to the EC affects properties of EC cells remains unclear. Second, the model proposed here demonstrates how sparse coding can learn spatiotemporal properties of hippocampal place cells, but whether the principle of sparse coding can be used to explain other aspects of hippocampal function, such as those involving memory consolidation, remains unclear. In addition, the spatiotemporal model of MEC grid cells is assumed here, and the question of how this MEC spatiotemporal grid cell structure originates is an active topic of ongoing research. Moreover, the model presented here uses rate-based neurons, and a spiking implementation together with spike-timing dependent plasticity of this model is left for future research."}
+{"text": "Connexins are assembled into dodecamer intercellular channels, a collection of which is termed a gap junction, and their canonical function allowing direct exchange of ions and metabolites has been unequivocally established. When initially assembled into undocked cell surface connexin hemichannels, healthy cells may also engage in cell signaling via a regulated small-molecule release. Recent advances in the field have led to an expanded view of the functional roles of intercellular channels and hemichannels in both physiology and pathology. As more of the 21-member human connexin family is intensely interrogated, mounting evidence points to the biological uniqueness of each member, and no longer can we confidently refer to all connexins engaging in the same cellular processes. Innovations in high-resolution cryo-electron microscopy have revealed important insights into the structure of functionally important domains of both hemichannels and channels. These and other studies have established a foundation of knowledge that should allow inhibitory smart drug design for situations where enhanced intercellular or hemichannel activity is at the root of a connexin-linked disease. Assessment of the connexin interactome, which varies widely for each connexin subtype, continues to provide regulatory insights into the assembly and function of connexins that exhibit a short half-life. As the most intensely studied, Cx43 is found in about 50% of all human cell types and is extensively regulated by multiple inhibitory and enhancing phosphorylation events that have direct implications on tissue function and outcomes of disease, including cancer. Here, we briefly discuss these advances and give our thoughts on where the field is headed. Gap junctions play both structural and intercellular communication roles to help regulate many cell processes, including cell migration, cell proliferation, embryonic development, differentiation, wound repair, and the coordinated contraction of heart and smooth muscle2. There is unequivocal evidence that, in humans, the channel-lining proteins of gap junctions are composed of proteins from the 21-member connexin gene family3  accelerated the structural revolution as high-resolution images of native lens gap junctions composed of Cx46 and Cx50 were obtained17. Here, the authors definitively proved the transmembrane domain arrangements and found open conformational states that were distinct from Cx2617, possibly revealing differences between alpha and beta connexin family members. This same team further resolved the structure of Cx46/50 channels in a dynamic aqueous-lipid context at near atomic resolution (1.9 \u00c5), where the connexin proteins were found to stabilize the local lipid microenvironment18. This level of molecular detail is now sufficiently informative to serve as a model for rational drug design for the possible treatment of any number of inherited connexin-linked diseases8 and the countless pathologies linked to connexin regulation in diseased tissue and cancer19.Interrogation by X-ray diffraction, freeze-fracture electron microscopy, nuclear magnetic resonance, and atomic force microscopy, together with sophisticated image analysis, revealed that the dodecameric arrangement of connexins was needed to form a complete intercellular channel20. In the years that followed, the community had to consider that connexins may, in fact, have a second canonical function in forming cell surface channels capable of releasing  metabolites or a variety of signaling molecules or both. While this core cellular function has molecular rivals, most notably pannexin channels22, there was no doubt that connexin hemichannel functions needed to be considered in both physiological and pathological contexts. The question continues to arise as to where and when connexin hemichannels play a role in normal healthy physiology and where and when aberrant hemichannel activity would lead to cell death or disease. No fewer than 1500 peer-reviewed papers have featured the existence and functional role of connexin hemichannels in virtually every organ of the human body. These studies range from Cx43 hemichannels engaging in a ventricular arrhythmogenic mechanism within microdomains of cardiac intercalated discs23 to Cx43 hemichannels in astrocytes serving as a therapeutic target in spinal cord injury24 and to Cx43 hemichannels in osteocytes playing a critical role in mechanical load-induced bone formation25. Intuitively, connexin intercellular channels tend to favor an open state, as supported by recent structural experimental evidence, but our view is that hemichannels must have a resting closed state under homeostatic conditions in order to protect the integrity of the cytosol30.While connexins form intercellular channels that permit the direct passage of metabolomes, the 1991 discovery that macrophages could release ATP, via what the authors termed \u201chalf-gap junctions\u201d or what today we would call hemichannels, expanded our view of connexin function31. Likewise, the evidence for functional Cx46 and Cx50 hemichannels in highly specialized lens tissue in the presence and absence of disease is compelling33. Still, other less well-studied connexins like Cx62 may have dual roles in platelets, functioning as both hemichannels and intercellular channels34. One must also consider that connexin hemichannels may only partially open, permitting the smallest of channel permeants to pass. Insight into this notion was provided when cryo-EM was used to solve the hemichannel structure of a connexin that appears unable to form functional gap junction channels, Cx31.331. Resolution analysis that exceeded 2.6 \u00c5 revealed that Cx31.3 hemichannels adapt to a partially closed state that would allow the passage of chloride ions through the 8 \u00c5 pore while preventing the passage of other cell metabolites27. Cryo-EM was also used to examine the open conformation of Cx26-N176Y mutant hemichannels within dynamic lipid bilayer nanodiscs28. Here, the alpha-helical structures found within the mutant Cx26 hemichannels were found to be the same as reported for Cx26 intercellular channels while the flexibility identified within the extracellular loops would probably serve to facilitate hemichannel docking in cases where complete gap junction channels are formed28. However, likely owing to their intrinsic disorder, we still know little about the structure of intracellular loop and C-terminal tail regions. Thus, there is no doubt that further high-resolution analysis of different connexin hemichannels and intercellular channels will continue to inform on their open and closed states.However, evidence continues to mount that hemichannels play a more universal and canonical functional role in non-diseased tissues. As an example, considerable evidence suggests that Cx31.3 may not even have the capacity to form traditional gap junction intercellular channels, raising the notion that their main role in cells may be to act as functional hemichannels36. Mutations in this class often lead to the loss of cell integrity and cell death, but other mechanisms of action need to be considered36. Blocking connexin hemichannels as a means of regulating the inflammatory response has given credence to the notion that this may be an effective strategy to treat chronic inflammatory eye diseases and eye injuries9. In fact, hyperactive or leaky hemichannels in disease or injury are somewhat ideal targets when considering commercialization and deployment of connexin hemichannel blockers37. Preferably, such hemichannel blockers should be specific to the aberrant hemichannel in question, a consideration that aligns with smart small-molecule design modeled from high-resolution connexin hemichannel structures, peptide mimetics that take into account both structure and domain flexibilities, and high-avidity antibodies. As an example, an antagonist antibody was shown to have efficacy in blocking leaky mutant Cx30 hemichannels in the treatment of Clouston syndrome38. Similarly, the hemichannel blocker flufenamic acid was found to inhibit aberrant Cx26-G45E hemichannel function, reducing the symptoms of keratitis ichthyosis deafness found in mutant mice39. As noted by others, several connexin-based therapeutics that have entered clinical trials need to consider the specificity of the connexin blocking agent and whether the pathological features of hemichannels can be selectively and effectively targeted40.To our minds, there remains little doubt that aberrant connexin hemichannel function is at the root of several inherited connexin-linked diseases and cases of tissue injury as well as in chronic and acute diseases. Many missense and truncating connexin-gene mutations lead to hyperactive or leaky hemichannels that appear especially prevalent in connexin-linked skin diseases41. In the case of Cx43, the interactome includes proteins involved in protein trafficking, connexin turnover, connexin assembly, connexin post-translational modification, scaffolding, and other functions that have been extensively reviewed elsewhere42. The gap junction scaffold is also functionally important for other connexins, as has been shown for Cx30 in the cochlea, where ephrin-B2 interacts at the periphery of the gap junction regulating gap junction turnover43 in a manner perhaps analogous to ZO-1 interaction with Cx4344. We believe the scaffold function of gap junctions will be found to play critical regulatory roles as more research is performed on other connexins, and we look forward to new revelations.Finally, while connexins are foundational molecules needed to assemble both hemichannels and gap junction channels, we would be remiss if we did not draw attention to the connexin interactome. The connexin interactome is highly dependent on the connexin family member as some interactomes are small , while others are in excess of 50 proteins 45. It is now clear that more than eight protein kinases phosphorylate Cx43 at most of the 21 serine residues and at least three of the six tyrosine residues found within the cytoplasmic exposed carboxy terminus  or to mimic a constitutively phosphorylated residue . Via this approach, casein kinase 1 phosphorylation of Cx43 was found to regulate several critical physiological events, including (a) proper cardiac beat rhythm58 and response to ischemia56, (b) efficient epidermal wound healing57, and (c) the effects of stromal fibroblasts on promoting pancreas cancer progression59, while (d) MAPK phosphorylation was shown to regulate neuroprotection during stroke60.Our current understanding of the regulation of gap junctions is heavily biased toward Cx43. Cx43 is expressed in most cell lines even if they originated from cells that do not typically express Cx43 . Furthermore, our analysis of the literature suggests that Cx43 is natively expressed in nearly half of the more than 200 cell types found in the human body. Many Cx43 post-translational modifications, including ubiquitination, acylation, hydroxylation, carboxylation, methylation, sumoylation, and nitrosylation, have been reported, but we know the most about the functional consequences of Cx43 phosphorylationterminus . We also45, but in some cases the specific residue or direct linkage of the phosphorylation event to an effect on connexin-linked physiology is lacking. We conclude that many of the alpha subfamily of connexins that have been studied have shown at least some evidence of regulation by phosphorylation. However, some connexins, particularly those of the beta subfamily that have short C-terminal regions, have not been shown to be specifically regulated by phosphorylation , although caution should be exercised as the effects of phosphorylation might be apparent only under very specific conditions. In any case, we believe new interest in the less common connexins that are expressed in diverse tissues will more clearly delineate the roles connexin phosphorylation plays in gap junction and hemichannel regulation.Our knowledge of phosphorylation of connexins other than Cx43 and the resulting physiological effects is in general less well-defined. There are reports that Cx31, Cx32, Cx36, Cx37, Cx40, Cx43, Cx46, Cx47, and Cx50 are phosphorylatedin situ. While connexin family members share considerable sequence homology, it is now clear that connexin members are remarkably diverse with unique structures, regulatory motifs, post-translational modifications, interactomes, cell expression profiles, and subcellular distributions that all contribute to diverse intercellular channel and hemichannel functions in cells and tissues.After an exhaustive review of over 180 peer-reviewed papers related to endogenous connexin expression, we conclude that the 21-member connexin family can be mapped to over 110 distinct cell types found within all 12 human body systems. On one end of this spectrum, it is unclear what human cells express Cx23, while at the other end, Cx43 has been convincingly shown to be expressed in 92 cell types, reflecting its dominance as the most widely distributed human connexin. After decades of intense investigation, the gap junction community not surprisingly is best equipped to describe where and when Cx43 functions regulate cell and tissue physiology. That said, the functional roles of lesser studied connexins  remain poorly understood, a situation made more difficult by the lack of reliable antibodies to specifically detect their expression 61, connexin expression at non-canonical sites such as exosomes62 and mitochondrial outer membranes63, as well as the role that connexins play in cancer invasion, metastasis, and resistance to treatment64. There remains no doubt that connexins regulate a variety of cellular functions in almost every human organ. However, the challenge remains to sort out the tissue-context roles played by hemichannels/channels, a task made more complicated by our relatively rudimentary knowledge of what metabolites pass through each channel subtype in situ and the fact that connexin subtypes can intermix to form heteromeric and heterotypic channels. As more and more connexins get linked to diseases, a broad spectrum of organ-specific investigators from around the world are being attracted to the gap junction field in search of new therapeutic targets. We wholeheartedly welcome these new investigators to the field and believe they will pave the way to a better understanding of the functional importance of connexin-based cellular communication in all tissues.New advances in understanding hemichannel functions, intercellular channel and hemichannel structures, genetic linkages to disease, and the connexin interactome all point to critical and canonical roles for connexin hemichannel and channel functions in both disease and normal physiology. Some new areas of intensive study include the role of hemichannels in cardiac conduction"}
+{"text": "CD4+ T cells were isolated from peripheral blood of dairy cows diagnosed as healthy or with ketosis, a common metabolic disorder of FA metabolism. Results revealed that levels of intracellular Ca2+ and reactive oxygen species (ROS) along with the abundance of store-operated Ca2+ entry (SOCE) moiety increased during ketosis. Further, plasma concentrations of inflammatory cytokines were elevated, the balance of Th17/Treg cells was disrupted, mitochondrial function impaired, and the abundance of mitophagy-related proteins in CD4+ T cells altered during ketosis. Molecular characterization of the direct effects of FA was evaluated in CD4+ T cells isolated from the spleen of 1-day-old calves. Enhanced supply of FA increased intracellular Ca2+ and ROS concentrations, upregulated the abundance of proteins associated with mitochondrial dynamics and ORAI1. Intermediates of mitophagy accumulated and the balance of Th17/Treg cells also was affected by the supply of FA. These negative effects were attenuated by silencing or inhibition of ORAI1 in CD4+ T cells. Together, data indicated that physiological states that lead to increases in systemic concentrations of FA could impact adaptive immunity negatively through ORAI1 regulated intracellular Ca2+, ROS balance, and increased effector functions of Th17 cells.The nutritional status of dairy cows and the metabolism of specific nutrients are critical regulators of immune cell function. Around the time of parturition, mobilization of body lipid and muscle helps compensate for the decrease in nutrient intake and the increased requirements of the mammary gland for lactation. An end-result of these processes is the marked increase in circulating concentrations of fatty acids (FA), which are a major risk factor for immune dysfunction. In food animal species such as dairy cows, any disturbance in nutritional or immunological homeostasis leads to deleterious feedback loops that can further risk health, efficiency of nutrient use, and compromise availability of safe and nutritious dairy foods for humans. Despite substantial progress with respect to regulation of innate immunity, such knowledge for adaptive immunity is scarce. To help bridge this gap in knowledge, we sought to study the role of calcium release-activated calcium modulator ORAI1 activation in T cells systemic immune function  Incidence of disease in food-producing animals is a major roadblock to a sustainable animal agriculture sector worldwide. Dairy cows, in particular, are highly-susceptible to both metabolic and infectious diseases around the period of parturition . In all + and CD4+  T cells. Further division of Th cells includes Th1, Th2, Th17, and T regulatory cells (Treg). The balance between pro-inflammatory Th17 cells and anti-inflammatory Treg cells determines the magnitude of immune responses that are critical for homeostasis and health of organisms  machinery, which includes stromal interaction molecule (STIM) and calcium release-activated calcium channel protein ORAI1, is key for cellular calcium homeostasis . Such events are followed by changes in mitochondrial morphology and initiation of mitophagy.Calcium and Caic cells . In immueostasis , 14. Chaammation \u201317. Furt2+ signaling, mitochondrial oxidative metabolism is a major source of ROS in the cell. In fact, Ca2+ concentrations can regulate cellular antioxidant defense systems  approved the animal use protocol.+ T cells and measurement of cytokines in serum.Lactating multiparous Holstein cows were selected from a 1,000-cow dairy farm with free-stall housing systems located in Daqing, Heilongjiang Province, China. We chose lactating Holstein cows with a similar number of lactations (2.53 \u00b1 0.11) and at a similar stage of lactation [11.86 \u00b1 0.44 day in milk (DIM) ]. Veterinarians classified cows as ketotic if milk yield and a nitroprusside test for ketone bodies in milk was positive . Accordiin vivo experiments, we placed the collected peripheral blood at 4\u00b0C and returned to the laboratory within 2 hours for cell isolation. Lymphocytes were isolated using a Bovine Peripheral Blood Lymphocyte Separation Kit  according to the manufacturer\u2019s protocols. After erythrocyte removal, CD4+ T cells were selected via MACS (MicroBeads sorting) isolation. In brief, lymphocytes were labeled with CD4 Monoclonal antibody  for 25 min at 4\u00b0C, then Anti-Mouse IgG2a+b MicroBeads (Miltenyi Biotec) was used for magnetic labeling for 15 min at 4\u00b0C. After labeling, CD4+ T cells were separated using MS Columns (Miltenyi Biotec). Subsequently, the column was washed two times with MACS Separation Buffer (Miltenyi Biotec) and CD4+ T cells collected through adsorption on the column.For in vitro experiments, 4 healthy female Holstein calves  from a commercial dairy farm  were used as spleen donors to isolate CD4+ T cells. Briefly, the calves were fasting and immediately transported to the laboratory within three hours after their birth. Subsequently, calves were humanely euthanatized by intravenous thiamylal sodium injection. Fresh spleen parenchyma was harvested aseptically from each calf. The spleen was washed twice with PBS containing 5000 UI/mL Amphotericin and Gentamicin for 10 min, then cut into small pieces before the splenic capsule was removed and ground to obtain splenocytes. The splenocyte suspension was then filtered sequentially with cell sieves of 50 (300 \u03bcm), 100 (150 \u03bcm), and 200 mesh (75 \u03bcm). After erythrocyte removal, the splenocyte suspension was washed twice with RPMI-1640 basic medium (Hyclone Laboratories) and centrifuged for 5 min at 500 \u00d7 g at 4\u00b0C and CD4+ T cells positively selected via MACS isolation. The labeling method was the same as that for in vivo experiments. Cell viability was assessed with the Trypan blue dye (Sigma-Aldrich) exclusion method after isolation. Only cells with viability >95% were used for further experiments. Primary calf CD4+ T cells were cultured in 6-well plates at 1 \u00d7 106 cells/mL using RPMI-1640 basic medium supplemented with 10% FBS, 1% Penstrep, 1% L-Glut and 55 \u03bcM \u03b2-mercaptoethanol and 1 \u03bcg/mL Concanavalin A for 60 h, and incubated at 37\u00b0C in 5% CO2.For + T cells were maintained in RPMI-1640 basic medium supplemented with 2% BSA and treated with vehicle only or a mixture of 1.2 mM fatty acids for 0, 6, 12, 24, 48, and 72 h. The concentration of fatty acids in the mixture developed was based on previous reports from dairy cows with ketosis  were seeded 24 h before the experiment in antibiotic-free medium. Cells were transfected with 40 pM cattle ORAI1 siRNA  and a non-target siRNA using siRNA-mate  according to the manufacturer\u2019s protocols. For inhibition, CD4+ T cells were pretreated with 50 mM of the store-operated Ca2+ Channel Inhibitor 2APB  for 2 h prior to fatty acid challenge. Ten mM of the OXS inhibitor N-Acetylcysteine , or vehicle (DMSO) was used for 2 h prior to fatty acid challenge.For silencing, 1 \u00d7 10Blood samples were collected use tubes containing sodium heparin anticoagulant from the tail vein and immediately centrifuged at 1,400 \u00d7 g for 10 min, and used for measurement of serum parameters within 2 hours. Serum IL-17, IL-10, TGF-\u03b21, and IL-6 concentrations were determined using commercially available kits according to manufacturers\u2019 instructions . Two measurements are made for the sample and the standard curve. All ELISA plates were read using a SpectraMax Plus384 microplate reader . Samples were run in duplicate, and duplicates with coefficients of variance (CV) less than 10% were included for analysis according to the instructions . To control for inter-assay variation, a pooled sample was run on each plate and duplicates with coefficients of variance less than 12% were included for analysis according to the instructions .3 cells/well in 96-well plates using RPMI-1640 basic medium supplemented with 10% FBS, 1% Penstrep, 1% L-Glut and 55 \u03bcM \u03b2-mercaptoethanol and 1 \u03bcg/mL Concanavalin A for 60 h, and incubated at 37\u00b0C in 5% CO2. After 60-h culture, cells were maintained in RPMI-1640 basic medium supplemented with 2% BSA and treated with or without 1.2 mM fatty acids for 0, 6, 12, 24, 48, and 72 h. After treatment, 20 \u00b5L of CCK-8 solution was added to each well and then incubated in the dark for 1 h at 37\u00b0C in 5% CO2. The optical density was measured at 450 nm on a spectrophotometer (Thermo Scientific Instruments Inc.). The experiments were repeated at least three times.Cell viability was determined with the Cell Counting Kit-8  according to manufacturer\u2019s instructions. Cells were seeded at 5 \u00d7 102O. The temperature program was: denaturation at 95\u00b0C for 3 minutes, a total of 40 cycles of amplification , and a final extension at 72\u00b0C for 5 min. Calculated mRNA abundance in each sample was normalized to GAPDH and ACTB. Relative abundance was quantified with the 2\u2212\u0394\u0394CT method. Reactions were performed in a Bio-Rad iCycler iQTM Real-Time PCR Detection System . Primer sequences used for qPCR are listed in Total RNA was isolated using TRIzol  according to the manufacturer\u2019s protocols. RNA was dissolved in UItraPure Distilled Water . Concentrations of RNA were determined by measuring absorbance at 260 nm and the purity of RNA was determined by calculating the ratio of absorbance at 260 and 280 nm with values ranging from 1.8 to 2.0. The isolated RNA was stored at -80\u00b0C. Reverse-transcription was performed with a PrimeScript RT reagent kit containing Gdna Eraser  as recommended by the manufacturer. The RNA was diluted to the optimal concentration and tested with qRT-PCR probes and primers. All qRT-PCR reactions were run in triplicate. The reaction system contained 10 \u03bcL of SYBR Green Master, 2 \u03bcL primers (1 \u03bcL forward primer and 1 \u03bcL reverse primer), 2 \u03bcL of cDNA templates, and 6 \u03bcL of RNase-free distilled H+ T cells using RIPA Lysis and Extraction Buffer  including protease inhibitors, placed on ice for 30 min, and centrifuged at 12,000 \u00d7 g for 5 min at 4\u00b0C. Protein concentrations were measured using the BCA protein assay kit .Total protein was isolated from CD4For western blot, after high temperature denaturation treatment, the same amount (30 \u03bcg/lane) of the protein sample was placed on a 10% SDS-PAGE for separation. Then, protein bands were transferred to a polyvinylidene difluoride (PVDF) membrane . The membrane was then blocked for 1 h at room temperature in blocking buffer (0.1% Triton-X/PBS containing 5% skim milk powder). Blocked membranes were incubated overnight at 4\u00b0C with specific antibodies for total OXPHOS , DRP1 , MFN2 , FIS1 , SQSTM1/P62 , Parkin , LC3B , Orai1 , ORAI3 , STIM1 , STIM2 , and \u03b2-actin . Subsequently, membranes were incubated with HRP-conjugated secondary antibodies  for 1 h at room temperature. Immunoreactive bands were detected using an enhanced chemiluminescence solution (Beyotime). Visualization of target proteins was performed using a ProteinSimple imager . Image Lab software  was used to analyze intensity of the bands and compared changes across the different treatments.2+ concentrations were analyzed by flow cytometry. Cells were washed and stained with 3 \u03bcM Fluo-3AM  in Tyrode buffer (pH 7.4), cultured at 37\u00b0C for 30 min.Cytosolic Ca5 cells were incubated with 10 \u03bcM carboxy-2\u2019,7\u2019-dichloro-dihydro-fluorescein diacetate probe to stain intracellular ROS in PBS for 30 min at 37 \u00b0C. Fluorescence was measured at 488 nm (excitation) and 525 nm (emission).To determine intracellular change in ROS generation in CD4+ T cells, they were stained using a ROS assay kit (Beyotime). A total of 1 \u00d7 10For cytokine staining, cells were re-stimulated with 1 \u03bcM ionomycin and 50 ng/mL PMA (phorbol myristate acetate) in the presence of 5 \u03bcM Brefeldin A (eBioscience) for 4 h. Cell surface staining was performed use CD4 Antibody (Invitrogen) in PBS, followed by permeabilization with Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and intercellular staining in permeabilization buffer (eBioscience). The following antibodies were used: anti-IL-17A  antibodies and anti-Foxp3  antibody.2) for 15 min protected from light at 37\u00b0C. Cells were then washed with warm modified HBSS buffer to remove residual dye. Samples were placed on a shaker at room temperature for 5 min. Opening of the mPTP was assessed via fluorescence using a laser confocal microscope .For calcein release, treated cells were transferred to a cell culture plate pre-coated with 1 x polylysine, and cells washed twice with a modified HBSS-buffered   saline solution preheated at 37\u00b0C. Then, samples were incubated with labeling solution , treated cells were adjusted to a density of 1 \u00d7 106 and transferred to a cell culture plate pre-coated with 1 \u00d7 polylysine. Then, cells were placed in a constant temperature incubator under 5% CO2, 37\u00b0C conditions for 20 min. Cell culture media was then removed and cells washed twice with a modified HBSS-buffered   saline solution preheated at 37\u00b0C for staining. For staining, cells were diluted with Ca2+ and Mg2+-containing HBSS buffer to a working solution with a final concentration of 5 \u03bcM Mito-SOX for ROS and incubated at 37\u00b0C for 10 min, or 4 \u03bcM Rhod-2-AM for Ca2+ for 40 min. Cells were then washed three times with warm modified HBSS buffer to remove residual dye. A laser confocal microscope was used to determine concentrations .To detect cell mitochondrial ROS  and Ca6 CD4+ T cells per well were spun onto poly-D-lysine coated seahorse 24 well plates and preincubated in Seahorse XF media (non-buffered RMPI + 10 mM L-glutamine + 10 mM sodium pyruvate + 25 mM glucose) at 37\u00b0C for a minimum of 30 min in the absence of CO2. OCR was measured under basal conditions, and after addition of the following drugs: 1.5 \u03bcM oligomycin, 1.0 \u03bcM flurorcarbonyl cyanide phenylhydrazone (FCCP) and 500 nM rotenone + 500 nM antimycin A. Measurements were obtained using a 24-well Extracellular Flux Analyzer .Oxygen consumption rate (OCR) was measured using the Seahorse XFe bioanalyzer. 1 \u00d7 10via unpaired Student\u2019s t-test or one-way ANOVA with a Duncan test for post hoc analysis. Only differences with P-value \u2264 0.05 were considered statistically significant and a P-value \u2264 0.01 was considered highly significant.Baseline characteristics of the dairy cows are reported as the median and interquartile range, and other data as the means \u00b1 SEM, with n denoting the number of independent experiments. Statistical analysis was conducted using SPSS 26.0 Software  and GraphPad Prism program . All data were tested for normality and homogeneity of variance using the Shapiro\u2013Wilk and Levene tests. For baseline characteristics of the dairy cows, data with skewed distribution were analyzed using a nonparametric statistical analysis using the Wilcoxon test . For oth+ T cells associated with inflammatory diseases, the levels of the inflammatory markers in serum were investigated firstly. Levels of serum IL-17 and IL-6 were greater and IL-10 lower, but TGF-\u03b2 did not differ in cows with ketosis  were lower in CD4+ T cells from ketotic cows   , 42. How(OXPHOS) . Thus, t2+ influx after TCR stimulation. It has been suggested that SOCE activates Ca2+ dependent enzymes and transcription factors and the nuclear factor of activated T cells (NFAT), which regulate the transcription of several cytokine genes including IL-17A, IL-21, IL-22 and IFN\u03b3 (2+. Deletion of the electron transport chain (ETC) complex III gene Uqcrfs1 reduced mitochondrial ROS levels and impaired T cell proliferation in mice . Under that scenario, electron leak would have increased along with ROS production. The end-result of altered mitochondrial metabolism and especially ROS production would further affect inflammatory cytokine secretion and immune cell activation.Activated SOCE provides the bulk of intracellular Caand IFN\u03b3 . A high and IFN\u03b3 . We obse in mice . The roloduction , 47. Thu+ T cells treated with fatty acids provided a direct link between these molecules (or indirectly via ROS) and induction of mitophagy. Mitophagy is an important process maintaining the stability of mitochondria and homeostasis of the intracellular environment, and works in concert with autophagic flux . Written informed consent was obtained from the owners for the participation of their animals in this study.Conceived and designed the experiments: BZ and CX. Performed the experiments and analyzed data: ML, WY, YY, JNW, JJW, YH, and SW. Drafted the paper: ML and BZ. Reviewed and edited: JL and QJ. All authors contributed to the article and approved the submitted version.The study was supported by grants from the National Natural Science Foundation of China  and Heilongjiang Natural Science Foundation (YQ2020C035).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Inflammasomes are multiprotein complexes of the innate immune system that play critical roles in activation of a variety of inflammatory responses. Many inflammasomes have been identified. Among of them, the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) has been well characterized  in treating PD and other neurodegenerative diseases . Using an MPTP-induced mouse model and 6-OHDA induced SH-SY5Y cell model, Que et al. investigated the therapeutic potential of Dl-3-nbutylphthalide (NBP) for treating PD. Their findings showed that NBP could rescue dopaminergic neurons by reducing NLRP3 inflammasome activation and the aggregation of a-Syn, leading to amelioration of mitochondrial impairments . Since the NLRP3 inflammasome is activated by amyloid \u03b2 (A\u03b2) and/or ATP via P2X7R in microglia, targeting P2X7R is a plausible approach to resolve neuroinflammation in AD patients. Islam et al. tested the effects of taurodeoxycholate  in inhibiting the activation of NLRP3 inflammasome and P2X7R expression. Their data provided important evidence that TDCA was also effective in decreasing A\u03b2 plaques and preventing neuronal loss, along with improving memory dysfunction in the 5xFAD mouse model of AD .This Frontier Research Topic has brought together a group of leading experts in the field to address these critical issues. Three of the articles in this Research Topic explored the underlying mechanisms of the NLRP3 inflammasome and their potential in PD and AD therapeutics by targeting the NLRP3 inflammasome. In a perspective article, Chen et al. compared the effects of DOR on the expression of miRNAs in hypoxic/ischemic-sensitive organs  and hypoxic/ischemic-insensitive organs (such as liver and muscle), and their impact on inflammatory injury. On the other hand, Xu et al. reported that UFP-512, a specific DOR agonist, inhibited hypoxia-induced microglial M1 activation and inflammatory activity, while knocking-down or inhibiting DOR promoted microglial M1 and M2 activations.Two papers on this Issue investigated the neuroprotective roles of \u03b4-opioid receptors (DOR) and DOR-mediated neuroinflammation signaling pathways (including NLRP3 inflammasome) in hypoxic/ischemic insults. Cheng and Wang introduced a novel approach to study the interactions of TXNIP and NLRP3 in three in silico models to evaluate the binding stability of the possible interaction of TXNIP/NLRP3. Their data suggest that the N-terminal of TXNIP is essential in allosteric regulation of NLRP3, even without directly binding to NLRP3, which provides an invaluable insight for future development of NLRP3 inflammasome inhibitors (Cheng and Wang).Thioredoxin-interacting protein  plays a regulator role in inflammation and various neurodegenerative diseases . Furthermore, the constructed models of TXNIP/NLRP3 interaction are also valuable for inhibitor development targeting the TXNIP/NLRP3 interaction during inflammasome activation (Cheng and Wang). We believe that this Frontier Research Topic will stimulate additional research by taking innovative approaches for further revealing the role of NLRP3 inflammasome in the pathogenesis of neurodegenerative diseases, and identifying new medications for the treatment of these disorders.In summary, studies published in this Research Topic provide an overview of the role of the NLRP3 inflammasome in the pathogenesis of PD, AD, hypoxia and ischemia. In particular, these articles investigated drugs that regulate NLRP3 inflammasome-related damage and potential therapies for these neurodegenerative diseases. Their findings provided important evidence to support inhibition of the NLRP3 inflammasome as a promising therapeutic target, including NLRP3 inflammasome inhibitors (such as MCC950 and NBP) for the treatment of PD, a GPCR19 ligand  for the treatment of AD, and \u03b4-opioid receptors for hypoxic/ischemic treatment (CD prepared the initial draft of the manuscript. CD, XC, KJ, and QW revised the manuscript. All authors contributed to the article and approved the submitted version."}
+{"text": "Inflammasomes are intracellular signaling complexes of the innate immune system, which is part of the response to exogenous pathogens or physiological aberration. The multiprotein complexes mainly consist of sensor proteins, adaptors, and pro-caspase-1. The assembly of the inflammasome upon extracellular and intracellular cues drives the activation of caspase-1, which processes pro-inflammatory cytokines IL-1\u03b2 and IL-18 to maturation and gasdermin-D for pore formation, leading to pyroptosis and cytokine release. Inflammasome signaling functions in numerous infectious or sterile inflammatory diseases, including inherited autoinflammatory diseases, metabolic disorders, cardiovascular diseases, cancers, neurodegenerative disorders, and COVID-19. In this review, we summarized current ideas on the organization and activation of inflammasomes, with details on the molecular mechanisms, regulations, and interventions. The recent developments of pharmacological strategies targeting inflammasomes as disease therapeutics were also covered. Innate immunity provides the most rapid and conserved defense against cellular damage caused by pathogenic infections, injuries, and cellular stresses. This response depends on the monitoring of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) through sensors called pattern recognition receptors (PRRs), located in both immune cells and non-immune cells ,2. DistiThe concept of the inflammasome was first defined by Dr. Jurg Tschopp in 2002 , upon idNLRs function as the primary sensors for inflammasome formation. Structurally, these family members are composed of an N-terminal pyrin domain (PYD), a central nucleotide-binding and oligomerization domain (NACHT), and C-terminal leucine-rich repeats (LRRs). While the NACHT and LRR domains are present in all NLR proteins (except NLRP10), the PYD determining the interaction partners is the most variable . Upon acAnother critical and direct substrate of caspase-1 is GSDMD, the primary executor of pyroptosis. Caspase-1 processes cytosolic GSDMD to release its N-terminal domain from an autoinhibited conformation ,37, a hiThis review summarized the recent advances in understanding the organization and activation mechanisms of the inflammasomes, the diseases relevant to their dysfunctions, and the natural and designed inhibitors targeting the inflammasome signaling for a therapeutic purpose.Bacillus anthracis lethal toxin [The NLRP1 inflammasome was the first one identified, initially described as a caspase-activating complex comprising caspase-1/5, Pycard/ASC, and NALP1 . Human Nal toxin , or a real toxin . In cont+ efflux caused by pyroptosis can further trigger the assembly of the NLRP3 inflammasome and promote NLRP3-caspase-1-dependent cytokine maturation [The NLRP3 inflammasome is the most extensively studied. Human NLRP3 comprises a PYD, a NACHT, and an LRR domain . The NACturation ,66,67. TNLRC4 was initially identified as a pro-apoptotic protein named the ICE-protease-activating factor (IPAF) based on its similarity to apoptotic protease-activating factor 1 (APAF1) . BesidesNucleic acids are predominant PAMPs and ubiquitous in living organisms. In addition to cytosolic DNA sensors such as cGAS and the critical roles of cGAS-STING signaling in cellular physiology ,78,79,80Clostridium difficile TcdB, a major virulence factor of Clostridium difficile, covalently modifies RhoA and inactivates these GTPases, which triggers the assembly of pyrin inflammasomes in an ASC-dependent manner [Pyrin  is encoded by the gene MEFV. In 1997, several groups disclosed that the mutations of the MEFV gene in humans are responsible for the monogenic autoinflammatory disease familial Mediterranean fever (FMF) . Later, t manner . Howevert manner ,90. NeveInflammasome activation is generally considered a two-step process, a priming step  and an activation step . Initially, the priming step is characterized by the transcriptional induction of NLRP3, pro-IL-1\u03b2, and pro-IL-18 through an inflammatory stimulus, including TLR ligands and cytokines such as TNF\u03b1 . The enh+ efflux, Ca2+ signaling, Na+ influx, and Cl\u2212 efflux), can also activate the NLRP3 inflammasome [Multi-faceted pathogenic and sterile inflammatory signals can activate NLRP3, such as foreign PAMPs from fungi, bacteria, and viruses, and host-derived molecules such as reactive oxygen species (ROS), extracellular ATP, and crystalline and particulate matters . Additionally, perturbing intracellular homeostasis, including lysosomal destabilization, mitochondrial dysfunction, and ion fluxes , leading to a K+ efflux and subsequent NLRP3 activation [+/H+ ionophore, is commonly used as an NLRP3 inflammasome agonist via the K+ efflux [+ is sufficient to activate the NLRP3 inflammasome, while the high extracellular concentration prevents it [+ efflux to trigger the NLRP3 inflammasome activation [+ efflux in NLRP3 activation, the exact nature underlying it is still not fully understood.The Ker DAMPs ,98. For tivation . Nigeric+ efflux . Notablyvents it ,101. A p2+, thus, inducing CaSRs signaling and leading to NLRP3 inflammasome activation in rheumatoid arthritis [Calcium-sensing receptor (CaSR) signaling is able to promote NLRP3 inflammasome activation by decreasing the intracellular cyclic AMP (cAMP) level ,103, as rthritis . ADP relrthritis .\u2212 efflux facilitates NLRP3 inflammasome activation have been presented. While some studies indicated that the Cl\u2212 efflux is responsible for the upstream signaling for NLRP3 activation, some groups showed that the Cl\u2212 efflux is required for ASC oligomerization [\u2212 level has previously been shown to promote IL-1\u03b2 secretion [Distinct opinions regarding how the Clrization ,108. Theecretion . Later, ecretion .2+ and Na+ influxes and mediates the K+ efflux through TWIK2 [2+ flux, regulated by the K+ efflux, is presumed to be critical in NLRP3 inflammasome activation [Additionally, ion fluxes have frequently been found coordinated in NLRP3 activation. For instance, ATP activates P2X7R, which promotes the Cagh TWIK2 . The Ca2tivation , althougtivation ,112. The+ efflux and Ca2+ influx [+ efflux in human monocytes [Studies have shown that particulate stimuli such as alum and cholesterol crystals activate the NLRP3 inflammasome by causing lysosomal rupture and the release of the particulates into the cytoplasm. This notion is supported by dipeptide Leu-Leu-OMe, a soluble lysosomotropic agent, which is sufficient to activate the NLRP3 inflammasome . Similar+ influx , emphasionocytes .Additionally, NLRP3 stimuli can promote the disassembly of the trans-Golgi network into the dTGN, which serves as a scaffold for NLRP3 aggregation and activation . AnotherBesides lysosomes, mitochondria are considered central organelles in regulating the NLRP3 inflammasome activation. Mitochondria can serve as the docking platform for the NLRP3 inflammasome assembly. Upon activation, NLRP3 translocates from the cytosol and ER to the mitochondria and mitochondria-associated membrane, and associates with the adaptor protein ASC ,62,63. SSmall molecules, such as imiquimod and CL097, can drive NLRP3 inflammasome activation by targeting mitochondria and inducing mtROS production . mtROS, + efflux [Several co-activators of NLRP3 have been identified, including dsDNA-binding protein PKR , guanyla+ efflux . Recentl+ efflux . HoweverEmerging evidence in recent years has suggested a critical role of NLRP3 PTMs in inflammasome priming and activation. Ubiquitination-mediated protein degradation controls NLRP3 inflammasome activation by regulating the availability of NLRP3. The lifespan of NLRP3 is coordinated by F-box/LRR-repeat protein 2 (FBXL2) and F-box-only protein (FBXO3) . FBXL2 uIntriguingly, ubiquitination has a dual role in NLRP3 inflammation activation. The deubiquitination of NLRP3 contributes to inflammasome activation. For instance, the BRCA2-containing complex subunit 3 (BRCC3) deubiquitinase complex interacts with NLRP3 and removes the K63-linked ubiquitin chain from the LRR domain of NLRP3, allowing its oligomerization and activation . By contPhosphorylation events control NLRP3 inflammasome assembly and activation, as exemplified by several cases. During the priming, c-Jun N-terminal kinase 1 (JNK1) directly phosphorylates human NLRP3 at Ser198 (mouse Ser194), which is critical for NLRP3 inflammasome assembly and function . The phoPhosphorylation also plays a negative role in regulating NLRP3 activation. For instance, the phosphorylation of human NLRP3 at Ser5 (mouse Ser3) by AKT inhibits NLRP3 inflammasome activation by disrupting the PYD\u2013PYD interaction , while pIn recent years, besides ubiquitination and phosphorylation, PTMs have also been shown to emerge in regulating the NLRP3 inflammasome. These NLRP3 modifications include SUMOylation ,164,165,+ efflux caused by GSDMD pores activates NLRP3 and initiates caspase1-dependent IL-1\u03b2 and IL-18 maturation. Galectin-3 can augment caspase-4/11 oligomerization and promote non-canonical inflammasome activation through LPS binding [In addition to the canonical pathway, the NLRP3 inflammasome can be activated via the direct sensing of cytosolic LPS by human caspase-4/5 or mouse caspase-11, the process of which is defined as the non-canonical pathway ,42,174.  binding . Notably binding . Besides binding ,179,180. binding .Acute inflammation is beneficial for pathogen clearance and tissue repair, while chronic inflammation is a pathological condition leading to tissue damage. A gain-of-function mutations study on the NLRP3 gene linked its functions to some inherited autoinflammatory diseases . MutatioBesides monocytes and macrophages, the NLRP3 inflammasome can also be activated in a wide range of endothelial, epithelial, and mesenchymal cells, thus, related to various inflammatory diseases in different organs such as the skin, brain, and liver. Thus, the NLRP3 inflammasome has been implicated in central nervous system (CNS) diseases, such as multiple sclerosis (MS), Alzheimer\u2019s disease (AD), and Parkinson\u2019s disease (PD) ,186, metNLRP3 is a drug target of significant interest in the pharmaceutical industry, with the potential for treating various inflammatory diseases. Small molecules directly targeting NLRP3 are developing rapidly . The mosBesides disrupting the NLRP3 inflammasome assembly, many inhibitors are developed to target its ATPase activity . RecentlN-acetyl-Phe-Leu-Thr-Asp-chloromethylketone (Ac-FLTD-CMK), was designed to target inflammatory caspase, such as caspase-1, -4, -5, and -11, but not the apoptotic caspases such as caspase-3. It was shown to inhibit GSDMD cleavage and suppress the pyroptosis downstream of both canonical and non-canonical inflammasomes [Other alternative strategies have also been attempted to inhibit the NLRP3 inflammasome, such as the blockade of ATP receptor P2X7, NF-\u03baB, caspase-1, IL-1\u03b2/IL-1R, and IL-18 . Avastinmmasomes . Notablymmasomes  and the mmasomes  and rilommasomes . Howevermmasomes  , was shown to disrupt the assembly of NLRP3 and NEK7 and subsequent NLRP3 oligomerization [In addition to synthetic compounds, herb-based medicine shows excellent potential for inflammatory disease treatment . An alkarization . Licocharization . Androgrrization ,230. Othrization , rpistimrization , pterostrization , and berrization , which aInflammasomes are dynamic protein complexes that multiple pathogenic and sterile inflammatory signals can trigger and induce infectious and non-infectious insults. Unlike other innate immune signalosomes that are generally membrane-anchored and less systemic in structure, inflammasomes appear unique. Given the large number of mutually unrelated upstream signals documented, inflammasome sensors seem to respond to general homeostasis stress, but are not a direct agonist. Characterizing the molecular mechanisms underlying inflammasome organization and cellular compartmentation would also be intriguing and critical to understanding this exquisite molecular machinery. Is there a key factor that integrates all the signals? How do the inflammasomes communicate with metabolism and nutrients and form special cellular compartments? Evidently, inhibitors targeting the inflammasome signaling components, such as those developed by studying cGAS-STING signaling , provide"}
+{"text": "The nucleotide-binding domain (NOD)-, leucine-rich repeat (LRR)-, and pyrin domain (PYD)-containing protein 3, NLRP3, is a multiprotein complex belonging to the innate immune system that can be activated by pathogens or danger-associated molecular patterns . The NLRFurthermore, emerging reports on SARS-CoV-2 suggested that NLRP3 inflammasome activation strongly contributes to the formation of the severe inflammatory cytokine storm, resulting in clinical and pathological manifestations of patients infected with COVID-19, especially in those with increased risk ,7,8.In view of these evidence, NLRP3 is actually considered as a new drug target with the potential for treating various inflammatory diseases, so small natural and synthetic molecule inhibitors targeting NLRP3 inflammasome signaling are currently developed rapidly, although the efficacies of these agents need further investigation .International Journal of Molecular Science titled \u201cThe NLRP3-Inflammasome in Health and Disease\u201d is devoted to collecting original research and reviews of the literature concerning the role of NLRP3-inflammasome in pathogenetic mechanisms leading to the onset and/or progression of different human chronic diseases, strengthening the observation that NLRP3-inflammasone could represent a new potential therapeutic target. New information has been added to this field by evidence provided by twelve articles, with eight original papers and four narrative reviews.The Special Issue of the The original papers described new molecular mechanisms by which low-grade chronic inflammation induced by NRPL3 inflammasome complex activation could contribute to the tissue damage observed in different models of chronic pathological conditions. The in vivo study of Burger et al. added new knowledge on the role of NLRP3 activation and IL-1\u03b2 production in atherosclerosis progression. Interestingly, the authors demonstrated that the infiltrating monocytes into the intima and hypercholesteremia, by triggering NLRP3 inflammasome activation and IL-1\u03b2 signaling, promoted the vascular smooth muscle cell\u2019s phenotypic switch to macrophages-like cells and the foam cell formation, causing destabilization and the rupture of the atherosclerotic plaques. Overall, the data suggested that they block inflammasome assemblies; thus, IL-1\u03b2 inhibition production could represent an effective tool for preventing major adverse cardiovascular events .Am\u00e9rico-Da-Silva explored, for the first time, the link between insulin-resistance (IR) and NLRP3 inflammasome activation, demonstrating an increased expression of NLRP3 inflammasome components in the skeletal muscle of obese insulin-resistant animals (HFD) compared to normal control diet mice (NCD). Furthermore, the authors observed, for the first time, that MCC950, a specific inhibitor of NLRP3 inflammasome activation, promoted increased insulin-induced GLUT4 translocation in skeletal muscle fibers from HFD mice and that the inhibitor increased GLUT4 translocation in the absence of insulin in both NCD and HFD muscle fibers. Collectively, the study supports the idea of the skeletal muscle tissue as an active component of inflammatory processes associated with obesity and that the NLRP3 inflammasome might play a physiological role in glucose metabolisms by regulating GLUT4 trafficking in skeletal muscles .Interestingly, Lee et al. provided evidence on the role of NLRP3 inflammasomes in the mechanisms by which glycemic variability is known to be a stronger predictor for microvascular and macrovascular complications than sustained hyperglycemia in patients with diabetes. The authors demonstrated that an acute glucose shift activated the NLRP3 inflammasome to a greater extent than constant high glucose, strengthening the current knowledge on the association between abnormal activation of the NLRP3 and onset and progression of diabetes ,13,14.Considerable studies have demonstrated the pathogenetic role of the NLRP3 complex activation in many kidney diseases, occurring both in an inflammasome-dependent and inflammasome-independent manner . In partThe in vivo model of rhabdomyolysis-induced acute kidney injuries proposed by Song et al. revealed that myoglobin-induced NLRP3 inflammasome activation strongly contributed to tubular renal injury, elucidating the not yet clear underlying mechanisms. Interestingly, the authors demonstrated that the lack of NLRP3 ameliorated renal tubular injury and, more importantly, that the main factor in decreasing inflammation is the attenuation of inflammatory responses in renal tubular cells rather than renal immune cells .Some studies conducted in patients affected by age-related macular degeneration revealed an abundant amount of NLRP3 in the retina and high levels of IL-1\u03b2 are in the vitreous body, addressing the pivotal role of NLRP3 machinery activation in the pathogenesis of this degenerative disease ,19. The The in vivo study of Mae et al. provided new evidence on the role of activated NLRP3 into the pathogenesis of periodontitis. The authors demonstrated that the activation of NLRP3 in macrophages stimulated with dental calculus, a common deposit in periodontitis patients, increased the local production of IL-1\u03b2 and IL-18 that, in turn, by promoting osteoclastogenic and anti-osteogenic effects, could trigger alveolar bone resorptions .The rat model of reserpine-induced fibromyalgia proposed by D\u2019Amico et al. provided evidence on the involvement of NLRP3 inflammasome activation in the pain states and neuroinflammation of patients affected by fibromyalgia. The authors showed that the pharmacological inhibition of purinergic P2X7 receptor (P2X7R) prevented the NLRP3 inflammasome\u2019s activation and, consequently, the release of IL-1\u03b2 and IL-18, which are involved in the activation of nociceptors in the spinal cord and brain tissues. The finding emerging from the study suggested that NLRP3 pathway activation could be involved in the heightened sensitivity to pain and contribute to the symptomatology of fibromyalgia . The studies investigating NLRP3 inflammasome activation in neuropsychiatric patients suffering from mood disorders showed increased serum levels of NLRP3 complex components, as well as of IL-1\u03b2 and/or IL-18 cytokine production, further supporting the role of inflammation in psychiatric diseases. The study of Zhou et al. pointed out that the techniques used in these studies are not sufficient for measuring NLRP3 activation. Therefore, the authors developed and validated an assay to measure the intracellular formation of ASC specks in activated PBMC and IL-1\u03b2 and caspase-1 levels in the periphery over 6 months in adolescents with bipolar and depressive disorders. Despite the small sample size, the results are interesting as, although all patients demonstrated significant increases in ASC specks and extracellular IL-1\u03b2 production, the authors observed an inter-individual difference in activation magnitudes, suggesting that NLRP3 inhibitors could be candidates for individualized early intervention and combination therapy in patients affected by mood disorders .The review article by Poli et al. highlighted studies investigating the role played by oxidative stress, inflammation, and NLRP3 inflammasome activation in varicocele disease, concomitantly describing some therapeutic antioxidant strategies that have recognized beneficial effects in counteracting the above-reported pathological conditions leading to varicocele-mediated hypofertility. Most evidence reported that the abnormal testicular function and failed spermatogenesis observed in patients affected by varicocele are caused by ROS-driven NLRP3 activation and the overexpression of cytokines that, in turn, further increase ROS production up to and exceeding the availability of antioxidant systems. A variety of antioxidants were assessed for their ability to counteract oxidative stress in the testes, and among them, the most effective antioxidant treatments are non-enzymatic factors such as resveratrol vitamins E and C, coenzyme Q10, and lycopene and therapeutical non-enzymatic drugs, such as Kallikrein, Pentoxifylline, and Cinnoxicam . The review of Ryan et al. aimed to highlight findings regarding the molecular mechanisms linking NLRP3 inflammasome activation and post-transplantation complications related to alloimmune injury and rejection. Despite the significant amount of studies, the authors concluded that it is mandatory to better understand how NLRP3 inflammasome activation is spatially and temporally regulated during the acute and chronic post-transplant period, and developing non-invasive biomarker surveillance that could be used as a risk factor stratification for poor clinical outcomes across solid organs is mandatory as well .Effendi et al. summarized the recent evidence focused on NLRP3 inflammasome activation in response to viral infection throughout the development and progression of idiopathic pulmonary fibrosis, the most common chronic-age-related respiratory disease rising from repeated micro-injury of the alveolar epithelium and aberrant epithelial\u2013mesenchymal transitions. The authors provided an overview of the mechanisms through which viral infection triggers the activation of the NLRP3 inflammasome, demonstrating that pyroptosis and IL-1\u03b2 and IL-18 production strongly contribute to robust inflammation, fibroblast to myofibroblasts differentiation, and the synthesis of the extracellular matrix and epithelial\u2013mesenchymal transition, leading to lung fibrosis. Furthermore, the authors highlighted that some recent evidence support the hypothesis that SARS-CoV-2 might directly activate the NLRP3 inflammasome, leading to cytokine storms and fibrosis and opening a new scenario for the management of COVID-19 .Overall, all studies included in this Special Issue provide an update on the role of NLRP3 inflammasome activations in the pathomechanism of several chronic diseases, highlighting that the NLRP3 signaling pathway is an interesting therapeutic target that may support or even replace traditional therapies in the future."}
+{"text": "Inflammation represents the innate immune response of the body tissues against invading microbes and cellular danger signals, and, in this way, it is beneficial . AlthougThis Special Issue invited original researches, reviews, and perspectives focusing on, but not limited to, the mechanisms of inflammasome regulation, the role of inflammasomes in inflammatory responses and disease, the identification and validation of novel molecules modulating inflammasome functions, and potential inflammasome-targeted therapeutics.The research article by Cho et al. explored the anti-inflammatory role of Korean red ginseng (KRG) saponins in caspase-11 non-canonical inflammasome-activated inflammatory responses, and the underlying molecular and cellular mechanisms in macrophages. A previous study by the same research group demonstrated that KRG has a strong anti-inflammatory effect by inhibiting caspase-11 non-canonical inflammasome in macrophages . Since gThe research article by Kang et al. identified a chemical compound, ODZ10117 (ODZ), and investigated the anti-inflammatory effect of ODZ by inhibiting NLRP3 inflammasome [Another research article by Kanwar et al. undertook a retrospective chart review of 29 consecutive ICU COVID-19 patients receiving dapsone and 30 not receiving dapsone to obtain an indication of whether dapsone can be used as part of the standard treatment of severe COVID-19 in patients entering the ICU . This stThe review article by Xia et al. highlights the studies investigating the post-translational modifications (PTMs) of the components of the NLRP3 inflammasome and the resultant effects on regulating its activity to provide references for the exploration of the molecular mechanisms by which the NLRP3 inflammasome is activated and controlled . This stIn conclusion, this Special Issue highlights the regulatory roles of inflammasomes and inflammasome-associated molecules in inflammatory responses and diseases and the underlying molecular mechanisms. This study also highlights the critical role of PTMs of the inflammasome components to\u00a0modulate\u00a0the\u00a0functions\u00a0of\u00a0the\u00a0inflammasomes\u00a0in\u00a0inflammatory\u00a0responses and various diseases. We hope that this Special Issue will attract the attention of researchers and urge them to further explore new roles of inflammasomes and inflammasome-associated molecules in inflammatory responses, as these underpin most human diseases. We further hope that this Special Issue will shed light on some of the research conducted to date in relation to the targeting of inflammasomes and inflammasome-associated molecules in the search for novel therapeutics."}
+{"text": "Cognitive impairment is a multifactorial and multi-step pathological process that places a heavy burden on patients and the society. Neuroinflammation is one of the main factors leading to cognitive impairment. The inflammasomes are multi-protein complexes that respond to various microorganisms and endogenous danger signals, helping to initiate innate protective responses in inflammatory diseases. NLRP3 inflammasomes produce proinflammatory cytokines (interleukin IL-1\u03b2 and IL-18) by activating caspase-1. In this review, we comprehensively describe the structure and functions of the NLRP3 inflammasome. We also explore the intrinsic relationship between the NLRP3 inflammasome and cognitive impairment, which involves immune cell activation, cell apoptosis, oxidative stress, mitochondrial autophagy, and neuroinflammation. Finally, we describe NLRP3 inflammasome antagonists as targeted therapies to improve cognitive impairment. Inflammasomes, first proposed by Tschopp et al. in 2002, play key roles in innate immunity. The inflammasomes consist of three main components: a sensor, an adaptor, and an effector .Some examples of inflammasomes include the absent in melanoma 2 (AIM2), the NACHT, LRR, and CARD domain-containing protein 4 (NLRC4), the NACHT, LRR, and pyrin domain (PYD)-containing protein (NLRP) 1, and the NLRP3 inflammasomes. Among them, the NLRP3 inflammasome is the most widely studied , 6.Cognitive dysfunction is a neurodegenerative disease that often leads to impairment of learning, memory, and sensory functions . Mild coAccumulating experimental evidence has suggested that the activation of NLRP3 inflammatory vesicles is strongly associated with neurodegenerative diseases , 16. MicThe molecular structure of inflammasomes was poorly understood until recent technological breakthroughs in low-temperature electron microscopy  .Like most other inflammasomes, the NLRP3 inflammasome is composed of a sensor NLRP3, an adaptor ASC, and an effector caspase-1 . With thNLRP3 inflammasomes are typically activated after an inflammatory response. The activation of the NLRP3 inflammasome is a highly regulated process that can be divided into classical, non-classical, and alternative pathways . The claThe activation stage induces activation and inflammasome formation. There have been relevant discussions about the specific mechanisms of activation, and there is a wealth of data describing the process. Since many of these data overlap, there is as yet no single unified account. NLRP3 can be activated by a large number of unrelated stimuli, and the upstream signals are considered to be independent of each other . HoweverThe non-classical pathway of NLRP3 activation is initiated by lipopolysaccharides (LPS) that exist in the gram-negative bacterial cell wall. This pathway involves upregulation of caspase-11 through the TLR4 pathway, and subsequent potassium efflux, which upregulates NLRP3 and IL-1\u03b2, and activates NLRP3 \u201345. In tThe alternative pathway is the direct activation of NLRP3, but the mechanism varies among species. In humans and pigs, for instance, TLR4 stimulation alone induces IL-1\u03b2 secretion, which activates NLRP3 , 48.After NLRP3 activation, oligomerization occurs, leading to the formation of a platform capable of accessing the CARD domain, which in turn recruits caspase-1, thus completing the assembly of the NLRP3 inflammatory vesicle complex. Furthermore, pro-caspase-1 can be enriched and activated to produce caspase-1, which then binds to ASC and functions as an effector .(PD) and AD [Autophagy is a crucial intracellular process of recycling and removing damaged proteins and organelles, and destroying intracellular pathogens , 50, mai) and AD , 55.Damaged or senescent mitochondria can be degraded in lysosomes by mitophagy , 56. As The NLRP3 inflammasome is a cytosolic polyprotein complex that serves as a platform for the activation and maturation of pro-inflammatory cytokines IL-1\u03b2 and caspase-1 . MitochoThere is an interplay between mitophagy and NLRP3 inflammasome. Caspase-1 activation inhibits autophagy induction, resulting in enhanced inflammatory response, which can be suppressed by removal of activators of NLRP3 inflammasome and inflammatory components . The balCell death can be classified as accidental cell death or regulated cell death (RCD), depending on whether the cell death process can be controlled or not , 68. RCDAlthough the role and the mechanism of pyroptosis in cognitive dysfunction are not fully understood, studies have shown that P2X7R/NLRP3, NLRP3/caspase-1, NLRP3/caspase-1/gasdermin D (GSDMD) and other signaling pathways related to pyroptosis are involved in the occurrence and development of cognitive dysfunction with different causes \u201374. In pPyroptosis is a common innate immune response observed in vertebrates . The innInflammasomes are components of the innate immune response . InflammOnce the NLRP3 inflammasome is activated, the active caspase-1 cleaves GSDMD at the aspartic acid residue of position 276 (human) or position 275 (mouse) into two fragments  , 78. TheIn addition to caspase-1, several other inflammatory caspases such as caspases-3, -6, -7, and -8 can also induce pyroptosis , 85. BesHypoxia and ischemia in brain tissues are major causes of cognitive impairment . The delObstructive sleep apnea (OSA) is characterized by the narrowing or collapse of the upper airway, which leads to multiple episodes of hypoxia during sleep . ChronicCerebrovascular disease is also a major cause of cognitive impairment. It includes acute ischemic stroke and chronic cerebral hypoperfusion (CCH) . The acuNLRP3 inflammasomes play a role in microglial activation and release of inflammatory factors. NLRP3 is a well-characterized sensory molecule that is activated in acute and chronic inflammatory conditions as well as in response to stress stimuli . PersistUnder pathological conditions, activation of the NLRP3 inflammasome can lead to caspase-1 cleavage and IL-1\u03b2 release  Cerebral ischemia has been reported to cause cell death, including neuronal death, resulting in the release of inflammatory cytokines and ROS, ultimately leading to the activation and infiltration of microglia . AdditioStudies have shown that cognitive impairment is often accompanied by inflammation and microglial activation, and that excessive activation of NLRP3 greatly aggravates the pathological process of cognitive impairment , 99, 102The typical NLRP3 inflammasome activation involves two steps: priming and activation  Fig.\u00a02)2). In thIn addition to GSDMD self-cracking, activated caspase-1 can also cleave GSDMD . Like moThe activated caspase-1 can release the N-terminal domain of GSDMD, which forms pores on the cell membrane, leading to the release of IL-1\u03b2 and IL-18 among cellular contents, inducing pyroptosis , 68 Fig.+, and this drop is sensed and converted by the NLRP3 inflammasome into a proinflammatory signal [P2X7R is a ligand-gated cation channel , which iy signal . The opey signal , 119. Poy signal . When thy signal . Simultay signal .Studies have shown that the P2X7R/NLRP3 signaling mediates inflammatory responses and cell death, including pyroptosis, and is associated with cognitive impairment in neurological diseases such as AD, and diabetes , 121\u2013123Among the distinct classes of PRRs, TLRs were the first to be discovered. They play key roles in immune function and inflammatory diseases , 125.TLR4 is an important mediator of the neuroinflammatory cascade in the CNS, which can activate the p65 subunit of NF-\u03baB to promote the transcription of NLRP3 components and increase the release of proinflammatory cytokines such as IL-1\u03b2 and IL-18, thereby activating the NLRP3 inflammasome \u2013128. StiNEK7 is a key component of the NLRP3 inflammasome, which acts downstream of potassium efflux and participates in the activation of NLRP3 . A previDDX3X, a member of the DEAD-box helicase family , is a ceDDX3X is a newly identified stress granule protein that interacts with NLRP3 to trigger NLRP3 inflammasome activation mediated by potassium efflux . Recent Previous studies have reported that DDX3X strongly interacts with the NACHT domain of NLRP3. Based on this, questions are raised whether DDX3X also interacts with NEK7, another NLRP3 chaperone, which directly binds to the NACHT domain of NLRP3 , 139. HoAs described previously, TLR4 can activate the p65 subunit of NF-\u03baB to promote the transcription of NLRP3 components and increase the release of proinflammatory cytokines such as IL-1\u03b2 and IL-18, thereby activating the NLRP3 inflammasome and participating in the process of neuroinflammation \u2013128.The proinflammatory function of NF-\u03baB has been extensively studied in macrophages . Under v+) and converts it into proinflammatory signals, which further promote neuroinflammation [The P2X7R pathway is involved in neuroinflammation. P2X7R activation by extracellular ATP allows potassium outflow from the cell and sodium and calcium influx, leading to NLRP3 inflammasome activation , 119. Atammation . Activatammation . Correspammation .The JAK/STAT signaling pathway plays a key role in various cellular responses such as inflammation, cell growth, metabolism, and gene transcription . FunctioThe expression of IL-13, an activator of the JAK-1/STAT signaling pathway, is down-regulated during autophagy\u00a0. NeutralROS/MAPKs can activate NF-\u03baB, which in turn promotes the activation of NLRP3 and participates in neuroinflammation . MAPKs cMiRNAs are small non-coding RNA molecules containing 17\u201324 nucleotides that play a regulatory role by base pairing with complementary sequences within target mRNAs . MiRNAs MiR-17 inhibits the translation of caspase-2 mRNA to regulate pro-apoptotic proteins in the mitochondrial apoptotic pathway . ThioredPrevious studies have shown that miR-7 inhibits \u03b1-synuclein (\u03b1-syn) expression and toxicity\u00a0that can activate the NLRP3 inflammasome via the release of lysosomal cathepsin B and accumulation of ROS . MoreoveMiR-9 inhibits the activation of the NLRP3 inflammasome and related inflammation via the JAK1/STAT1 pathway . InhibitMiR-20a targets TXNIP, inhibiting TXNIP expression and TXNIP-mediated NLRP3 inflammasome formation, and decreasing IL-1\u03b2 release .MiR-133a-1 inhibits the activation of NLRP3 inflammasome by inhibiting the mitochondrial uncoupling protein 2 .Expression of miR-223 reduces the differentiation of monocytes into macrophages while increasing NLRP3 inflammasome protein levels . HoweverImmune system activation and inflammation are common factors leading to cognitive impairment , 162. ThPrevious studies have suggested that the NLRP3 inflammasome is a persistent source of neuroinflammation that drives progressive dopaminergic neuropathology . In neurThe complex signaling cascade of the NLRP3 inflammasome offers multiple targets for inhibition, such as inhibiting the activation of the NLRP3 inflammasome, inhibiting upstream signal transduction, blocking inflammasome assembly and caspase-1 activation, and inhibiting or neutralizing inflammatory cytokines produced by the NLRP3 inflammasome , 166. ThMCC950 is small molecule that selectively inhibits NLRP3 activation , but notIn a recent study, pharmacological inhibition of NLRP3 inflammasome activation by oral MCC950 treatment prevented dopaminergic degeneration in multiple rodent PD models . These rThus, the specific inhibitory effect of MCC950 provides the possibility of treating conditions involving classical and/or non-classical NLRP3 inflammasomes.Studies have shown that the ATPase activity of NLRP3 is a potential drug target for the NLRP3-related diseases .C172, the inhibitor of cystic fibrosis transmembrane conductance regulator channel, has significant inhibitory effects on NLRP3 inflammasome activation . As a C1Importantly, CY-09 exhibits favorable pharmacokinetic profiles in terms of safety, stability, and oral bioavailability. Thus, CY-09 is the first NLRP3 inflammasome-specific inhibitory compound identified both in vitro and in vivo with identified inhibitory mechanisms. Although further studies are required to reveal its effects on other inflammasomes, CY-09 provides a novel approach to inhibiting NLRP3 inflammasome activation.OLT1177, an active \u03b2-sulfonyl nitrile compound, was originally formulated as a topical treatment for degenerative arthritis . OLT1177OLT1177 has been shown to inhibit caspase-1 activity and reduce IL-1\u03b2 production in monocytes of patients with cryopyrin-associated periodic syndrome. Animal studies showed that it can decrease the severity of LPS-induced systemic inflammation. Importantly, treatment with high concentrations of OLT1177 in humans for 8\u00a0days had no adverse biochemical or hematological effects . SimilarThe specific inhibitory effect of OLT1177 extends the possibility of treating NLRP3 inflammasome-related diseases, and no adverse effects were observed in humans over an 8-day period. Meanwhile, OLT1177, an oral inhibitor of the NLRP3 inflammasome, has therapeutic potential for neuroinflammation. However, its effect on the activation of other inflammasomes, such as NLRP1 and pyrin, requires further study. To achieve better therapeutic effects, future studies are needed to clarify whether OLT1177 interferes with other inflammasomes during its pharmacological action.Tranilast is an analog of a tryptophan metabolite and was originally recognized as an antiallergic agent for the treatment of various inflammatory diseases . FurtherExperimental studies have identified tranilast as a specific inhibitor of the NLRP3 inflammasome . TranilaStudies in mouse models have shown significant therapeutic and preventive effects of tranilast on NLRP3-related diseases. Considering its safety and specific inhibitory effects on the NLRP3 inflammasome, tranilast may be potentially used for treatment of NLRP3 inflammasome-related diseases in the future.Oridonin exerts anti-inflammatory, antitumor, and pro-apoptotic effects \u2013178. PrePrevious studies have elucidated the underlying mechanisms underlying the anti-inflammatory effects of oridonin. Oridonin specifically inhibits the activation of NLRP3 inflammasome while having no effect on the activation of AIM2 or NLRC4 inflammasome, or LPS-induced expression of NLRP3 and IL-1\u03b2 as well as TNF-\u03b1 production .Oridonin directly binds to cysteine 279 of NLRP3 NACHT domain through a covalent bond, thereby preventing the NEK7-NLRP3 interaction and subsequent activation of NLRP3 inflammasome . OridoniCognitive dysfunction is a challenging and prominent problem in the aging society. New treatment strategies for cognitive dysfunctions are required. NLRP3, an inflammatory vesicle, plays a key role in neuroinflammation and thus influences the onset and development of cognitive dysfunction.In this review, we demonstrate that NLRP3 inflammatory vesicles are involved in mitochondrial autophagy impairment, cellular scorching, glial activation and oxidative stress, thereby triggering neuroinflammation and cognitive dysfunction. In addition, we have refined and summarized the currently published NLRP3 signaling pathways involved in pyroptosis, including NLRP3/caspase-1, P2X7R/NLRP3, NLRP3/caspase-1/GSDMD, TLR4/NF-\u03ba\u0392/NLRP1/3, and DDX3X-NLRP3; as well as those involved in neuroinflammation, including TLR4/NF-\u03baB/NLRP3, P2X7R/NLRP3, JAK1/STAT/NF-\u03baB/NLRP3 and ROS/MAPKs/NF-\u03baB/NLRP3 pathways.However, current evidence for NLRP3 inflammasome as therapeutic targets is limited and insufficient. The NLRP3-related signaling pathways are a key component of the human immune system. How to reduce NLRP3 activity while avoiding damage to the immune system is a serious challenge. In addition, there have been few animal and clinical studies testing the use of NLRP3 as a therapeutic target for the treatment of cognitive impairment, and the safety and efficacy of such therapeutic strategies need to be evaluated in future. In future experiments, we hope that these miRNAs can exert pro-inflammatory effects by regulating signaling pathway components. Fully exploring the drug properties of miRNAs may provide new directions for the treatment of NLRP3-related diseases."}
+{"text": "Inflammasomes are multiprotein signaling platforms in the cytosol that senses exogenous and endogenous danger signals and respond with the maturation and secretion of IL-1\u03b2 and IL-18 and pyroptosis to induce inflammation and protect the host. The inflammasome best studied is the Nucleotide-binding oligomerization domain, leucine-rich repeat-containing family pyrin domain containing 3 (NLRP3) inflammasome. It is activated in a two-step process: the priming and the activation, leading to sensor NLRP3 oligomerization and recruitment of both adaptor ASC and executioner pro-caspase 1, which is activated by cleavage. Moreover, NLRP3 inflammasome activation is regulated by posttranslational modifications, including ubiquitination/deubiquitination, phosphorylation/dephosphorylation, acetylation/deacetylation, SUMOylation and nitrosylation, and interaction with NLPR3 protein binding partners. Moreover, the connection between it and metabolism is receiving increasing attention in this field. In this review, we present the structure, functions, activation, and regulation of NLRP3, with special emphasis on regulation by mitochondrial dysfunction-mtROS production and metabolic signals, i.e., metabolites as well as enzymes. By understanding the regulation of NLRP3 inflammasome activation, specific inhibitors can be rationally designed for the treatment and prevention of various immune- or metabolic-based diseases. Lastly, we review current NLRP3 inflammasome inhibitors and their mechanism of action. In recent years, the relationship between the innate immune system, inflammation, metabolism, and metabolic diseases has gained significant attention \u20134. It isIn this context, inflammasomes, multiprotein signaling platforms, have emerged as critical players linking metabolism and inflammation , 9. InflThere exist different types of inflammasomes. Nucleotide-binding oligomerization domain (NOD), leucine-rich repeat-containing (LRR) (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is the most studied. NLRP3 mutation have been associated with cryopyrin-associated periodic syndrome (CAPS), a group of autoinflammatory disorders characterized by recurrent fevers and systemic inflammation .This review aims to provide a comprehensive overview of the current understanding of the interplay between metabolism and NLRP3 inflammasome activation. We will delve into the molecular mechanisms through which metabolic factors influence NLRP3 inflammasome signaling, emphasizing the role of mitochondrial dysfunction-mitochondrial reactive oxygen species (mtROS) production, metabolites and metabolic pathways in this process. Furthermore, we will explore the therapeutic opportunities that arise from targeting metabolic pathways to modulate NLRP3 inflammasome activation, offering potential strategies for the development of novel anti-inflammatory therapies.2Inflammasomes are composed of the sensors/receptors of the danger signals, the adaptor and the effector. There are a variety of sensors/receptors that classify the distinct inflammasomes which are activated by different stimuli: NLRP1, NLRP3, NLRC4, AIM2 or pyrin . The ada3NLRP3 canonical function consists in the sensing of danger signals, pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), in the cytoplasm of cells as a pattern recognition receptor (PRR) of the innate immune system and respond with the activation and assembly of NLRP3 inflammasome . NLRP3 i3.1Pyroptosis is a form of programmed cell death that is characterized by a highly inflammatory response. It is mediated by inflammatory caspases, which are caspase-1 in human and mice, caspases-4 and -5 in human and the ortholog caspase-11 in mice . NLRP3 i3.2The activated form of caspase-1 cleaves pro-IL-1\u03b2 and pro-IL-18 at D116 and D36, respectively . IL-1\u03b2 aDespite the mechanism is less well understood, NLRP3 inflammasome is also involved in the secretion of IL-1\u03b1 , 71, hig4The activation of NLRP3 inflammasome is well regulated because it is activated in a two-step process, the priming and the activation steps  see Fi. The pri+ or Cl- efflux, Ca2+ flux, lysosomal disruption, mitochondrial dysfunction and reactive oxygen species (ROS) generation, metabolic signals and trans-Golgi disassembly  that only requires signal 1 to activate NLRP3 inflammasome via TLR4-TRIF-RIPK1-FADD-CASP8 signaling upstream of NLRP3, leading to IL-1\u03b2 secretion but no to pyroptosis , 98.Lastly, pathogenic pollutants and cigarette smoke activate NLRP3 inflammasome via ROS and mtROS generation, contributing to chronic inflammation and fibrosis in the respiratory system, which plays a crucial role in the pathogenesis of respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD) and lung fibrosis . Fine paThe role of cigarette smoke (CS) in the activation of NLRP3 inflammasome remains controversial. On the one hand, constituents of CS are involved in the activation of NLRP3 through oxidative stress, mitochondrial dysfunction MyD88/NF-\u03baB, HMGB1, endoplasmic reticulum stress, lysosomal destabilization and TLR/NF-\u03baB signaling pathway \u2013108. On Because the aim of this review is to connect the inflammasome activity and metabolism, we will take a closer look at the regulation by mitochondrial dysfunction-ROS generation and metabolic signaling.4.1The mitochondria are the organelles in charge of adenosine triphosphate (ATP) generation. However, it also regulates cellular processes, including the cell death by apoptosis, through the intrinsic pathway . It is c+ efflux (mtROS are increased during cellular stress and mediated through voltage dependent anion-selective channel (VDAC) . Inhibit+ efflux , 114. Ho+ efflux  and the + efflux  and inhi+ efflux  of NLRP3Likewise, mtDNA binds to and activates NLRP3 inflammasome , 120. AcLastly, when mitochondrial cardiolipin (mtCL), a unique phospholipid of the inner membrane (IMM), translocate to the outer membrane (OMM) binds the LRR domain of NLRP3 . Recentl4.2Metabolic reprogramming refers to the alterations in cellular metabolic pathways that occur in immune cells, enabling them to adapt their energy production and biosynthetic processes to support immune responses effectively . Indeed,4.2.1First, glucose has been shown to regulate the NLRP3 inflammasome. High glucose (HG) in endothelial cells increases expression of E74 like ETS transcription factor 3 (ELF3) and decreases SET8, a methyltransferase, leading to up-regulation of microtubule affinity regulatory kinase 4 (MARK4) . MARK4 aPyruvate is a key intermediary metabolite, the end product of glycolysis that can be transformed into acetyl-CoA, lactate or alanine. In experiments, ethyl pyruvate prevents NLRP3 inflammasome activation while preserving mitochondrial integrity. Pyruvate inhibits ATP- and nigericin-induced accumulation of electron-dense mitochondria, maintains mitochondrial membrane potential, and prevents ATP- or nigericin-triggered release of mtDNA into the cytoplasm . In micrHexokinase 1 (HK1) catalyzes glucose entry into glycolysis in the cytosol. In macrophages, NLRP3 inflammasome activation is regulated by mTORC1-induced up-regulation of HK1-dependent glycolysis . FurtherGlucose-6-phosphate dehydrogenase (G6PD) catalyzes the entry of glucose into the pentose phosphate pathway. One of its main functions is to control the redox state of the cell . Yen et\u00a04.2.2Regarding TCA, there are two intermediates that lead to opposite effects in terms of NLRP3 inflammasome activation. On the one hand, hypoxic induction of TGF-\u03b21 in the synovium of rheumatoid arthritis (RA) rats resulted in elevated succinate accumulation. This was caused by inhibition of succinate dehydrogenase (SDH) activation and led to NLRP3 inflammasome activation, which was dependent on HIF-1\u03b1 induction. Moreover, citrate-derived itaconate has gained attention as an anti-inflammatory modulator in macrophages . Hooftma4.2.3Lipid metabolism plays a crucial role in a variety of cellular processes, such as energy production, membrane synthesis and signaling pathways. New research has revealed a significant connection between lipid metabolism and NLRP3 inflammasome activation/inhibition. First, the accumulation of fatty acids palmitate and ceramide activate the NLRP3 inflammasome through the AMPK-autophagy-mtROS signaling pathway , 149. InCholesterol crystals are a common feature of atherosclerotic plaques . Cholest5The recognition of the pivotal role of inflammasomes in the development and progression of metabolic diseases opens up exciting possibilities for therapeutic interventions , 163. Ta+/Cl- efflux, P2X7 and mtROS, effectors such as GSDMD, interaction binding partners such as NEK7 and, obviously, the NLRP3 inflammasome components NLRP3 receptor, ASC and caspase-1. Currently, the ATPase activity of the NACHT domain in NLRP3 is the target of the majority of NLRP3 inflammasome inhibitors under clinical and preclinical research (see Developing small-molecule inhibitors that specifically target NLRP3 inflammasome components, such as NLRP3 or caspase-1, represents a promising avenue . Inhibitarch see Table\u00a02.6The interplay between the innate immune system, inflammation, metabolism, and metabolic diseases has revealed a complex and intertwined relationship. Inflammasomes, particularly the NLRP3 inflammasome, have emerged as critical mediators linking these processes together. The activation of inflammasomes triggers the release of proinflammatory cytokines IL-1\u03b2 and IL-18 and the cell death by pyroptosis. The activation of NLRP3 inflammasome is under complex regulation involving multiple signaling networks. Mitochondrial dysfunction-mtROS production as well as metabolic signaling link the bioenergetics and metabolism status of the cell with cell death by pyroptosis, inflammation and innate immune functions through NLRP3 inflammasome activation.Future studies should focus on elucidating the specific mechanisms by which inflammasomes contribute to metabolic dysfunction, identifying potential therapeutic targets within the inflammasome pathway, and developing interventions to modulate inflammasome activation and subsequent inflammation in metabolic diseases, and ultimately, improve patient outcomes and quality of life.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication."}
+{"text": "Genomic data are subject to various sources of confounding, such as demographic variables, biological heterogeneity, and batch effects. To identify genomic features associated with a variable of interest in the presence of confounders, the traditional approach involves fitting a confounder-adjusted regression model to each genomic feature, followed by multiplicity correction.This study shows that the traditional approach is suboptimal and proposes a new two-dimensional false discovery rate control framework (2DFDR+) that provides significant power improvement over the conventional method and applies to a wide range of settings. 2DFDR+ uses marginal independence test statistics as auxiliary information to filter out less promising features, and FDR control is performed based on conditional independence test statistics in the remaining features. 2DFDR+ provides  valid inference from samples in settings where the conditional distribution of the genomic variables given the covariate of interest and the confounders is arbitrary and completely unknown. Promising finite sample performance is demonstrated via extensive simulations and real data applications.https://github.com/asmita112358/tdfdr.np.R codes and vignettes are available at  One central theme of genomic data analysis is identifying genomic features associated with a variable of interest, such as disease status. Due to the constraint of clinical sample collection, the variable of interest is often correlated with other variables, which may confound the associations of interest. For example, when identifying microbiome biomarkers for endometrial cancer, the age of patients acts as a confounder, as older patients tend to have more malignant tumors, and the female reproduction tract microbiome changes with age. Controlling for confounders is crucial for successful validation, cost reduction, and faster translation of discoveries to clinical tests. However, confounder adjustment in genome-scale association tests exacerbates the low statistical power and inflates the type I error rate.P-values for multiple testing using FDR control  distribution of the conditional and marginal independence test statistics of 2DFDR.It enables different methods for estimating the conditional distribution of the covariate given the confounders, including permutation/bootstrap, simulation, MCMC, and conditional GAN.It improves FDR control by explicitly modeling the relationship between the variable of interest and confounders, especially in the presence of strong confounding effects.q, we can show that in the worst-case scenario, the asymptotic power loss for 2DFDR+ compared to the 1D procedure is at most q, see Theoretical analysis demonstrates that 2DFDR+ provides asymptotic FDR control and retains the same number of rejections as the corresponding 1D procedure. A unique feature of 2DFDR+ is that it lets the data decide the usefulness/informativeness of the auxiliary statistic. If the auxiliary statistic provides helpful information, 2DFDR+ has significant power gain, otherwise, it reduces to the corresponding 1D procedure. When the FDR is controlled at level Section 2 describes the problem setup and a two-dimensional (2d) rejection region based on a primary statistic for testing the conditional independence between the omics feature and the covariate of interest given the confounders and an auxiliary statistic for testing the marginal independence between the omics feature and the covariate. Section 3 introduces an oracle FDR-controlling procedure, where the conditional distribution of the covariate given the confounders is assumed to be known. Sections 5 and 6 are devoted to numerical studies and real data analyses, respectively.n i.i.d. samples iY that are dependent of We formulate the feature selection problem by allowing the omics variables to depend on the covariate of interest and confounders arbitrarily. To state the problem and the procedure carefully, suppose we have for jY and Our idea to resolve this issue is to use two statistics jointly, namely a primary statistic for testing the conditional independence specified in m independent generalized linear models:As a motivation, we consider g is a known link function, j\u03b8 is the canonical parameter, for Associated with both the covariate of interest and confounders: Solely associated with the covariate of interest: Solely associated with the confounders: Not associated with either the covariate of interest or confounders: Dimension 1. Use the marginal independence test statistics to determine a preliminary set of features Dimension 2. Reject We note that: (i) Although marginal dependence does not imply conditional dependence, it can be leveraged to increase the signal density and reduce multiple testing burden in the second dimension. More precisely, the usefulness of the marginal dependence test is due tothe marginal dependence test statistics screen out a large number of noises in Category D and thus ease the multiple testing burden in the second dimension;the marginal dependence test statistics are more effective in detecting signals from Category B as the conditional dependence test causes over-adjustment, reducing the signal strength.Example 1.Z and \u03b40 denotes a point mass at 0. t-statistic for testing t-statistic for testing MT) statistic against the conditional (CT) statistic for various confounded scenarios. The standard approach performs (1D) FDR control based on the conditional statistic (CT) only (we refer it as 1DFDR). When the correlation between the variable of interest and the confounder . For 2DFDR+, it first uses MT to exclude a large number of irrelevant features . Next, a lower cutoff  is used to achieve the same FDR level. As a result, it achieves significant power improvement, and the improvement increases with the correlation between the variable of interest and the confounder.Consider the following data generating process:We illustrate the rational behind 2DFDR+ through the following example. A detailed description of the method is provided in the next section.We introduce an oracle FDR-controlling procedure, where we assume that the conditional distribution of t1, t2) such that the FDR is controlled at a desired level while the number of rejections is as large as possible. Let Our goal here is to develop a principled way of finding the cutoff values  be the jth order statistics of\u2002(and\u2002). Given some integer\u2002, we define\u2002and\u2002, where\u2002denotes the integer part of a.Compute\u2002where the maximization is over all\u2002with\u2002[see]. Reject the jth hypothesis if\u2002and\u2002which can be regarded as John Storey\u2019s version of the 2DFDR+ procedure.Theorem 1.UnderAssumptions 1\u20133 in the where\u2002is defined inEquation (2).To state the main theorem, we use a list of assumptions defined and justified in the Corollary 1.Let\u2002be the corresponding value of\u2002based on\u2002sampled from\u2002. Defineand the corresponding cutoffs as:Then underAssumptions 1\u20135 in the Supplementary Material,In practice, Additional theoretical results on FWER control and power analysis for the algorithm have been relegated to the j\u03b1 and j\u03b2 independently over j from the mixture distribution \u03b40 denotes a point mass at 0. We vary the following factors in the simulations:We conduct comprehensive simulations to evaluate the performance of 2DFDR+ and compare it to competing methods. We generate \u03c1 determine the strength of association between \u03c1 in each simulated model.Degree of confounding: Let Signal density: Signal effect: We report the empirical FDR and power averaged over 100 simulation runs for all possible combinations of the three factors.We compare the finite sample performance of the following seven methods.t-statistics for testing z-statistic  corresponding to the coefficient of X for a full model fit.MS-1DFDR: The 1D procedure based on the RV-1DFDR: The 1D procedure based on the conditional RV coefficient. To account for the potential nonlinearity in the underlying relationship between HSIC-1DFDR: The 1D procedure based on the cHSIC described in 2DFDR: The 2DFDR procedure proposed in t-statistics for testing MS-2DFDR+: The proposed 2DFDR+ procedure with RV-2DFDR+: The proposed 2DFDR+ procedure with HSIC-2DFDR+: The proposed 2DFDR+ procedure with The 1D procedure can be viewed as a special case of the corresponding 2d procedure by forcing the cutoff of the auxiliary statistic to be zero. As the 2d procedure is searching over a larger rejection region , the proposed 2d procedure is guaranteed to make more rejections in finite sample.n to be 100, and the number of hypotheses m (i.e. the number of features) to be 1000. In the main paper, we consider two cases, remaining cases and detailed models have been discussed in Throughout the simulations, we set the sample size Linear/Nonlinear models with continuous X and Z.X and Z are associated with each other through the following model:\u03c1 controls the degree of confounding and X to dissociate any possible entanglement between signal strength and the degree of confounding. This type of simulation setup has been used in Models 1\u20134 to explore the effect of the relations among X, jY, and Z on the FDR and power. The empirical FDR and power of RV-1DFDR, HSIC-1DFDR, 2DFDR, RV-2DFDR+, and HSIC-2DFDR+ are summarized in where Binary response. The following logistic regression model has been considered:with X and continuous Z; (2) Linear models with discrete X and Z; (3) Count response; (4) FWER control; (5) global null; (6) dependent errors, and (7) separating the effects of the densities of the signal of interest and the confounder signal.In X and Z, when the underlying models between Y and , and X and Z are both linear . The microbiome composition was profiled using 16S rRNA gene-targeted sequencing and processed using the QIIME bioinformatics pipeline  in a linear regression model with the IR and body mass index (BMI) being the predictors. The numbers of rejections at different FDR levels are shown in For illustration purposes, the OTU abundances were treated as both continuous and binary outcomes. The results for the binary outcomes are given in the m\u2009=\u20091201). The BMI of a subject is a confounding factor as the IR of a subject is largely influenced by the BMI , No Pouchitis, Acute Pouchitis, Chronic Pouchitis, and Crohn\u2019s Disease like Inflammation. As the variable of interest is nominal, we did not use the RV coefficients in this case. Finally, we consider a Pouchitis dataset  for performing multiple hypotheses testing while adjusting for confounding effects. Within this new framework, the conditional distribution of the omics features given the variable of interest and confounders can be arbitrary and completely unknown. The framework is flexible by allowing the joint use of any conditional and marginal independence tests, continuous/binary/count/multivariate responses, and various ways of modeling the conditional distribution of the variable of interest given the confounders. As a general methodology, 2DFDR+ can be applied to multiple types of omics data. In view of the numerical results, we recommend using RV-2DFDR+ (based on the spline-transformed variables) under most scenarios due to its robustness and efficiency. In cases where the RV-based statistics are not applicable, for instance, when either of btad563_Supplementary_DataClick here for additional data file."}
+{"text": "Giardia lamblia is the causative agent of giardiasis, a neglected infectious disease in humans. In this study, we used a bioinformatics approach to examine the structural features of GTOR, a G. lamblia TOR-like protein, and predict functional associations. Our findings confirmed that it shares significant similarities with functional TOR kinases, including a binding domain for the FKBP-rapamycin complex and a kinase domain resembling that of phosphatidylinositol 3-kinase-related kinases. In addition, it can form multiprotein complexes such as TORC1 and TORC2. These results provide valuable insights into the structure\u2013function relationship of GTOR, highlighting its potential as a molecular target for controlling G. lamblia cell proliferation. Furthermore, our study represents a step toward rational drug design for specific anti-giardiasis therapeutic agents.TOR proteins, also known as targets of rapamycin, are serine/threonine kinases involved in various signaling pathways that regulate cell growth. The protozoan parasite  TOR proteins, also known as targets of rapamycin, are a class of Ser/Thr kinases that play critical roles in regulating cell growth by integrating environmental and nutritional signals. This kinase family is conserved from yeast to humans and comprises proteins with a canonical domain organization: HEAT\u2013FAT\u2013FRB\u2013PIKKc\u2013FATC ,2,3. RapGiardia lamblia is the protozoan parasite that causes human giardiasis, an intestinal infection that can lead to severe diarrhea. It ranks among the top ten human parasites worldwide [orldwide ,9,10. Giorldwide . Moreoveorldwide .Metronidazole and other nitroimidazole derivatives are usually prescribed to treat human giardiasis ,14. HoweG. lamblia TOR protein (GTOR) using a biocomputational approach to gain further insights into its structural features and potential for inhibition. Our research findings demonstrated that it has domains with a highly similar structure to that of functional TOR kinases. Consequently, it seems feasible to presume that GTOR represents a viable target for developing specific therapeutic agents using a rational approach such as structure-based drug design.A critical initial step toward developing new or improved drugs to treat infectious diseases is identifying reliable molecular targets, such as unique virulence factors or well-known proteins involved in essential processes ,18,19. TG. lamblia. Primary structure analysis revealed that GTOR exhibits a domain organization similar to that of active TOR kinases  to its human and yeast counterparts. Further primary and secondary structural analyses revealed two known motifs, catalytic and activation loops, and the expected LST8 interface . MoreoveHomology-based modeling provided additional insights into the structure\u2013function relationship of the PIKKc domain. The best model A confirmG. lamblia metabolism and cell proliferation.Inhibition of the kinase activity of TOR proteins is a feasible approach for blocking the functions of both TORC1 and TORC2 complexes, as evidenced by promising results from studies on treating certain types of cancers with ATP-competitive inhibitors of mTOR kinase activity ,27,28,29G. lamblia proteins involved in TOR-linked signaling pathways  network provided further data regarding the ability of GTOR to bind or interact with putative TORC components or other pathways .G. lamblia.GTOR can potentially interact with various proteins, including two putative TORC components, LST8- and RAPTOR-like proteins, and a PPIase , whiG. lamblia, denoted as GTORC1 and GTORC2. Both complexes must contain GTOR (the only TOR-like protein encoded by this human protozoan). Moreover, as observed in their mammalian counterparts [G. lamblia.Supplementary biocomputational analyses provided further information regarding TOR complexes in terparts , they coG. lamblia cells with 36 \u03bcM rapamycin reduced encystation, the process by which the parasite evolves from the replicative form (trophozoite) to the dormant stage (cyst) and induces cell death at higher concentrations (EC50 of 65\u201370 \u03bcM) [Several studies have provided insights into the structural and functional regulation of TOR signaling, its multiprotein complexes, and the crosstalk with other signaling pathways ,35,36,375\u201370 \u03bcM) . Althoughttps://www.uniprot.org/; accessed on 1 July 2022) [https://web.expasy.org/protparam/; accessed on 1 July 2022) [https://prosite.expasy.org/scanprosite/; accessed on 1 July 2022) [http://cbdm-01.zdv.uni-mainz.de/~munoz/rep/; accessed on 2 July 2022) [https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml; accessed on 1 July 2022) [https://pfam.xfam.org/; accessed on 2 July 2022) [https://www.ebi.ac.uk/Tools/msa/clustalo/; accessed on 5 July 2022) [https://toolkit.tuebingen.mpg.de/; accessed on 5 December 2022) [The GTOR polypeptide sequence (entry code A8BIV9) was obtained from UniProtKB (ly 2022) . The phyly 2022) , while tly 2022) ,45,46. Tly 2022) , while tly 2022) ,49. The ly 2022) , and thely 2022) . The secer 2022) .First, a comparative analysis of the 3D models generated using different resources for protein structure prediction, I-TASSER ,54, IntFhttps://zhanggroup.org/I-TASSER/; accessed on 10 December 2022), one of the most widely used servers for automatic homology-based modeling. The top-ranked 3D structures were further improved using ModRefiner and FG-MD on the I-TASSER server. The accuracy of the best 3D structures was validated utilizing MolProbity , which combined the all-atom contact analysis with updated versions of more traditional tools to validate geometry and dihedral angle combinations [https://prosa.services.came.sbg.ac.at/; accessed on 12 December) were used for further validation. Unless otherwise stated, 3D structures were analyzed using the UCSF Chimera as a molecular visualization system [The 3D structure of two conserved and potentially functional GTOR domains (FRB and PIKKc) was predicted using I-TASSER , which predicts the binding site of target proteins through comparisons with ligand-containing PDB templates [https://zhanggroup.org/COACH/; accessed on 15 January 2022), a consensus approach to ligand binding site prediction that combines the results of five individual algorithms via the support vector machine (SVM) training [https://prankweb.cz/; accessed on 24 January 2022), a machine learning-based method for the prediction of ligand binding sites from protein structures [The putative rapamycin binding site was detected by primary and tertiary structure analysis of the FRB domain (2717\u20132815 residues) utilizing three bioinformatics tools. The IntFOLD suite . The STRING database collects, scores, and integrates all available data on known and predicted PPIs [https://www.ebi.ac.uk/interpro/; accessed on 20 February 2023) [The potential PPI partners of GTOR were detected using the STRING web resources  ."}
+{"text": "No significant difference in survival rates was observed between groups . LGL lymphocytosis was observed in almost 10% of autologous HSCT recipients. In contrast to allogeneic HSCT, the duration of LGL was shorter and no significant improvement in survival was observed.Expansion of large granular lymphocytes (LGLs) is sometimes observed in allogeneic hematopoietic stem cell transplantation (HSCT) recipients, and is reported to be associated with a favorable transplant outcome. LGLs are also observed after autologous HSCT, but their clinical implications have not been well investigated. We retrospectively reviewed peripheral blood smears of consecutive autologous HSCT recipients. LGL lymphocytosis was defined as the observation of LGLs in the peripheral blood (>\u200920% white blood cells) in at least two consecutive blood tests. We evaluated the clinical impact of LGL lymphocytosis on autologous HSCT recipients. LGL lymphocytosis was observed in 18 of 197 patients (9.1%) who received autologous HSCT, at a median of 49\u00a0days after transplantation, with a median duration of 120.5\u00a0days. Incidence of cytomegalovirus reactivation was significantly higher in patients with LGL lymphocytosis than those without (16.7% vs. 3.3%,  Large granular lymphocytes (LGLs) are characterized by an abundant cytoplasm with azurophilic granulation, composed of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). LGLs have been reported to increase in various settings, including viral infection , during In addition to these well-known causes of increased LGLs, LGL lymphocytosis was reported in various clinical settings in patients with hematologic malignancies , 8. AmonWe retrospectively reviewed the peripheral blood smear of consecutive patients who underwent their first autologous HSCT at the Department of Hematology & Oncology of the University of Tokyo Hospital between January 2007 and April 2021. LGL lymphocytosis was defined as the predominance of large lymphocytes containing typical azurophilic granules with a reniform or round nucleus with mature chromatin in the peripheral blood (>\u200920% WBCs) at least two consecutive blood tests. Some samples were subjected to immunophenotyping by flow cytometry. LGLs with CD3\u2009+\u2009, CD8\u2009+\u2009, and CD57\u2009+\u2009phenotypes were defined as T-LGLs, whereas those with CD3\u2013CD16\u2009+\u2009and CD56\u2009+\u2009phenotypes were defined as NK-LGLs .p value of\u2009<\u20090.05 was considered statistically significant. EZR  was used for statistical analysis [The day of stem cell infusion was defined as day 0, and overall survival (OS) and progression-free survival (PFS) were defined to be calculated from day 0. We analyzed the association between LGL appearance and patients\u2019 clinical characteristics  for aggressive lymphoma, ISS  for myeloma, HCT-CI (hematopoietic cell transplantation-specific comorbidity index), CMV serostatus, infused CD34\u2009+\u2009cell dose, febrile neutropenia, viral infection) using Fisher\u2019s exact test and Student\u2019s t-test, whereas OS and PFS probabilities were compared using the log-rank test. CMV reactivation was defined as the detection of three or more CMV pp65 antigen-positive cells on two glass slides using CMV C10/C11 assay after autologous HSCT in patients who CMV IgG positivity had been confirmed before transplantation. Febrile neutropenia was defined as a condition marked by fever (axillary temperature 37.5\u2103 or higher) during a period of neutropenia (<\u2009500/\u00b5L) after autologous HSCT. To eliminate immortal bias, landmark analysis was performed; LGL expansions were defined as the appearance of LGLs within 100\u00a0days from transplantation, and patients who died within 100\u00a0days from transplantation were excluded from the analysis. PFS was analyzed excluding patients whose disease progressed within 100\u00a0days after transplantation. Only the first autologous HSCT was included and was censored when the patient received the 2nd autologous HSCT. However, in the case of tandem transplantation for MM, the 2nd transplantation had been planned in advance and was performed not for relapse, and was not censored in the analysis. A analysis . The stuA total of 197 patients who received their first autologous HSCT and survived\u2009>\u2009100\u00a0days from transplantation were identified. The median follow-up was 4.7\u00a0years , and 3-year OS and PFS were 90.8%  and 74.7% (95% CI 67.4\u201380.5%), respectively. Diseases included 113 aggressive lymphomas, 23 indolent lymphomas, 7 Hodgkin lymphomas, 48 multiple myelomas, 4 acute promyelocytic leukemias and 2 POEMS  syndromes. Ten MM and one DLBCL patient underwent the 2nd autologous HSCT; among MM patients, 4 were tandem transplantation.p\u2009=\u20090.392). LGL expansion was significantly correlated with the high or high-intermediate risk of the international prognostic index (IPI) in lymphoma . CMV reactivation was checked in 135 out of 197 patients and observed at a median of 30.1 (range 10\u201337) days from transplantation in the overall cohort. CMV reactivation post-transplantation was significantly frequently observed in patients with LGLs . Among them, one and two cases of CMV colitis were observed in patients with and without LGLs, respectively. Febrile neutropenia occurred at a median of 5 (range 0\u201326) days from transplantation. Although there was no significant difference, the incidence of febrile neutropenia tended to be frequently observed in patients with LGLs . Notably, all these outcomes including febrile neutropenia and CMV reactivation were observed before the LGL appearance. The median periods from CMV reactivation and FN to LGL appearance were 7 (range 0\u201314) days and 42.5 (range 16\u201391) days, respectively. Eleven patients among 48 MM cases had started lenalidomide maintenance therapy within 100\u00a0days after autologous HSCT, and LGL lymphocytosis was observed in two of them (18%). One patient developed treatment-refractory LGL leukemia 3.8\u00a0years after autologous HSCT for DLBCL. The LGL phenotypes were determined by flow cytometry of the peripheral blood in six patients: three with T-cell phenotypes and three with the coexistence of T- and NK-cell phenotypes.Eighteen patients 9.1%) were found to have persistent LGLs in the peripheral blood within 100\u00a0days of transplantation. Their diseases included nine diffuse large B-cell lymphomas (DLBCL), two mantle cell lymphomas, one follicular lymphoma, one Hodgkin lymphoma and five multiple myelomas. Between cases with and without LGLs, there was no significant difference in the ratio of lymphoma to MM and aggressive to indolent lymphoma, or whether the objective of transplantation was upfront consolidation or salvage at relapse. LGL expansion was observed in none of the four patients who underwent tandem transplantation for MM. LGLs appeared at a median of 49 (range 20\u201393) days post-transplantation, and all patients showed LGL expansion after engraftment . The median duration of LGL appearance was 120.5 (range 3\u20131373) days. Baseline characteristics between autologous HSCT recipients are shown in Table % were fop\u2009=\u20090.793; 3-year PFS 82.5 vs. 77.5%, p\u2009=\u20090.803, respectively). Similarly, in patients with multiple myeloma, no significant differences in OS and PFS were observed between patients with LGL expansion and the control .The outcomes are shown in Fig.\u00a0This is the first study to explore the clinical features and prognoses of patients with LGL lymphocytosis post-autologous HSCT. LGL lymphocytosis was confirmed in 9.1% of patients undergoing autologous HSCT, and CMV reactivation and febrile neutropenia were more frequently detected in the cases with LGLs. The effect of LGL appearance on the prognosis of hematologic malignancies after autologous HSCT was not observed.The time to LGL appearance and its duration were both shorter than those of allogeneic HSCT , which mAs clinical outcomes, OS or PFS improvement was not observed in autologous HSCT recipients with LGL lymphocytosis in either malignant lymphoma or multiple myeloma, which was inconsistent with allogeneic HSCT recipients. One possible reason may be that the shorter duration of LGL lymphocytosis than in allogeneic HSCT might have attenuated its impact on disease prognosis. Another reason may be the difference in antigen stimulation. In contrast to allogeneic HSCT, there was no alloantigen in autologous HSCT. It was possible that alloantigen stimulation was necessary for expanded LGLs to exert the anti-tumor effect.Notably, among patients with malignant lymphoma, the proportion of patients with LGL expansion was significantly higher in patients with higher IPI, which suggested a population with a poor prognosis. Considering the similar prognosis with or without LGLs in patients with malignant lymphoma, increased LGLs may have potentially improved the prognosis in malignant lymphoma. Further large-scale studies were needed to confirm this notion. The possible mechanism for the correlation between higher IPI and LGL lymphocytosis may be that patients with higher IPI tend to be older and with advanced stages based on its scoring system, which are also risk factors for infection . TherefoAs a mechanism for the LGL appearance, immune stimulation by CMV reactivation was estimated. Furthermore, the fact that febrile neutropenia was more frequently observed before the appearance of LGLs suggests that evident infections and some other undiagnosed subclinical infections stimulate the cytotoxic immune reaction in those immunosuppressed hosts, which leads to LGL appearance. Additionally, in our institution, evaluation of CMV reactivation is not routinely performed, but frequently monitored especially in cases with undetermined fever. Because of this, the expansion of LGLs after CMV reactivation may partially be affected by other undetermined infections. Although alloantigen stimulation does not exist unlike in allogeneic HSCT, it is suspected that immune reconstruction after autologous HSCT might stimulate LGL expansion, which should be investigated further in the future.This study has some limitations. First, phenotypes and clonality of LGLs were evaluated in a limited number of patients, and the immunological characteristics remain unclear. Moreover, Because the reconstruction of NK cells and CD8\u2009+\u2009T cells, which constitute LGLs, is reported usually earlier than that of CD4\u2009+\u2009T cells and B cells , the effIn conclusion, LGL lymphocytosis was confirmed in 9.1% of patients undergoing autologous HSCT. In contrast to allogeneic HSCT recipients or patients during TKI therapy, LGL expansion was not significantly associated with preferable outcomes in autologous HSCT recipients."}
+{"text": "Mastitis is one of the most common diseases of the dairy industry and with it brings important economic losses. The most prevalent form of the disease is subclinical mastitis, which leads, in the absence of clinical signs, to decreased milk production, increased somatic cell count, and an increased risk of clinical mastitis during lactation. With the increasing public health concerns for antimicrobial use and its relationship with the development of antimicrobial resistance, nation-specific regulations and general pressure to reduce group-level prophylactic use of antimicrobials have been established. Selective dry therapy reserves antimicrobial treatment for cows or quarters suspected of having an intramammary infection. The treatment is administered after the last milking, while uninfected cows or quarters do not receive antibiotherapy. Since selective dry cow therapy was introduced, different methods of selecting infected cows or quarters have been reported. The aim of this article is to describe the management of mastitis in dairy cows and the main tools for its diagnosis, with a specific focus on on-farm instruments.Mastitis is one of the most important diseases in dairy cattle farms, and it can affect the health status of the udder and the quantity and quality of milk yielded. The correct management of mastitis is based both on preventive and treatment action. With the increasing concern for antimicrobial resistance, it is strongly recommended to treat only the mammary quarters presenting intramammary infection. For this reason, a timely and accurate diagnosis is fundamental. The possibility to detect and characterize mastitis directly on farm would be very useful to choose the correct management protocol. Some on-field diagnostic tools are already routinely applied to detect mastitis, such as the California Mastitis Test and on-farm culture. Other instruments are emerging to perform a timely diagnosis and to characterize mastitis, such as Infra-Red Thermography, mammary ultrasound evaluation and blood gas analysis, even if their application still needs to be improved. The main purpose of this article is to present an overview of the methods currently used to control, detect, and characterize mastitis in dairy cows, in order to perform a timely diagnosis and to choose the most appropriate management protocol, with a specific focus on on-farm diagnostic tools. Mastitis, the inflammation of the mammary gland, is one of the most frequent diseases affecting dairy cows worldwide. It is responsible for approximately 60\u201370% of all antimicrobials administered on dairy farms ,2. It leIt also represents a public health concern, considering that approximately 62% of isolated mastitis-causing agents are resistant to at least one antimicrobial agent, and that some of them are zoonotic agents ,8,9. ForMastitis can be classified, according to its etiology, into environmental and contagious or, according to symptoms, into clinical and subclinical .Being a multifactorial disease, mastitis susceptibility is given by multiple factors such as age, parity, lactation stage, milk yield, and udder anatomical dispositions. Two of the main factors are immunological condition and reactivity of the mammary gland. Consequently, the clinical manifestations as well as its further course depend on the interplay between the innate resistance and adaptive immunity of the dairy cow and the type, concentration, and virulence of udder pathogens .Staphylococcus aureus, Streptococcus agalactiae, and Mycoplasma bovis. Environmental mastitis pathogens include a wide range of organisms, such as coliforms , environmental streptococci , Trueperella pyogenes, non-aureus staphylococci (NAS), and others such as Pseudomonas, Proteus, Serratia, Aerococcus, Listeria, yeast, and Prototheca ..101].Recently, several studies have also established a significant positive correlation between SCC and udder temperature. Performing thermal imaging, it is important to consider that IRT images can be influenced by several factors, such as mechanical brushing of the udder, direct solar radiation and wind speed, parity, stage of lactation, and pregnancy. It has also been shown that the temperature of the thermal images of the forequarters region of the udder had a greater correlation with SCC. This is because the rear region of the udder is more exposed to climatic variables and physical damage during milking, which may overestimate udder temperature during the IRT analysis. For this reason, it is fundamental to calibrate the thermal imager and to establish a standardized distance and acquisition method ,102. ComNowadays, mastitis still represents one of the most frequent diseases of dairy cows, having well-recognized detrimental effects on animal wellbeing and farming economy, and being one of the main causes of antimicrobial use in dairy farms. Due to the growing concern for antimicrobial resistance, regulations have been implemented nationally and internationally to reduce unnecessary antibiotic use. Not all IMIs require antibiotic treatment, and their administration must be based on the culture and sensitivity results rather than empirical therapy. Moreover, therapy success depends on several factors such as causal agent, parity, stage of lactation, other systemic diseases, and mammary parenchyma alterations. The management of mastitis must be under constant and continuous control of the veterinarian. This could certainly be facilitated by the possibility of establishing which animals are prone to have an improvement in treatment success; an improved treatment success would also result in a reduction in antimicrobial administration. Some on-farm diagnostic tools are already being routinely used to apply SDCT, such as CMT and OFC. IRT is a noninvasive and rapid method and can be of great value in early mastitis detection for optimum response from treatment. Even if it is an indirect measure, it has relatively good sensitivity, specificity, and positive and negative predictive values. Additionally, it can perceive changes in skin surface temperature in response to varying degrees of severity of the mammary gland infection. Ultrasound mammary evaluation has proved to be effective in identifying the presence of lesions or alterations of the mammary parenchyma and the teat. Such alterations might affect the prognosis of the animal. Also, blood gas analysis would provide interesting parameters to evaluate on farm and in real time the clinical conditions of the affected cows. These diagnostic techniques are still emerging in this field, and their application to detect mastitis and establish whether to treat a case of mastitis or not certainly has to be improved. Further studies are needed in order to establish objective parameters of detection and prognostic index."}
+{"text": "COVID-19 disproportionately affects those with preexisting conditions, but little research has determined whether those with chronic diseases view the pandemic itself differently - and whether there are differences between chronic diseases. We theorized that while individuals with respiratory disease or autoimmune disorders would perceive greater threat from COVID-19 and be more supportive of non-pharmaceutical interventions (NPIs), those with autoimmune disorders would be less likely to support vaccination-based interventions.We conducted a two-wave online survey conducted in February and November 2021 asking respondents their beliefs about COVID-19 risk perception, adoption and support of interventions, willingness to be vaccinated against COVID-19, and reasons for vaccination. Regression analysis was conducted to assess the relationship of respondents reporting a chronic disease and COVID-19 behaviors and attitudes, compared to healthy respondents adjusting for demographic and political factors.In the initial survey, individuals reporting a chronic disease had both stronger feelings of risk from COVID-19 as well as preferences for NPIs than healthy controls. The only NPI that was still practiced significantly more compared to healthy controls in the resample was limiting trips outside of the home. Support for community-level NPIs was higher among individuals reporting a chronic disease than healthy controls and remained high among those with respiratory diseases in sample 2. Vaccine acceptance produced more divergent results: those reporting chronic respiratory diseases were 6% more willing to be vaccinated than healthy controls, while we found no significant difference between individuals with autoimmune diseases and healthy controls. Respondents with chronic respiratory disease and those with autoimmune diseases were more likely to want to be vaccinated to protect themselves from COVID-19, and those with an autoimmune disease were more likely to report fear of a bad vaccine reaction as the reason for vaccine hesitancy. In the resample, neither those with respiratory diseases nor autoimmune diseases reported being more willing to receive a booster vaccine than healthy controls.It is not enough to recognize the importance of health in determining attitudes: nuanced differences between conditions must also be recognized.The online version contains supplementary material available at 10.1186/s13223-023-00791-6. The COVID-19 pandemic disproportionately affects individuals with comorbidities , but comUnderstanding acceptance of NPIs and vaccination is critical to ongoing response efforts, especially as uptake of vaccines and boosters has stalled. In earlier phases of the pandemic concerns were raised about the risk of developing severe COVID-19 for individuals with chronic respiratory and autoimmune diseases. The latter\u2019s potential contraindication for vaccine candidates was also of concern, and these individuals remain under-addressed in CDC vaccine guidance due to lack of data  but are Our study identifies the relationship between an individual\u2019s self-identified chronic illness, how they perceive COVID-19 risks, engage with individual and community-level NPIs, and weigh the benefits and risks of vaccination in the US context. Self-identification is important as these categories do not strictly track medical diagnoses, but rather the relationship between medical indications, physical or cognitive capacities, and social function . For exaOur follow-up survey, conducted in the same population, assessed participants\u2019 fatigue with the pandemic, their continued willingness to undertake individual and community-level NPIs, vaccine enthusiasm, and the persistence of these beliefs from pre- to post-COVID-19 vaccine availability. We hypothesized that those with chronic respiratory and autoimmune disease would be both more concerned about COVID-19 and more likely to support risk-mitigation than healthy individuals. Conversely we posited that those with autoimmune disease would be less likely to vaccinate due to the risk of possible symptom exacerbation post vaccination , 16, theParticipants were contacted through Prolific\u2019s survey platform, which recruits a large and diverse pool of potential participants through social media, physical flyers, and referrals. While this is a non-probability convenience sample, it is more diverse than most convenience samples and researchers have successfully replicated established studies through the platform , 19. UnlIn both samples, participants were asked about their beliefs about the risk of COVID-19 to their health, to the public\u2019s health, and whether the risk of COVID-19 is overblown. These risk perceptions were collected on a five-point scale ranging from \u201cstrongly agree\u201d to \u201cstrongly disagree,\u201d and then normalized to a  interval where 1 indicates strong agreement. The full survey is available in the Supplement. In the resample, we additionally assessed \u2018pandemic fatigue.\u2019 First, questions based on Johansson et al.\u2019s mental fatigue scale  assessedAdoption of individual risk mitigation measures were collected on a six-point true/false scale normalized to a  interval where 1 indicates greatest adoption of that measure. Individual risk mitigation measures were reducing trips; mask wear; working from home; handwashing; and maintaining physical distance. In the resample, participants were asked about the same set of individual risk mitigation measures, excluding working from home. Approval of community-level NPIs  were measured on a six-point approve/disapprove scale, normalized to a  interval where 1 indicates strong approval. In the resample, participants evaluated the same set of policies, with the addition of two questions about a vaccine mandate and vaccine \u2018passports\u2019.In the first sample, respondents indicated whether they were willing, unwilling, or had already received a COVID-19 vaccine on a seven-point scale, then provided reasons for their vaccine decision. For those willing to be or already vaccinated, reasons included protecting themselves, protecting others, belief the vaccine had been fully tested, the vaccine was safe, a desire to get back to normal, or a need to be vaccinated for work or other activities. For those unwilling to get a vaccine, potential reasons included belief that COVID-19 is not a serious health threat, concern about a bad reaction to the vaccine, belief the vaccine had not been fully tested, the vaccine was not safe, or opposition to vaccines in general. Participants could select multiple reasons or write their own. Willingness to receive the vaccine was normalized to a  interval where 1 indicates either that participants would \u201cdefinitely get the vaccine\u201d or had already been vaccinated. In the resample, participants were asked if they were vaccinated, unvaccinated, or partially vaccinated. They then indicated whether they were willing, unwilling, or had already received a COVID-19 vaccine booster on a seven-point scale.Age, race/ethnicity, sex, household income, educational level, geographic region, rural or urban residence, partisanship, employment, and education demographics were collected for the first sample Table\u00a0. These rWe conducted regression analyses to evaluate the association between disease status and COVID-19 behaviors or attitudes, as well as the change in attitudes by disease from survey 1 to survey 2 using R version 4.0.2 and STATA version 17 , controlRegression coefficients and odds ratios can be seen in Table\u00a0R)\u2009=\u20090.12, 95% confidence interval (CI)\u2009=\u20090.10\u20130.15; Autoimmune (BA)\u2009=\u20090.11, CI\u2009=\u20090.08\u20130.14) and to a lesser extent the public\u2019s health . In both cases, the effect of disease status on perception of threat to the respondent was significantly higher than the effect on perception of threat to the public . Respondents reporting chronic respiratory or autoimmune diseases were also less likely than healthy controls to think the threat of COVID-19 was overblown . In the resample, while both groups still thought that COVID-19 was a threat to themselves , those with autoimmune disease were no longer more likely to believe it was a threat to the public\u2019s health, and neither group was any less likely to believe that that COVID-19 was overblown compared to healthy controls. Both chronic condition groups felt more emotional/mental fatigue than healthy controls , but neither felt more need to get back to \u201cnormal\u201d.In the initial sample, respondents reporting chronic respiratory or autoimmune diseases were significantly more likely than healthy controls to report that COVID-19 was a threat to themselves , physically distance , and decrease trips outside the home . Interestingly, the only NPI that was still practiced significantly more compared to healthy controls in the resample was limiting trips outside of the home .Acceptance of NPIs in the first sample was broadly concordant with COVID-19 risk perceptions: individuals reporting a chronic disease had stronger preferences for NPIs. They were more likely to wear masks outside the home , broad lockdowns , mask mandates , and school closures . Only limits on in-person worship services diverged: while respondents reporting chronic respiratory diseases were significantly more likely than controls to support limits , individuals reporting autoimmune diseases were no more or less likely to prefer those limits than healthy controls . In the resample, those with respiratory diseases still supported limits on in-person worship services, mandatory mask wearing in public, and school closures, and also supported vaccine mandates  and passports , but those with autoimmune diseases showed no difference in support compared to healthy controls.Support for community-level NPIs was higher among individuals reporting a chronic disease than healthy controls and remained high among those with respiratory diseases in sample 2. In sample 1, both groups were more likely to support prohibitions on indoor dining . When assessing reasons for being willing or having been vaccinated, respondents with chronic respiratory disease and those with autoimmune diseases were more likely to want to be vaccinated to protect themselves from COVID-19 . Respondents reporting a chronic respiratory disease were also more likely to want to safely return to work . Individuals with autoimmune diseases were the only group to have a significant association with a particular cause for vaccine hesitancy: they were more likely to report fear of a bad vaccine reaction as the reason for unwillingness . Respondents with autoimmune diseases were also less likely to say that their unwillingness was due to not seeing COVID-19 as a threat . In the resample, neither those with respiratory diseases nor autoimmune diseases reported being more willing to receive a booster vaccine than healthy controls.Vaccine acceptance produced more divergent results. Respondents who reported chronic respiratory diseases were 6% more willing to be vaccinated than healthy controls (CI\u2009=\u20090.03\u20130.09), while we found no significant difference between individuals with autoimmune diseases and healthy controls (Bitself was rarely responsible for changing attitudes and behaviors (Table\u00a0Acceptance of certain behaviors within disease groups did change from sample 1 to sample 2, but disease status rs Table\u00a0. For exaOver two nationwide surveys, we found individuals with self-reported chronic respiratory or autoimmune conditions were significantly more likely to be concerned about COVID-19\u2019s threat to the public and, to a significantly greater extent, more concerned about their personal threat from COVID-19 compared to respondents without a chronic illness. This highlights the significant internalization of risk messaging in these communities which could provide a basis for choosing strategies to communicate public health information based on self or community interests.Compared to studies evaluating individuals with medically confirmed chronic illnesses , our resThe relationship between chronic illness and identity may be important as part of policies directed at changing health behaviors. Chronic illness and disability, when viewed as a medical indication, may diverge from an individual\u2019s experience of barriers to social participation, membership in a community, or as part of collective action to achieve policy aims . AppealiIndividuals reporting chronic respiratory diseases, but not autoimmune diseases, were more willing to receive their primary vaccine series than controls. While few vaccine uptake studies have focused on individuals with respiratory diseases, one study found that individuals with medically confirmed autoimmune diseases were equally willing to be vaccinated as healthy controls .Among those willing or already vaccinated in the initial survey, both chronic disease groups reported wanting to protect themselves as a motivating factor for vaccination, confirming previous COVID-19 vaccine attitude studies , 27\u201329. There was no difference in willingness to get a booster between either disease group and healthy controls. Recent work has argued that elevated risks among individuals with chronic illnesses will likely affect their booster vaccine intentions , but we The heterogeneity in key aspects of health behaviors, most significantly in vaccination, is an important finding for individual and public health communication. If specific patient populations are more likely to be vaccine hesitant than others, medical specialty groups could focus on patient engagement for vaccine uptake earlier in a pandemic. The Kaiser Family Foundation has noted that for those Americans who are still \u201cwait and see\u201d over the decision to be vaccinated , the effOver time, these trends were maintained or became more pronounced. Those with chronic illnesses were more fatigued by the pandemic, but less likely to believe it was time to get back to normal. Thus, while pandemic fatigue is defined by the WHO as \u201cexpress[ing] itself as emerging demotivation to engage in protective behaviors and seek COVID-19-related information and as complacency, alienation and hopelessness,\u201d  our findBoth chronic illness groups remained more likely to believe COVID-19 was a threat to their personal health but diverged in what they believed should be done about it. Those with respiratory disease viewed COVID-19 more as a general threat, to be controlled with vaccination, masking, and limits on gatherings. Those with autoimmune disorders, however, viewed COVID-19 more as a personal threat, to be avoided with fewer trips outside the home. While those with respiratory disease were more likely to be fully vaccinated against COVID-19 than healthy controls, those with autoimmune disorders were not. When we controlled for disease status and response to survey 1, we found that behavior and attitude change was not dependent on having a chronic disease.Adherence to or support for NPIs are self-reported. Social pressure on respondents to report greater adherence or support may have influenced responses at the time of sample. We note however that the measurement of relative difference between groups was highly significant across a variety of NPIs. It is difficult to imagine why these pressures would differ between groups; or why social pressures would be greater on individuals reporting chronic diseases than those without to the point that they confound otherwise insignificant results.Representativeness of our sample may be limited as we utilized a convenience sample using quotas based on reported disease state. It would be extremely difficult to recruit sufficient chronic disease populations while maintaining representative sampling, however. This represents a necessary trade-off between our ability to test our key hypotheses about chronic respiratory and autoimmune diseases, and our ability to make statements about the general population. Samples also reflect the real-world social dimensions of these diseases, such the higher prevalence of these diseases among women.Finally, when we compared the demographics of participants in survey 2 and survey 1 who did not participant in the resample, individuals with autoimmune diseases, non-Hispanic whites, women, people in the south, older respondents, and Republicans were more likely to participate in the second survey, while we were less likely to recontact students and Asian-Americans. Our analysis controls for these factors, however, and so the slight imbalance is unlikely to have impacted our results significantly.This research provides insight into how vulnerable individuals conceive of COVID-19 risk and adjust their behavior based on their disease status, which has implications for patient care and public health in general. The relationship between disease and acceptance of NPIs can shape how practitioners build support with individuals and communities for social and personal pandemic interventions. In maintaining public health measures in the long term for COVID-19 and other infectious disease outbreaks, individuals with chronic illnesses are likely to be more receptive and enduring supporters of public health interventions. Understanding the relationship between disease and vaccine acceptance allows us to address concerns of specific subpopulations to further promote vaccination against COVID-19.Below is the link to the electronic supplementary material.Supplementary Material 1"
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