Rheumatoid arthritis (RA) is a chronic, heterogeneous autoimmune disease. Its high disability rate has a serious impact on society and individuals, but there is still a lack of effective and reliable diagnostic markers and therapeutic targets for RA. In this study, we integrated RA patient information from three GEO databases for differential gene expression analysis. Additionally, we also obtained pan-cancer-related genes from the TCGA and GTEx databases. For RA-related differential genes, we performed functional enrichment analysis and constructed a weighted gene co-expression network (WGCNA). Then, we obtained 490 key genes by intersecting the significant module genes selected by WGCNA and the differential genes. After using the RanddomForest, SVM-REF, and LASSO three algorithms to analyze these key genes and take the intersection, based on the four core genes  that we found, we constructed an RA diagnosis. The nomogram model showed good reliability and validity after evaluation, and the ROC curves of the four genes showed that these four genes played an important role in the pathogenesis of RA. After further gene correlation analysis, immune infiltration analysis, and mouse gene expression validation, we finally selected CYFIP2 as the cut-in gene for pan-cancer analysis. The results of the pan-cancer analysis showed that CYFIP2 was closely related to the prognosis of patients with various tumors, the degree of immune cell infiltration, as well as TMB, MSI, and other indicators, suggesting that this gene may be a potential intervention target for human diseases including RA and tumors. RA is a chronic, symmetrical, autoimmune disease that is aggressive and involves multiple joints in the body. The worldwide prevalence is approximately 5 per 1,000 and the incidence in women is usually 2 to 3 times higher than in men  and macrophages (RASM) are key factors in the development of RA , 5. B ceThe main treatments for RA are anti-inflammatory drugs, analgesic drugs, and DMARDs.The first two can only relieve the symptoms of RA but do not stop the further development of RA. Since the immune response is the main pathogenesis of RA, disease-modifying antirheumatic drugs (DMARDs) have become the primary choice for RA. Although DMARDs have shown good efficacy in reducing RA, there is still a possibility of treatment failure with DMARDs for some patients . In the in vivo experiment, CYFIP2 was filtered out and verified via pan-cancer analysis, which illustrated a strong correlation with various tumors.In this article, we used the R tool and the limma package to statistically analyze the four data sets and analyze the differential expression of mRNAs. The WGCNA R package was then used to calculate the association of gene significance (GS) and module membership (MM), analyze the correlation between modules to construct a weighted gene co-expression network, and merge DEGs with key module genes for functional analysis. The feature genes were also identified by the algorithm, and LASSO regression analysis was performed to narrow down the range of feature genes. To further validate the selected signature genes, we used GSEA analysis, interaction analysis, ROC analysis, and studied the level of immune cell infiltration in the RA group, and finally calculated the relationship between signature genes and immunity. For further selection, by integrating multiple datasets, we aimed to screen the signature genes that play a key role in the development of RA and various cancers. Combining with the immune infiltration analysis and https://www.ncbi.nlm.nih.gov/geo/) database, and information on these datasets is supplied in GSE1919 , GSE5545The data were evaluated using functional enrichment to confirm the possible functionalities of prospective targets. Gene ontology (GO) is a popular technique for assigning functions to genes, particularly molecular functions (MF), biological pathways (BP), and cellular components (CC). KEGG enrichment analysis can be used to analyze gene functions as well as related high-level genomic functional information. The “GOplot” package and the “cluster profiler” in R were used to examine the GO function of prospective mRNAs and to enhance KEGG pathways to better understand the carcinogenic role of target genes .The WGCNA method aids in the investigation of gene set expression. Through the following main phases, the WGCNA R package was used at various stages for the development and modularization of various gene networks. To determine if there were any significant outliers, the samples were placed in clusters. Following that, automated networks were used to create co-expression networks. The modules used hierarchical clustering and dynamic tree cutting function detection. Module membership (MM) and gene significance (GS) were estimated to connect modules with clinical characteristics. Hub modules were designated as those with the highest Pearson module membership correlation (MM) and a p absolute value of 0.05. High module connection and clinical importance were denoted by MM >0.8 and GS >0.2, respectively. The gene information for the corresponding module was advanced for further investigation .The above genes were used to isolate the feature genes that were used to diagnose RA. SVM is a regression or classification-supervised machine learning technique that requires a training set with labels . SVM-RFEhttp://www.genemania.org) is a website for building protein–protein interaction (PPI) networks, which can be used to generate gene function predictions and locate genes with comparable effects. Physical interaction, co-expression, co-localization, gene enrichment analysis, genetic interaction, and site prediction are some of the bioinformatics methods used by the network integration algorithm. GeneMANIA was used to analyze PPI networks of signature genes in this study.GeneMANIA  curves to determine the area under the curve (AUC) for screening signature genes and evaluating their diagnostic value .To investigate the various levels of infiltration of immune cell types between RA tissue and normal tissue. To analyze the association between immune cells and distinctive genes, the “corrplot” package was used to obtain the Spearman rank correlation coefficient.Using deep forest plots, the “foresrplot” R program was used to perform univariate cox regression analysis and display p-values, HRs, and 95% CIs.We used TIMER, XCELL, QUANTISEQ, MCPCOUNTER, and EPIC algorithms to explore the relationship between AXIN1 expression and immune invasion in all TCGA tumors.The Animal Care & Ethics Committee of Jinan University’s First Hospital approved all animal care and experimental operations. We also followed the Guide for the Care and Use of Laboratory Animals that was established by the National Institutes of Health. The ARRIVE criteria were followed for reporting animal experiments , 20. MicFor immunohistochemistry, synovial tissue sections were stripped and then incubated with 5% serum in PBS for 2 h to block nonspecific binding and with 3% H2O2 for 10 min to block endogenous peroxidase activity. The expression of CYFIP2 and ST8SIA1 was determined by staining with polyclonal rabbit anti‐mouse CYFIP2 and ST8SIA1 antibodies overnight at 4°C. As controls, irrelevant isotype-matched antibodies were used. A polyclonal goat anti-rabbit antibody was detected with diaminobenzidine using goat anti-rabbit antibodies labeled with HRP.in vitro experiments were conducted with a minimum of five independent experiments. Therefore, the results were expressed as mean + SEM. Blinding was used in the experimental procedures or treatment and data analysis. We normalized immunoblots, glucose uptake, and mRNA expression for quantitative analysis to reduce baseline variations between independent experiments. Comparing the two groups was done using Student’s t-test. The one-way ANOVA was applied to three or more different groups. If F exceeded 0.05 and the variance in homogeneity was not significant, all results were discarded. Two post hoc tests were applied: Dunnett’s post hoc test on each group compared with the control group or Sidak’s post hoc test on multiple groups compared together. To analyze the data normalization, a non-parametric statistical analysis was performed. Data with non-parametric characteristics were analyzed with the Kruskal-Wallis test or Wilcoxon test two-sample. The statistical analyses of the data were conducted using SPSS 13.0 software. A P-value of less than 0.05 was considered significant.Data collection and analysis complied with pharmacology’s recommendations for experimental design and analysis . The in The data are standardized in a box plot, where different colors represent different data sets, rows represent samples, and columns represent gene expression values in samples  using the Rms package . We took the CYFIP2 gene to the next level of analysis after combining the ssGSEA results. Since the immune response is crucial not only in RA but also in cancer, we used overlapping immune genes to see if there is any link between the two diseases. CYFIP2 was identified to be highly expressed in BRCA, CHOL, HNSC, PRAD, THCA, and low expressed in BLCA, BRCA, COAD, ESCA, GBM, KICH, KIRC, KIRP, LUAD, LUSC, and PAAD in the TCGA data , disease-specific survival (DSS), and progression-free survival (PFS). In the OS analysis, cox regression of 33 tumors revealed that CYFIP2 expression was substantially linked with OS in six cancers: KIRC, LGG, PAAD, SKCM, and THYM as protective factors, and UCEC as a risk factor , a common systematic autoimmune disease, has gained increasing attention around the world recently. The main symptoms of RA include musculoskeletal pain, swollen joints, and stiffness, which can severely impair motor function and quality of life . UsuallyRecent decades have witnessed the rapid development of molecular research and bioinformatics techniques. By enrichment analysis through molecular function, biological processes, and cellular components, molecular biology could provide us with a comprehensive and further investigation of how gene variation and co-expression influence protein function and disease progression. Meanwhile, the emerging weighted gene co-expression network analysis (WGCNA) has gradually been used in the association between diseases along with related phenotypes and clusters (modules) of highly corrected genes . SeveralGiven these, we developed a comprehensive and in-depth evaluation system to analyze and verify hub genes and molecular pathways involved in RA patients through bioinformatics, especially WGCNA and protein–protein interaction (PPI) techniques, aiming to broaden the horizons into physiopathology and molecular mechanisms of RA and provide novel therapeutic targets for clinical treatment.In this study, we screened 891 differentially expressed genes (DEGs) and found 427 genes were upregulated and 464 were downregulated. Subsequent GO enrichment analysis showed all DEGs mainly associated with cell–cell adhesion, components of the plasma membrane, and cytokine receptor activity, while KEGG enrichment analysis showed some correlation with hematopoietic and T cells, along with chemokine signaling pathways. WGCNA analysis showed 33 cluster samples and 24 modules. No significant correlation verified the reliability of dividing parts. Critical machine algorithms and LAASO regression analysis found 4 hub genes, then validation datasets confirmed that BTN3A2, CYFIP2, ST8SIA1, and TYMS were highly expressed in RA, and the first three genes were highly similar in biological function.Horsburgh et al. illustrated that CpG-specific methylation at RA might become a marker of treatment response. Most notably, one of the CpG sites in the BTN3A2 genes was strongly associated with treatment response  is an important enzyme in the lication . To pred therapy . Another therapy , and Lim results . This coTo illustrate the action of hub genes in RA one step further, we conducted a GSEA analysis, and the results showed that the primary immunodeficiency was significantly enriched in the high-expression subgroup of all 4 hub genes, and the intestinal immune network for IgA was enriched in CYFIP2 high-expression and ST8SIA1 high-expression subgroups, while hematopoietic cell lineage, Th1 and Th2 cell differentiation were enriched in ST8SIA1 high-expression and TYMS high-expression subgroups at the same time. Subsequent ROC analysis showed that all hub genes played a critical role in RA, indicating a potential diagnostic value in clinical treatment.Finally, further immune infiltration analysis showed the mast cells in the RA group were higher than those in the control group. Mast cells could stimulate osteoclast differentiation in monocytes and then stimulate osteoclastogenesis, which is a mechanism of inflammatory and tissue destruction effects in RA patients . The assin vitro validation using RA mice, it was found that the expression levels of both genes were increased, which further confirmed our previous research inferences. Combined with ssGSEA analysis, CYFIP2 was highly correlated with more immune cells and immune response processes compared with ST8SIA1. Therefore, we used CYFIP2 as the target gene for further analysis.To further explore the core genes among the four key genes, we chose to download immune genes from the InnateDB database and found two overlapping genes (CYFIP2 and ST8SIA1) at the intersection of the four characteristic genes. After Interestingly, some existing studies point to a relationship between RA and a variety of cancers. On the one hand, RA has been pointed out to have a relationship with the risk of cancer, including lung cancer , lymphomIn our study, CYFIP2 was a prognostic protective factor for KIRC, LGG, and PAAD, and a risk factor for UCEC, but there is still a lack of relevant studies to prove it. From the overall situation of the current research, CYFIP2 has been studied more in digestive system cancers. For example, the study by Mongroo et al. . showed in vivo or in vitro via human samples are warranted to confirm our results. Secondly, because our data are from a database, some aspects like sex, age, and complications are not considered in our research, and further clinical investigation is needed.Even though this is a comprehensive and novel evaluation system to explore hub genes and related signaling pathways in RA patients, even in pan–cancer, there were also several limitations in our study. Firstly, although we performed validation of gene expression in mice, due to the innate restrictions of bioinformatics techniques, more experiments To explore specific hub genes for the association between immune infiltration and RA as well as pan–cancer, we conducted a comprehensive and in–depth analysis to analyze related genes and pathways. The 2 hub genes (CYFIP2 and ST8SIA1) we discovered would broaden our insights into molecular mechanisms and bring more potential therapeutic targets for clinical treatment, which also needs more research to verify and develop. For further pan–cancer analysis, CYFIP2 was considered the most potential target both in RA and 33 kinds of tumors, which may shed the hoping light on the therapy of human immune–related diseases and even cancer.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The animal study was reviewed and approved by the Experimental Animal Ethics Committee of Jinan University.ZYZ, SJH, and XCY planned the research concept and designed it, made provisions for study material, collected data and analyzed them, wrote and approved the manuscript. XFL and ST searched for data and wrote programming code. MHW, MMA, and XQH collected pictures and graphs as well as edited them. SZ and DSZ collected data and analyzed them, wrote and approved, and helped correct the manuscript. All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This research is funded by the Shenzhen Key Laboratory of Musculoskeletal Tissue Reconstruction and Function Restoration and Shenzhen People’s Hospital (Project number: ZDSYS20200811143752005), the Guangzhou Science and Technology Project  model in mice, the National Natural Science Foundation of China (Project number:81401766), the Fundamental Research Funds for the Central Universities (Project number: 21619348), the National Natural Science Foundation of China (Project number: 81901650), and the Science and Technology Projects in Guangzhou (Project number: 2021020200460).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.The reviewer Z–WG declared a shared parent affiliation with the author MEA to the handling editor at the time of the review.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.


Background: The purpose of the study was to investigate the levels of amino acids and acylcarnitines in newborns of the Tibet Autonomous Region for the first time and to provide an experimental basis for the diagnosis of genetic metabolic diseases.Methods: We detected concentrations of 43 kinds of amino acids, acylcarnitines and succinylacetone in the dried blood spots of 18482 newborns using liquid chromatography tandem mass spectrometry and diagnose the case by gene sequencing. We compared the indexes between Tibet and our lab, where most data come from an inland area and Han Chinese people. Then we compared amino acid and acylcarnitine levels of seven regions in Tibet and explored their impact factors.Results: We described the levels of amino acids and acylcarnitines in Tibet newborns using 95% confidence intervals. The distribution of amino acid and acylcarnitines were different in Tibet.Conclusion: This study has contributed to filling in the blanks of Tibet newborn screening, which should be considered in the newborn metabolic disease screening in this area. Inherited metabolic diseases (IMD) are caused by genetic mutations that interfere with typical metabolism. This genetic mutation tends to result in a deficiency of an enzyme defect, leading to a lack of the enzyme’s products as well as an accumulation of the enzyme’s substrates, which then causes a series of clinical symptoms . At presOwing to the high morbidity, mortality and strong risk of recurrence in affected families, neonatal IMD screening is an important element of modern preventive medicine as it allows the identification of both potential and asymptomatic infants as early as possible. Meanwhile, neonatal screening programs are an effective measure to reduce birth defects and improve the health of a population.At present, the technique liquid chromatography-tandem mass chromatography (LC-MS/MS) has become the ideal analysis technique for IMD screening, due to its high sensitivity, signal-to-noise ratio, high specificity and high selectivity  Tu et a. In ChinIn 2020, the Chinese National Center for Clinical Laboratories (NCCL) launched a nationwide study to investigate the dynamic pattern of 35 MS/MS NBS biomarkers and establish accurate and robust reference intervals, in which 4,714,089 Chinese neonates were tested in participating centers/laboratories . HoweverWe performed the detection and statistics of amino acid and acylcarnitine levels in neonates from the Tibet Autonomous, with the aim of providing a preliminary reference data in this region.Samples were taken from 18482 live born infants from seven provinces of Tibet Autonomous Region between October 2015 and February 2021. Heel blood was collected after 72 h after the infants were born and fully breastfed, and then dropped onto filter paper (Whatman 903). Blood spot samples were placed in the shade to dry and were sealed and stored at -4°C prior to detection.Small molecule metabolites of dry blood spots were extracted using underivatized amino acids, carnitine and succinylacetone assay kits  according to the manual. Concentrations of 43 types of amino acid, carnitine and succinylacetone were detected by liquid chromatography and tandem mass spectrometry  and then analyzed by the software Masslynx  and Neolynx. .tgex.genecards.org).The diseases were diagnosed by combining with clinical manifestation, laboratory examination and gene sequencing. Genomic DNA of peripheral blood leukocytes from patients and their parents was extracted using a QIAamp DNA Blood Mini Kit . The genes of patients were sequencing by next-generation sequencing technology , then the mutations of the parents were confirmed by Sanger sequencing. The data was analyzed and reported by software TGex .Between-group comparisons were performed by independent-samples t-tests and ANOVA with Bonferroni posthoc multiple comparisons test for normally distributed data or Mann-Whitney U test for non-parametric sample distribution. All statistical analysis was performed using SPSS (version 21.0), and two-tail This study was approved by the Ethics Committee of the Chinese PLA General Hospital (No. S2018-025-01) and this trial has been verified by Chinese Clinical Trial Registry (No. ChiCTR1800016903). All participants provided informed written consent for sample collection, as well as permission for the samples’ use in research.A total of 18482 neonatal screenings were conducted between 2015 and 2021, including 10719 males and 7,763 females. Some 99.67% of these babies (18421/18482) were Tibetan. The cases were distributed in seven prefecture-level cities in the Tibet Autonomous Region, including 6,586 in Lhasa, 2,850 in Naqu, 1,155 in Changdu, 1,079 in Shannan, 5,629 in Xigaze, 593 in Ngari and 590 in Nyingchi. The distribution of neonates from the Tibet Autonomous Region is depicted in Before this study, there was little data on neonatal screening from the Tibet region and Tibetan people. As shown in The levels of amino acids and acylcarnitines in neonates from the Tibet Autonomous Region were described based on the P0.5%∼P99.5% of the values . If we rThe screening area covers seven prefecture-level cities. A total of 99.67% of the babies (18421/18482) were Tibetan. Due to local limited medical conditions and nomadic customs, the mean blood collection time was 30 days after birth. Most babies had been given supplemental food from the birth, according to the local custom, which caused differences of small metabolites molecules between this area and inland areas. For example, babies in Xigaze region were fed by breast milk and zanba (highland barley) and were also given a small amount of barley wine and buttered tea. Therefore, we initially explored the impact of the special dietary habits in different areas of Tibet on the small molecule metabolites indicators.As shown in In order to explore effects of climate and altitude on tandem results, we compared the results of Nyingchi and Ngari, which have similar diets. Ngari is located in the southwest border of China and has a mean altitude of more than 5,000 m. Due to its high altitude, the climate is cold and dry, annual rainfall is relatively low, the difference between day and night temperature is large, and winter is long and cold. In contrast, Nyingchi, which is located in the southeast of Tibet, has a mean altitude of 3,100 m, while its lowest point is only 900 m, lower than other areas of Tibet. Due to the Indian ocean current, the climate is warm and comfortable. Annual rainfall is approximately 650 mm, the average annual temperature is 8.7°C, average annual sunshine is 2022.2 h, and the frost-free period is 180 days. Therefore, while the two regions have similar population composition and eating habits, their altitudes, climates and other factors are different.As shown in In the past 10 years, MS/MS was widely used for neonatal hereditary metabolic disease screening in China, improving the ability and efficiency of newborn screening . The NCCHere we investigated the amino acid and acylcarnitine levels in 18482 neonates from the Tibet Autonomous Region, providing preliminary data in this region for the first. There were some unsatisfactory factors, such as the time of blood collection and follow up. According to the technical specifications for neonatal disease screening (2010 version), blood sampling time is 72 h after birth, within 7 days, and full breastfeeding. However, in the actual situation, due to the inconvenient transportation, limited medical conditions, nomadic habits, special eating habits, and weak awareness of medical screening, the blood collection time in Tibet could not meet the requirements of the standard, with an average of 30 days. Therefore, the sampling time was one factor that affect the levels of small molecule metabolites. Considering the particularity of Tibet and the blank of data, we think it is very necessary to set up a new cut-off value according to the actual situation.in vivo and reflected the metabolic level in Tibetan newborns, which may be caused by special diet during newborn, such as high-protein food, like yak milk. Interestingly, in the metabolism pathway of Phe and Tyr, Tibetan healthy newborn showed a higher level of Phe, and lower level of Tyr. Phe, one of special indexes for diagnosing hyperphenylalaninemia, had a maximum value (95%CI) of 130 μmol/L in Tibet, which is higher than our regular cutoff 120 μmol/L  to the National Center for Examination and took part in interventricular quality assessment twice every year, which ensures that results are valid. The preliminary reference range is only applicable to the current neonatal disease screening in Tibet. After the popularization of the new screening education, the improvement of medical conditions, more standardized blood collection and the expansion of sample size, more suitable cutoff value is required in the future.By examining data from more than 18,000 newborns, this study investigated the amino acid and acylcarnitine levels in neonates of the Tibet Autonomous for the first time. Data reflect the metabolic level of newborns in Tibet and fill in gaps in the region. This study suggested an independent cut-off value of neonatal screening should be established in different regions, especially in high altitude areas, which will greatly improve the diagnostic efficiency for neonatal screening.


Flavobacterium and Pseudomonas were the dominant genera. The climatological parameters that were most correlated with the bacterial communities and their functional profiles were pH, DO, ORP, turbidity, TN, EC, NH4+, and NO3–. The bacterial communities displayed variations in their functional composition for nitrogen, phosphorus, and sulfur metabolisms during the sampling months. The bacterial communities within the lake are highly susceptible to nutrient loads and low DO levels during the rainy season. Bacterial communities had a higher relative abundance of genes associated with denitrification, nitrogen fixation, assimilatory sulfate reduction, cysteine, SOX system, and all phosphorus metabolic pathways. The results obtained here enrich our understanding of the bidirectional interactions between bacterial communities and major biogeochemical processes in eutrophic subtropical lakes.Lakes in subtropical regions are highly susceptible to eutrophication due to the heavy rainfall, which causes significant runoff of pollutants  to reach surface waters, altering the water quality and influencing the microbial communities that regulate the biogeochemical cycles within these ecosystems. Lake Cajititlán is a shallow, subtropical, and endorheic lake in western Mexico. Nutrient pollution from agricultural activity and wastewater discharge have affected the lake’s water quality, leading the reservoir to a hypereutrophic state, resulting in episodes of fish mortality during the rainy season. This study investigated the temporal dynamics of bacterial communities within Lake Cajititlán and their genes associated with the nitrogen, phosphorus, sulfur, and carbon biogeochemical cycles during the rainy season, as well as the influences of physicochemical and environmental variables on such dynamics. Significant temporal variations were observed in the composition of bacterial communities, of which  Water samples were collected from Lake Cajititlán at five sampling stations  and at two monitoring depths  , using a States) . The mon States) .Genomic DNA of the samples was extracted from two different pore sized cellulose nitrate filters to retain different microbial fractions. First, each replicate was filtered through a membrane with pore sizes ranging from 20 to 25 μm. The filtrate was then passed through a second membrane with a pore size of 0.45 μm. Each of these two filters was then cut into pieces with sterile scissors, and 100 mg of each was weighed and placed into a lysing matrix to perform a DNA extraction and purification of the samples using the FastDNA Spin Kit for Soil , following the manufacturer’s protocol. The extracted DNA samples were quantified using a NanoDrop ND-1000 UV-Vis spectrophotometer . The 16S rDNA regions V3-V4  were amplified following the Illumina protocol for 16S Metagenomic Sequencing Library Preparation .® MiSeq sequencer  for 300-bp paired-end sequencing at the facilities of Tecnológico de Monterrey, Campus Monterrey.The amplified DNA was verified by 1.0% agarose gel electrophoresis in 1X TAE buffer. Sequencing libraries were cleaned using magnetic beads from the AM Pure XP kit  and quantified with a Qubit 2.0 fluorometer . Finally, 96 samples were loaded onto the Illuminaview.qiime2.org.We used QIIME 2.0 v.2021.8  to process raw 16S rRNA gene sequence data . Using D1.The software, PICRUSt 2.0 v.2.4.1 was used for the predictive functional analysis of bacterial communities . RepreseAll statistical analyses were carried out using the R v. 3.5.3 software unless otherwise stated . All barPrincipal coordinate analysis (PCoA) was performed to observe microbial community differences (β-diversity) and to reveal functional differences in the metabolism of the biogeochemical cycles  between sampling points and months. Distances were based on Bray-Curtis dissimilarities using the vegan package and the cmdscale function. Additionally, an analysis of variance was performed using distance matrices (ADONIS) through the vegan package and the Adonis pairwise function to test the significance of spatial and temporal differences in the composition of the bacterial communities of Lake Cajititlán .Bar charts of relative abundance based on the metagenomic compositions of the KOs (relative abundance KOs) were made using the phyloseq and vegan packages . To undeFinally, using the “corrplot” package, Mantel tests based on Spearman’s correlation between the abundant taxa of bacteria and the physicochemical and environmental factors were performed, and another correlation matrix was created to evaluate the relationships between the physicochemical and environmental factors with the metabolism (set of metabolic pathways) of the biogeochemical processes analyzed .4+ and NO3– decreased over time (from July to September) whereas those of DO, ORP, and chlorophyll-a increased. Additionally, pH, turbidity, and BGA-PC all displayed similar and significant temporal patterns, with the highest levels observed in July, followed by a decrease in concentration in August and, finally, an increase in the last month , revealed that most of the physicochemical parameters monitored showed significant temporal fluctuations when comparing the values reported for the three different months of the rainy season . The const month . Ultimatst month .No significant differences were observed in the environmental parameters  analyzed during the study period. However, the climatological parameter that displayed the greatest variation was PREC, and July was the month that displayed the highest amount of rain . SimilarFlavobacterium and Pseudomonas were most abundant in all months and at all sampling points. Similarly, the groups unknown (Un.) Oxyphotobacteria and Un. Cyanobacteria presented high abundances; however, these could not be classified to the genus level  in the composition of bacterial communities were found over the study period when comparing the bacterial compositions within the samples taken during the different sampling months   .R = 0.003, P = 0.008; R = 0.0054, P = 0.29; A total of 1,416 KOs detected within the bacterial communities of Lake Cajititlán, which 26.84% (380 KOs) were related to the nitrogen, carbon, sulfur, and phosphorus biogeochemical cycles. The functional composition of the bacterial communities related to nitrogen, phosphorus, and sulfur metabolism was highly different among the sampling months and particularly different for the month of July , whereas no differences between the metabolic pathways were detected for carbon . In this case, the bacterial functional composition displayed a significantly higher relative abundance of genes associated with the denitrification and the nitrogen fixation pathways, and July was the month with the highest gene abundance related to these pathways. Comammox and anammox were also slightly higher in July (compared to August and September), although these differences were not significant. The gene abundance levels related to dissimilatory nitrate reduction to ammonia (DNRA), assimilatory nitrate reduction to ammonia (ANRA), and nitrification processes were similar among the sampling months. Within these metabolic pathways, DNRA displayed the highest gene abundance during all sampling months .We found 23 KOs related to sulfur metabolism (ko00920 in the KEGG database). In July, all metabolic pathways displayed a higher relative gene abundance, with the assimilatory sulfate reduction (ASR) pathway displaying the highest gene abundance, followed by the cysteine, the sulfur oxidation system (SOX system), and the dissimilatory sulfate reduction (DSR) pathways. All metabolic pathways displayed similar abundance proportions in August and September, except for the SOX system, which was greatest in August . AccordiOverall, 34 KOs were found to be related to the phosphorus cycle. All phosphorus pathways examined were most abundant during July. The organic P-mineralization (OPM) pathway was the most abundant pathway, followed by P-starvation response regulation (PSRR), (PUT), and inorganic P-solubilization (IPS). Similar fractions were found between August and September for all metabolic pathways .Physicochemical and environmental factors are important determinants of microbial communities and their functional composition in Lake Cajititlán. The data matrix of the physicochemical and environmental parameters generated was used to explore the influence of these parameters on the bacterial communities’ functional composition during the study period . 4+, NO3–, TN, pH, and WT, whereas chlorophyll-a was most correlated with the taxonomic composition in September  imply that higher values of these parameters negatively affect the prevalence of the bacterial populations over the research period  correlated with the genes of the bacterial communities involved in biogeochemical cycling (4+ and NO3–) suggest that biogeochemical cycling activities are enhanced under these higher concentrations, whereas negatively correlated parameters  may hinder biochemical cycling.According to the RDA results, the physicochemical parameters that were most correlated with the functional composition of the bacterial communities of July were ORP, NHeptember . The fireptember ; pH, ORP cycling . PositivFlavobacterium and the group Un. Oxyphotobacteria were positively correlated to OD, whereas the Pseudomonas genus was positively correlated to chlorophyll-a and all environmental variables  but negatively correlated to ORP, NO3–, and TN, and the groups Un. Oxyphotobacteria and Un. Cyanobacteria were negatively correlated with TN. The correlation matrix between the biogeochemical metabolisms and the physicochemical and environmental variables  were highest in July because of nutrient runoff from the agricultural lands surrounding the lake, where significant amounts of nutrients accumulated in the fields are washed off after the onset of the rainy season and as a result of the high evaporation rates (mean ∼9 mm) throughout the hot-dry season (February to May), and the decrease in nutrient concentrations throughout the study period (July to September) could be the consequence of a dilution process throughout the rainy season   . Some spbacteria , could bbacteria . The DO bacteria ; it alsobacteria .Flavobacterium is strongly positively correlated with this parameter, as shown in this study  have already been reported to be caused by the natural geological conditions of the lake’s basin . The pH is study  and as ris study . Finallyis study .Previous research on eutrophic freshwater lakes has focused on the dynamics of phosphorus in shallow lake sediments . Based o4+, and NO3–  are produced in relatively greater quantities because these processes are linked to electron transport , no temporal differences were observed. However, a higher relative abundance of DNRA-associated genes was observed during all months of the study in comparison with other pathways , suggestransport . Based oransport , it can ransport , which cransport .During the 3 sampling months, the functional composition related to the carbohydrate metabolism pathway was most abundant within all carbon metabolism pathways, followed by CFPP, methane metabolism, CFPO, and lipid metabolism. However, no temporal differences were observed among them .Microorganisms play an important role in the production, transformation, and mineralization of organic matter . As a reBoth ORP and DO are critical water quality parameter for the carbon cycle, as reducing conditions and low DO levels can enhance methane synthesis, whereas methane-oxidizing and sulfate-reducing bacteria contribute to carbon fixation . SulfateBacillus, Pseudomonas, Azotobacter, Rhodococcus, Serratia, Bradyrhizobium, Salmonella, and Thiobacillus, among others  features of Lake Cajititlán. The most abundant bacterial genera found were Flavobacterium and Pseudomonas. Bacterial communities displayed a higher relative abundance of genes associated with denitrification, nitrogen fixation, ASR, cysteine, SOX system, and all phosphorus pathways in July, whereas the remaining pathways and months showed similar patterns in abundance as bacterial communities are highly influenced by nutrient loads during the onset of the rainy season (July) and by low DO levels. A limitation of PICRUSt inferences is that they are based on evolutionary modeling of the gene content of known reference genomes. Therefore, the accuracy of any given sample type will depend heavily on the availability of appropriate references. On the other hand, the input data for the standard PICRUSt workflow are partial 16S rRNA gene sequences, and therefore, eukaryotic, or viral contributions to the metagenome will not be predicted. Future research will use shotgun sequencing to comprehensively analyze genes from all organisms present in a sample. This can provide more accurate knowledge of the metabolic mechanisms that may be taking on to allow these organisms to adapt to their environment. Also, these data would allow the further exploration of the functional and taxonomic microbial diversity in freshwater environments and the potential impacts of water quality and climate change (rainy season) on the functioning of these ecosystems.This study provides valuable information on ecological interactions between water quality and bacterial taxonomic and functional composition in a subtropical and hypereutrophic lake during the rainy season. Results showed significant temporal variations over a short period of time, induced by the rainy season, causing high seasonal surface runoff and, therefore, significant changes in the physicochemical  can be found below: OD-T, MG-H, and CS-G conceived the study, designed the methodology, and led the writing of the manuscript. OD-T, JA, AP, and MG-H collected the data. OD-T and CY-M analyzed the data. MG-H, CS-G, OL-M, and AP were responsible for funding acquisition. All authors contributed critically to the drafts and gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to develop a deep convolutional neural network (DCNN) model to classify molecular subtypes of breast cancer from ultrasound (US) images together with clinical information.A total of 1,012 breast cancer patients with 2,284 US images (center 1) were collected as the main cohort for training and internal testing. Another cohort of 117 breast cancer cases with 153 US images (center 2) was used as the external testing cohort. Patients were grouped according to thresholds of nodule sizes of 20 mm and age of 50 years. The DCNN models were constructed based on US images and the clinical information to predict the molecular subtypes of breast cancer. A Breast Imaging-Reporting and Data System (BI-RADS) lexicon model was built on the same data based on morphological and clinical description parameters for diagnostic performance comparison. The diagnostic performance was assessed through the accuracy, sensitivity, specificity, Youden’s index (YI), and area under the receiver operating characteristic curve (AUC).Our DCNN model achieved better diagnostic performance than the BI-RADS lexicon model in differentiating molecular subtypes of breast cancer in both the main cohort and external testing cohort . In the main cohort, when classifying luminal A from non-luminal A subtypes, our model obtained an AUC of 0.776  for patients older than 50 years and 0.818  for those with tumor sizes ≤20 mm. For young patients ≤50 years, the AUC value of our model for detecting triple-negative breast cancer was 0.712 . In the external testing cohort, when classifying luminal A from non-luminal A subtypes for patients older than 50 years, our DCNN model achieved an AUC of 0.686 .We employed a DCNN model to predict the molecular subtypes of breast cancer based on US images. Our model can be valuable depending on the patient’s age and nodule sizes. JAMA demonstrated that 63% of the overall mortality reduction of breast cancer was associated with treatments of chemotherapy, hormone therapy, and trastuzumab, and the associations varied by breast cancer molecular subtypes  on molecular typing of breast masses by morphological features –7. HowevAt present, several studies combined medical images with machine learning or deep learning (DL) approaches, which are more accurate and convenient to predict molecular subtypes preoperatively based on US, mammography, and MRI images –11. DiffBased on pathophysiological and epidemiological results, triple-negative breast cancer occurs more frequently in women who are younger, and the tumors are usually larger in size. Furthermore, in China, the peak ages of breast cancer are in the 40–50-year age group , 15. As in situ hybridization (FISH) should be taken for final diagnosis. Ki-67 expression was graded as low (<14%) or high (≥14%). Breast cancers were categorized into four subtypes on the basis of their receptor status. The subgroups defined were as follows: luminal A , luminal B , HER2-positive , and triple-negative  . Informed consent was waived by the board. The inclusion criteria of this study were as follows: 1) all the breast masses were pathologically proven, 2) patients with complete information of US examinations, 3) the patients had not undergone other treatments, and 4) complete immunohistochemical (IHC) marker  result on histopathology. The exclusion criteria were as follows: 1) US images with poor quality and 2) the pathological results and IHC markers were incomplete for molecular subtyping. ER and PR positivity was defined as the detection of ≥1% positive staining in tumor nuclei. HER2 status was graded as 0, 1+, 2+, or 3+. A score of 3+ staining was defined as HER2 positive, and scores of 0 and 1+ were considered as HER2 negative. As for 2+ staining, HER2 gene amplification by fluorescence d HER2−) Figure 1The US images in this study were acquired by several different US equipment in our medical center, including Philips , Aixplorer , GE Healthcare , Hitachi , Esaote , and Siemens  with linear transducers of frequency 5–12 MHz. Moreover, clinical data on patient age, US tumor size (short diameter and long diameter), and aspect ratio were also collected for feature extraction.https://github.com/tzutalin/labelImg) was used to crop the regions of interest (ROIs) from the original breast US images with rectangular boxes. The annotation was manually processed and confirmed by a breast radiologist with over 5 years of experience in breast US interpretation.The labeling tool of LabelImg  optimizer accomplished in a computer with two NVIDIA 2080Ti graphic processing units (GPUs) and 256 GB of random access memory. To solve the problem of unbalanced samples, the focal loss strategy was used. The batch size, learning rate, and maximum iterations were set to 16, 0.005, and 100, respectively. The processing flow of our DCNN Architecture is shown in Morphological characteristics of each mass were classified by two radiologists who were blinded to the clinical and pathological results of the breast lesions. If there was no initial agreement, the final result will be reached after discussion. The two radiologists had 7 and 20 years of experience in breast US interpretation.In accordance with the American College of Radiology Breast Imaging Reporting and Data System (BI-RADS) lexicon, the morphological description included the following parameters: the shape , orientation , margin , echo pattern , posterior acoustic features , surrounding tissue , calcification , and vascularity distribution . These BI-RADS parameters were included as variables for BI-RADS lexicon modeling. Other included parameters were age, lesion number, lesion location, tumor size, and lymph node status in US .Then we constructed a BI-RADS lexicon model based on the above parameters. The solver, penalty, and maximum iterations of the model were all adjusted for the best result during the training period.The diagnosis performance was assessed according to the classification accuracy (ACC), sensitivity (SEN), specificity (SPC), Youden’s index (YI), and area under the receiver operating characteristic curve (AUC). The diagnosis performance was evaluated based on the patient level, not the image level. The bootstrapping analysis was performed on the AUC values of each group of experiments to obtain the 95% CIs. The paired t-test was applied to AUC values to measure the performance differences. Statistical analysis was computed using the Python (3.6) programming language and R Studio software (3.5.2). p < 0.05 was regarded as statistically significant.As shown in As shown in As shown in Medical imaging explored the characteristics from cellular to molecular levels and even the microscopic views to identify the different types of breast cancer to aid in clinical practice. Breast cancer with different molecular subtypes displays diverse, subtle, and overlapping imaging features, which makes accurate diagnosis difficult , 17. In Based on BI-RADS terminology for US, after standard morphological feature classification, we established a BI-RADS lexicon model for molecular subtype diagnosis to compare with our DCNN model. The BI-RADS lexicon model analysis has been widely applied to tumor characterization, cancer recurrence prediction, and detection of breast cancer for fine-needle aspiration cytology , 21. It Our dataset has a considerable sample size, which is leveraged by our DCNN model to fully mine the hierarchical information from breast cancer US images, and thus the DCNN could cope with a variety of tasks including the stratification analysis of the ages and tumor sizes. According to the breast cancer tumor node metastasis (TNM) stage system, the tumor size is a key reference factor for surgical method selection. A small tumor (tumor size ≤20 mm) is difficult to diagnose by US images, because its morphological characteristics are not obvious, and it is easy to be misdiagnosed as a benign nodule . DelayedSeveral studies have shown that the patients with TN breast cancer are younger (<40 years) than those with non-TN subtypes . TherefoOverall, our DCNN models performed moderately in stratified studies based on ages and nodule sizes. This could be attributed to the large variety of image acquisition machines in this study and the single modality of US images. However, from another point of view, it also shows the good applicability of our model to different machines. Due to the small amount of data, the external testing results of the subgroups of our DCNN model were unstable. In the subsequent experiments, we will try to add multi-modal information based on larger sample size for exploration, and the diagnostic performance is expected to be further improved.There are several limitations to this study. Firstly, compared with other imaging examinations, in the application research of artificial intelligence technology, the high noise, low resolution, non-uniform image standard, and other deficiencies have seriously affected its performance on this tough task of differentiating breast cancer molecular subtypes. Secondly, based on the retrospective collection of two-dimensional US image datasets, there are still deficiencies of inconsistent image quality and limited sample size in this study.In conclusion, our study has demonstrated that the DCNN model based on US images has the potential to provide a non-invasive method to preoperatively predict the breast cancer molecular subtypes, which could be a reference for clinical treatment arrangement. Specifically, our model could be more valuable in the classification of luminal A from non-luminal A subtypes for patients older than 50 years or whose tumor sizes ≤20 mm. For young patients ≤50 years, our model could be helpful in detecting triple-negative breast cancer more accurately. Future work on expanding the sample size and imaging modalities could further optimize our model and eventually assist radiologists and oncologists in real-world clinical practice.https://wurong7111@163.com.The data analyzed in this study is subject to the following licenses/restrictions: The dataset is private and involves patient privacy. Requests to access these datasets should be directed to Rong Wu, CL, SS and JC collected the images and cilinical data. QZ and RW designed the study. HH and YC performed the image processing and the statistical analysis. CL, HH and QZ contributed to the manuscript drafting and editing. All authors approved the manuscript.This work was supported by the National Natural Science Foundation of China , program for Shanghai Outstanding Medical Academic Leaders (2019LJ18), the interdisciplinary program of Shanghai Jiaotong university (ZH2018ZDA17), the program from Science and Technology Commission of Shanghai Municipality (No. 20Y11912400), and the 2019 clinical research innovation team of Shanghai General Hospital (No. CTCCR-2019B05).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.


Respiratory disorders result in rehospitalization and premature death of patients with cervical spinal cord injuries (CSCI). Community game-based respiratory muscle training (RMT) programs could reduce secondary complications.1), peak expiratory flow (PEF), vital capacity (VC), inspiratory capacity (IC), inspiratory reserve volume (IRV), expiratory reserve volume (ERV), maximum inspiratory pressure (MIP), maximum expiratory pressure (MEP), and peak cough flow (PCF) were measured.We examined the feasibility and preliminary efficacy of RMT as a community-based exercise program. Among the 10 included participants , four, one, one, and four reported C3, C4, C5, and C6 complete injuries, respectively . Their mean age was 43 ± 12.3 y. The time since injury was 10 ± 6.7 y. The participants completed an RMT program for 60 min/day, twice weekly, for 8 weeks. The participants were trained in the use of a newly developed game-based RMT device. The device provides consistent pressure for respiratory muscle strength and endurance training. Seven RMT devices were modified to allow 10 game-based RMT programs. Forced vital capacity (FVC), forced expiratory volume in 1 s , PEF , VC , IC , IRV , MIP , MEP , and PCF . The participants reported a positive impact of the program.There were improvements after RMT compared to pre-RMT in FVC (Community game-based RMT for individuals with CSCI appears to be safe and feasible. Community exercise with RMT use may have a positive impact on the respiratory measures for patients with CSCI who are vulnerable to respiratory compromise.KCT0005980. Spinal cord injury (SCI) has become increasingly widespread . Falls, Dysfunctions owing to cervical SCI (CSCI) include complete or partial impairment of motor control and sensory function . CSCI diA common consequence of CSCI is the defective innervation of the inspiratory and expiratory muscles . This deIn the initial stage of spinal shock, pulmonary function is reduced due to flaccid paralysis of all the muscles and paralysis of the respiratory muscles . In patiAlthough the implementation of RMT interventions is crucial to prevent respiratory complications following CSCI, participation in irregular RMT intervention may lead to obstructive pulmonary disease and worsening of respiratory failure . In partAccording to a Cochrane review , severalThis study was approved, and all methods were carried out in accordance with relevant guidelines and regulations by the National Rehabilitation Hospital’s Institutional Review Board NRCIRB 2016-03-029). In this feasibility and preliminary study, participants were recruited on a voluntary basis from a rehabilitation sport (RS) class. This study was conducted following the principles of the Declaration of Helsinki. The study protocol was registered and assigned the number KCT0005980 (first registration 09/03/2021). Both verbal and written consent for study participation was obtained from each participant prior to the commencement of the study. All the participants were informed of the objectives, procedures, and potential risks or discomfort associated with study participation. The RS class was completed at The Korea National Rehabilitation Institute Project , a community-based organization that provides exercise opportunities for people with disabilities. The RS class was specifically designed for patients with CSCI and included all such cases irrespective of their age, time since injury, and injury levels. All the participants in the RS class were allowed to participate in this study , game-based RMT program, and arrangement exercise (breathing assistant muscle stretching).The participants were trained in the use of a developed game-based RMT device . The gamAn exercise instructor and physical therapist were trained in the administration of the RMT devices; the initial training level was set such that they could complete 15 breaths/set without exhibiting symptoms of hyperventilation. The RMT program (60 min/session) was performed twice a week (8 weeks) using the developed RMT devices. Each training day program consisted of warm-up (10 min) with stretching muscles around the neck, cool-down (10 min), and game-based RMT (40 min).This training protocol was reported to be feasible and effective in patients with CSCI . The par1), peak expiratory flow (PEF), vital capacity (VC), inspiratory capacity (IC), inspiratory reserve volume (IRV), and expiratory reserve volume (ERV) were measured.The outcome measures were evaluated before and after the 8-week program. Before the evaluation, the participants were taught regarding the test method. The respiratory function was measured using a digital respiratory function measuring device  . For accThe respiratory muscle strength was evaluated by the maximum inspiratory pressure (MIP) and the maximum expiratory pressure (MEP) measurement using the Pony FX (COSMED) .The peak cough flow (PCF), which measures the ability to produce an effective cough, was assessed using a PCF meter . The participants were instructed to inhale to the maximum capacity and then cough as strongly as possible . Three mAll the statistical analyses were performed with SPSS version 21.0 . The means and standard deviations of each variable were obtained using descriptive statistics. The number of samples (n = 10) was < 30. The normality test was performed using the Shapiro–Wilk test; however, the non-parametric test was conducted, since it satisfied the significance level (p < 0.05). The Wilcoxon test was conducted to assess the differences between the pre- and post-exercise measurements of the participants’ performance. In addition to this null hypothesis testing, the data were also assessed for clinical significance using an approach based on the magnitudes of change. We calculated the magnitude of the size of differences by the effect size (ES) . We consIn total, 10 participants  with CSCI consented to participate, completed the training, and were included in the analysis. All participants had complete cervical level injuries Table . Their ap = 0.027, 10.62%, 0.22 ES), PEF , VC , IC , and IRV  were significantly improved. MIP  and MEP  significantly improved after the game-based RMT. The PCF values  also significantly improved.The mean difference for all measures across participants demonstrated an overall improvement in all the outcome measures Table . In the There was no study related to adverse events. Respiratory variables were examined for the 10 participants who completed 16 training sessions during the 8 weeks Table .In this study, we reported that game-based RMT significantly improved the respiratory outcomes in patients with CSCI. This is the first study to demonstrate how a novel approach to game-based RMT with a community exercise program could be an encouraging intervention strategy for patients with CSCI.A functional, effective method of RMT is required to support the repetitive, intensive training warranted for the respiratory rehabilitation of patients with CSCI. Additionally, participants’ active participation throughout the lengthy rehabilitation.1 and ERV.RMT should easily attract the interest of participants. Patients with CSCI experience weakened respiratory muscles, leading to ineffective cough or sputum removal capacity . FurtherThe program was conducted in two 60-min sessions per week, for 8 weeks. To examine the effectiveness of RMT, pre- and post-intervention evaluations of the participants’ respiratory function, respiratory muscle strength, and cough ability were evaluated. An observation of the study findings revealed significant improvements in the FVC, PEF, VC, IC, IRV, MIP, MEP, and PCF throughout the 8-week RMT intervention period.1 increased. The pulmonary function test (FVC and FEV1) is the simplest and most comprehensive respiratory functional assessment for the diagnosis and evaluation of airway diseases. The FVC and FEV1 measure forceful expiration following maximum inspiration and forceful expiration in one second, respectively [After training, both the FVC and FEVectively . Pulmona1. The observed significant improvements suggested that game-based RMT interventions are effective in increasing the FVC in patients with CSCI.The observed significant improvement in FVC was consistent with the results of a previous study  that repAfter RMT completion, PEF, VC, IC, and IRV were significantly improved.These results were similar to those of previous studies that reported a significant improvement in the PEF, VC, and IC after RMT in patients with SCI , 16, 28.Evaluation of respiratory muscle strength is used to determine respiratory failure  and to eFurthermore, the provision of game-based RMT for 8 weeks among patients with CSCI showed a significant improvement in the PCF. This finding was similar to that of a previous study that reported a significant improvement in the PCF following 4 weeks of RMT among patients with CSCI . HoweverConventional RMTs, such as diaphragmatic breathing, isocapnic hyperpnea training, air stacking exercise, pursed-lip breathing, and air-shifting, are repetitive hospital-based interventions, which could result in the loss of patients’ interest and in abandonment of the rehabilitation process . The intSince this was a preliminary pilot study, there were some limitations that should be acknowledged. First, it had a single group pre- and post-design, and there was no control group to compare the exercise effects. It is necessary to study the effects of RMT in future studies based on the principle of random allocation of participants with a control group. Second, the study had a small sample size and the participants were mostly male; thus, the results may not be generalizable to all patients with CSCI. Further studies should include more women to examine sex differences in the results of the respiratory function tests performed in patients with CSCI. Third, it is necessary to examine the effect of age, smoking status, location of injury , and onset of injury on the participants’ respiratory function. Fourth, the results did not indicate whether the improvements resulted from the performed exercise programs, as all the participants partook in various programs. Further studies with larger numbers of individuals with CSCI considering various injury characteristics  and clinical information  are warranted to identify the factors contributing to pulmonary function improvement and, ultimately, obtain favorable rehabilitative outcomes.To our knowledge, this is the first feasibility and preliminary study to suggest the use of RMT in combination with a community exercise program for patients with CSCI. Overall, the participants demonstrated improvement in all the respiratory outcomes. With increased education and expansion of these types of programs, compliance with an RMT program may increase.Finally, we judged that the safety verification for the mechanical part should be carried out for the generalization of the developed RMT devices.


Evidence from longitudinal studies has shown that influenza infection is linked to an increased risk of arrhythmia. Therefore, we aimed to assess the role of influenza vaccination in arrhythmia prevention.The PubMed, Embase, and Cochrane Library databases were searched to identify studies that investigated the potential effects of the influenza vaccine on arrhythmia risk published until October 25th, 2021. The study was registered with PROSPERO (CRD42022300815).P = 0.20] in patients after myocardial infarction or those with high-risk stable coronary heart disease. A meta-analysis based on observational studies showed that vaccination was associated with a significantly lower risk of arrhythmia . Additionally, subgroup analysis showed a decreased risk of atrial fibrillation (AF)  and a non-significant but positive trend concerning ventricular arrhythmias (VAs)  after influenza vaccination.One RCT with 2,532 patients and six observational studies with 3,167,445 patients were included. One RCT demonstrated a non-significant benefit of the influenza vaccine against arrhythmias , identifier [CRD42022300815].[ Cardiac arrhythmias, including atrial fibrillation (AF), atrial flutter, ventricular flutter, ventricular fibrillation, and heart arrest, are a common kind of cardiovascular disease, resulting in frequent hospitalizations, hemodynamic abnormalities, and thromboembolic events. Various infections, either by bacteria or viruses, can cause cardiac injury and secondary dysfunction of the cardiac conduction system –5. AmongGiven this background, it is not clear whether influenza vaccination is associated with reduced cardiac arrhythmias. Thus, in the present study, we aimed to (1) clarify the relationship between the flu vaccine and cardiac arrhythmias and (2) further explore the associations of specific types of arrhythmias  with the flu vaccine.1 The protocol was registered with PROSPERO .This study was performed according to preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines .1 The prTwo authors (ML and WL) independently searched the PubMed, Embase and Cochrane Library databases for published articles without language restrictions through October 25th, 2021. The following MeSH terms were used for all databases: (“Influenza vaccination”) AND . We also checked the conference abstracts and bibliographies of related literature to obtain other articles that might meet the requirements. Any discrepancy was resolved through discussion (ML and WL) until a consensus was reached.Studies were included if they met the following criteria: (a) were designed as randomized controlled trials (RCTs) or observational studies; (b) assessed the relationship between influenza vaccination and the risk of arrhythmia; and (c) reported estimate effects as adjusted relative risks (RRs)/hazard ratios (HRs)/odds ratios (ORs) with the corresponding 95% confidence intervals (CIs) or other measures that could be used to compute these values.According to the population, intervention, comparison, outcome, and study design (PICOS) framework, the inclusion criteria were as follows: (1) Participants: adults (aged > 18 years); (2) Exposure and comparator: vaccinated vs. Unvaccinated people or those beyond the period of efficacy after influenza vaccination; (3) Outcomes: the risk of arrhythmia  and estimate effects reported as RRs/HRs/ORs with the corresponding 95% CIs or other measures that could be used to compute these values; and (4) Types of studies: RCTs or observational studies. We selected the most recent publication if multiple studies used the same population. Certain publication types  or studies with unavailable data were excluded from this analysis.The study information and the basic characteristics of the articles were extracted, including the first author, year of publication, country, study design, study participants, sample size, age, sex%, method of vaccine exposure, length of follow-up, reported outcomes, risk estimates, 95% CIs and adjustments. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of the included observational studies. Studies with an NOS score > 6 were considered high-quality studies.I2-test  was used to represent the degree of heterogeneity across the studies   software and Stata software  were applied for analysis. ORs and 95% CIs were used to estimate the effects. The P < 0.05 was considered statistically significant.The random-effects model was applied considering the potential heterogeneity. Egger’s test and Begg’s test were used to detect publication bias. A two-tailed A systematic search of online databases revealed 360 articles . After excluding duplicates and screening the titles/abstracts, 24 articles were subjected to a more detailed full-text assessment, after which seven articles with 3,169,977 patients were included in the analysis , 17–20 . The excThe basic characteristics of the included studies are shown in P = 0.20) in patients after myocardial infarction or those with high-risk stable coronary heart disease after 12 months of follow-up. A meta-analysis based on observational studies showed that compared with the unvaccinated group, in the vaccinated group, the risk of arrhythmia decreased by 18% , with high heterogeneity   .P = 0.006), with low heterogeneity  (P = 0.12), with high heterogeneity  (P for subgroup difference = 0.65), although there was a borderline reduced risk of arrhythmia in the retrospective cohort studies , but there was no statistically significant difference in the case-control studies   ; however2 = 85%) . We also2 = 85%) .P = 0.452) and Egger’s test (P = 0.222) showed no statistically significant potential publication bias, although correcting for publication bias was not recommended considering that the number of included studies was limited (N < 10).As shown in Although an RCT reported a non-significant association between influenza vaccination and arrhythmias, pooled results from observational studies showed that the risk of arrhythmia decreased by 17% in vaccinated individuals compared with unvaccinated individuals, thereby demonstrating that influenza vaccination might play an important role in the prevention of arrhythmia. Subgroup analysis showed that influenza vaccination decreased the risk of AF by 6%. To our knowledge, this is the first meta-analysis to examine the association of influenza vaccination with the risk of arrhythmia.Pooled results from observational studies showed a decreased risk of arrhythmia with influenza vaccination; however, an RCT showed a non-significant benefit. The inconsistent results between the RCT and observational studies should be interpreted with caution. Several reasons may account for this inconclusive result. First, arrhythmias were a secondary endpoint in the RCT, and thus, the effect of influenza vaccination on arrhythmias might be below the power to detect a significant difference. Second, there was a very large magnitude of effect  for arrhythmias after influenza vaccination in the RCT. We cannot exclude the possibility that the risk estimates would reach significance if more patients or studies were included. Finally, the duration of the trial was only 12 months, which was relatively short and might have an impact on the outcome of hospitalization rates for arrhythmias. Overall, we supposed that the influenza vaccine may benefit against arrhythmias.The subgroup analysis showed that there was a significant benefit for AF rather than VA. The incidence of VA is much lower than that of AF, which might reduce the statistical power to detect a significant difference in the benefit against VA. Moreover, the measure of VA varied among the studies; two studies defined VA as the use of ICD therapies, one study defined VA as primary cardiac arrest, and one study did not state the definition clearly. The above point might be responsible for the non-significant association. Interestingly, the study of Chen et al. . DemonstP = 0.004) (P = 0.13) and reduced the frequency of coronary ischemic events in nearly 1 year of follow-up  (P = 0.004)  . The FLU= 0.047) . Another= 0.004) , and two= 0.004)  or coron= 0.004) . This re= 0.004) . TherefoAlthough the exact mechanism remains to be elucidated, there are several possible explanations . The mecSecond, the flu virus induces arrhythmias by remodeling the structure of the heart and the connections between cells. TNFα promotes fibroblast activation, leading to collagen III deposition and eventually contributing to ventricular and atrial fibrosis . TNFα al+/H+ exchanger, causing H+ transport out of the cell in exchange for Na transport into the cell (resulting in increased intracellular Na). This, in turn, causes cell swelling and Ca2+ overload, which is secondary to the activation of Na+/Ca2+ exchangers operating in the opposite mode (Na+ is transported out in exchange for Ca2+). These metabolic changes are accompanied by electrophysiological disturbances, including membrane depolarization that causes the inactivation of Na+ channels and a reduction in fast Na+ currents, consequently leading to a slowing of conduction that can result in subsequent arrhythmias.Last, infection with the influenza virus also affects the metabolism of cardiomyocytes. Studies have shown that inflammatory cytokines and CRP induced by influenza infection are also related to the occurrence and aggravation of myocardial ischemia, especially in individuals with comorbidities –45. The The results of this review suggest a positive effect of influenza vaccination on the risk of arrhythmia. Furthermore, the 2006 American and 2019 European secondary cardiovascular prevention guidelines recommend influenza vaccinations  to prevent influenza infection from exacerbating the condition of patients with cardiovascular disease , 46, 47.Our study has several limitations. First, most of the included studies were observational, and therefore, we cannot prove causality. Additionally, the most articles that we included were retrospective cohort studies and case-control studies. The benefit of influenza vaccination on arrhythmia were not significant in the subgroup analysis, which may be limited by the reduced sample size. More prospective studies may be needed to confirm the effect of the flu vaccine on the risk of arrhythmias. Second, all the observational studies were retrospective, and recall bias is unavoidable. Furthermore, some studies did not adjust for several clinical confounders, such as age or comorbidities. Third, substantial heterogeneity was found in the main results. When stratified by arrhythmia type, the heterogeneity was reduced to 0, but it remained moderate in VAs. The difference in the baseline clinical characteristics across studies, such as age, comorbidities, the measurement of VA, and study design, might be responsible for the heterogeneity. Finally, the population included in this article had different health conditions, and the participants were highly heterogeneous. Further studies are needed to investigate the effects of the flu vaccine on the risk of arrhythmias in people with different health conditions, especially patients with CVD.Based on the current evidence, we found that influenza vaccination may be linked to a reduced risk of arrhythmias, especially AF. Our results need to be interpreted with caution, and more prospective studies are needed to confirm our findings. Moreover, the effect of influenza vaccination on the risk of VAs and arrhythmias in individuals at low risk for cardiovascular diseases should be further studied.The original contributions presented in this study are included in the article/ML and ZY contributed to the study concept and design and revised the draft. WL, PY, and YZ performed the search strategy and contributed to database research, acquisition of data, and statistical analyses. All the authors participated in data analysis, reviewed, and approved the final manuscript.


The inequality caused by circumstances is known as "inequality of opportunity" (IOp). Many scholars have studied IOp in the health field, but few studies have quantified contributors to the IOp of health service utilization among middle-aged and elderly people. This study measured the IOp of health service utilization and decomposed the contributors to IOp present among Chinese middle-aged and elderly people.Data were obtained from the China Health and Retirement Longitudinal Study (CHARLS) in 2013, 2015 and 2018. A mean-based regression method was adopted to measure the IOp of health service utilization. Shapley–Shorrocks decomposition was used to analyze the main contributors to IOp seen among the middle-aged and elderly.Although the absolute IOp of health service utilization decreased over time, IOp still explains the total inequality to a large extent. The absolute IOp and relative IOp were greatest in the areas of self-treatment and inpatient care utilization, respectively. Shapley decomposition results showed that the out-of-pocket (OOP) ratio contributed most to the IOp of outpatient care utilization; and the residence area highly explains the IOp of inpatient service utilization. Meanwhile, social and economic factors such as work status and income contribute more to the IOp of inpatient care utilization than outpatient and self-treatment.Strategies aimed at achieving equal opportunities remain necessary to ensure the fairness of health service utilization. Policies and measures should further adjust the medical insurance compensation policies, and pay more attention to the middle-aged and elderly residents in rural areas, optimize health resource allocation, improve the social security systems, and narrow the socioeconomic gap between urban and rural areas in China. In addition to improving the health level and efficiency, equality has increasingly become one of the main goals of health reform efforts around the world . Health Many scholars have studied the inequality of health service utilization. However, most previous studies focused on analyzing the inequality of outcomes of health service utilization , 5–7. EqEquality of opportunity can be traced back to a new egalitarian method proposed by the political philosopher Rawls during the last century. This method regards personal responsibility as an important limiting factor of the ethically expected equality, arguing that opportunities should be equally open to all people, regardless of race, religion, or other status, and individuals should also be responsible for their own choices or preferences. Egalitarian theory has gradually developed from equality of results to equality of opportunities , in whicThe most common way to understand equal opportunity in health service utilization is from the concept of horizontal equity, which involves providing equal access to health services for those who have the same needs for health services. However, as health service utilization depends upon access conditions and personal preferences, even if access conditions are equal, it is impossible for people with the same demands to use equivalent health services . In viewScholars around the world have conducted research on IOp in health care. International scholars have carried out theoretical studies on the applicability and application principles of equal opportunity in the field of health care –24. For According to the results of China's seventh census, by the end of 2021, China's population aged ≥ 60 years had reached 267.36 million people, accounting for 18.9% of the country's total population, and those aged ≥ 65 years accounted for 14.2% of the total population . With thBased on Roemer's "equal opportunity" theory, this study measured the IOp in outpatient, inpatient, and self-treatment utilization among Chinese middle-aged and elderly people. Specifically, we first used a regression method to estimate the related factors of the utilization of different health services among the middle-aged and elderly. Then, an ex-ante method was used to measure the IOp of health service utilization. In addition, Shapley decomposition was used to evaluate the contributions of associated factors to the IOp. Finally, we proposed strategies to alleviate the IOp of health service utilization among middle-aged and elderly people, and provide new evidence for improving health service equity in China.The study data were obtained from the China Health and Retirement Longitudinal Survey (CHARLS), which were conducted by the National Development Research Institute of Peking University. To ensure the impartiality and representativeness of the sample, CHARLS conducts sampling at 4 levels, which are county (district), village, household, and individual. Probability proportional to size (PPS) is adopted at the county and village levels, and random sampling is adopted at the household and individual levels. CHARLS was conducted in 2011, 2013, 2015, and 2018 in 150 counties and 450 communities (villages) in 28 provinces of China, targeting individuals aged ≥ 45 years and their family members. The survey questionnaire covers personal basic information, family structure and financial support, health status, physical measurement, medical service utilization and medical insurance, work, retirement and pension, income, consumption, and assets. This database is the most representative national database of health and medical service utilization of middle-aged and elderly people in China, which can provide high-quality micro-data support for our research. After excluding samples with missing information on circumstance or effort variables, we constructed a longitudinal dataset of 13,769 consecutive participants surveyed in 2013, 2015, and 2018 to analyze the IOp of outpatient, inpatient, and self-treatment utilization. Figure Previous studies have shown that demographic characteristics, education, occupation, insurance status, residential area, etc., are related factors of health service utilization , 31–35. Some researches measure health service utilization by 2-week outpatient rate, hospitalization rate, etc. , 37, andCircumstances are considered "illegal" factors of inequality of health service utilization, that is, the non-need factor of health service utilization, such as medical service supply, medical financing, etc. . ResidenEffort is a personal choice and individuals should be responsible for their own preferences . IndividWe included age and gender as the main demographic variables for health service utilization because these 2 predisposing factors are major elements affecting health service utilization and have been analyzed in many previous studies , 41. FurFollowing Roemer's framework, personal outcomes or achievements depend on circumstances , efforts , and demographics (including gender and age). Therefore, the health service utilization result where In previous studies, many researchers discussed parametric and non-parametric approaches to evaluate IOp , 15, 42.The ex-ante approach assumes that, if all middle-aged and elderly people are facing the same opportunities before making efforts and making use of health services, then equality of opportunity exists . Roemer'where the residual term After organizing Eqs. 2 and 3, the production function of health service utilization can be rewritten using the following equation:where In order to measure the IOp, we used an inequality index——Gini coefficient, which is widely used to measure the IOp in many fields such as income and education –46. In tIf IOp does not exist, the Gini coefficient of the predicted value of the regression model should be the same as that of the original sample. For the convenience of observation and comparison, we calculated the ratio of IOp to total inequality, which can be regarded as relative IOp , and expShorrocks proposed the Shapley decomposition method based on the "Shapley value" to decompose inequality . The regTable Regarding circumstance factors, the number of middle-aged and elderly people with basic medical insurance increased (from 89.75% to 95.30%) during the period of 2013–2018. The OOP ratio of self-treatment was the highest (95.96%–96.68%), followed by that of outpatient (86.79%–87.97%) and inpatient (56.72%–60.47%) expenses. In addition, the vast majority of respondents (67.25%–64.05%) had only received a primary education. Most of the respondents were engaged in agricultural work (48.46%–57.60%) or were unemployed and retired (22.80%–39.49%).As for effort factors, from 2013 to 2018, the self-reported health status of middle-aged and elderly people showed a slight deterioration (from 2.99 in 2013 to 3.01 in 2018), while their objective health status improved, with a reduction in the number of chronic diseases from 1.29 in 2013 to 0.68 in 2018. The proportion of middle-aged and elderly people having supplementary medical insurance remained low, but the awareness of physical examinations has gradually increased: 34.39% chose to participate in physical examinations in 2013, which increased to 45.97% in 2018.P < 0.1) and inpatient services (P < 0.05) in 2013 than those aged 45–54 years. Male patients' hospitalization expenditures were significantly higher than that of female patients in 2013 (P < 0.1) and 2018 (P < 0.01), while female patients' self-treatment expenditures were significantly higher than those of male patients in 2015 (P < 0.01) and 2018 (P < 0.05).We first conducted a regression analysis on the associated factors of health expenditures (see Table P < 0.01). The absolute regression coefficient of OOP ratio is the largest in outpatient service utilization, indicating that OOP ratio has greater impact on outpatient service utilization than that of hospitalization and self-treatment, Distance had a greater influence on the utilization of inpatient services in 2013, while it was greater on the utilization of outpatient services in 2015 and 2018. Residence, education, and work status were also significant factors affecting the utilization of health services. Respondents who lived in urban areas used more outpatient care services in 2015 and 2018 (P < 0.01), more inpatient services in 2018 (P < 0.01), and more self-treatment in all waves (P < 0.01) compared with rural residents. People with higher education used more self-treatment . Those who were unemployed and retired also had higher medical expenses (P < 0.01). In addition, the results of 2013 and 2015 showed that, the higher the income, the more the hospitalization and self-treatment expenses (P < 0.05).Among the circumstances, middle-aged and elderly people with lower OOP ratios or who were traveling further to receive medical treatment had higher medical expenses (P < 0.01) or who participated in physical examinations (P < 0.01) in 2013 showed more outpatient utilization, and those with more chronic diseases also had significantly increased outpatient (P < 0.01) and inpatient (P < 0.01) spending in 2018. Supplementary medical insurance had a positive effect on the utilization of outpatient and inpatient services (P < 0.05) in 2015.All effort factors except for supplementary medical insurance significantly affected the utilization of self-treatment services among middle-aged and elderly people in 2013, 2015, and 2018. Respondents with worse self-reported health status , residence (5.57%–14.59%), and work status (0.93%–7.84%) were also factors that led to the IOp of outpatient expenditures, and their contributions were all increased. From 2013 to 2018, residence area highly explained the IOp of inpatient service utilization and its contribution increased over time (from 10.45% in 2013 to 30.55% in 2018). Work status (6.00%–24.40%), OOP ratio (5.98%–15.24%) and income (2.53%–21.20%) are also the main contributors to the IOp of inpatient service. Among all circumstance factors, the contributions of OOP ratio (7.00%–13.42%), residence (6.11%–13.74%), and work status (5.16%–13.40%) to the IOp of self-treatment expenditures were relatively slight but all increased from 2013 to 2018. The contribution of basic medical insurance to IOp of three health services utilization remained low in all waves.At present, China is under the realistic background of huge differences in the utilization of medical services and the policy background of the government constantly promoting the equalization of medical services . DeepeniHere, we found that the utilization of outpatient, inpatient, and self-treatment services among Chinese middle-aged and elderly people improved from 2013 to 2018, but differences in health service utilization among individuals still existed. The empirical results showed that residence area, OOP ratio, medical distance, work status in circumstance factors and physical examination in effort factors are important explanatory variables of the 3 types of health service utilization among middle-aged and elderly people (Table The IOp of outpatient care, inpatient care, and self-treatment utilization among the middle-aged and elderly overall declined over time Table : the IOpShapley decomposition revealed that the contribution of circumstance factors to the unequal utilization of various health services is gradually expanding; however, the contribution of individual effort factors is gradually weakening. Among circumstance factors, OOP ratio contributed the most to the IOp of outpatient care. This may indicate that differences in individual co-payment levels may account for much of the inequality in outpatient care, which is also confirmed by prior literature , 53. At In contrast, some socioeconomic variables in circumstance factors, such as work status and income, have limited contributions to the IOp of outpatient and self-treatment but a great impact on the IOp of inpatient service, respectively. This suggests that occupational and income factors play an important role in inpatient utilization, and these differences in socioeconomic status greatly contribute to the IOp of hospitalization utilization. Inpatient care utilization is sensitive to individuals' socioeconomic factors due to the higher costs associated with it. The middle-aged and elderly with informal jobs or lower incomes are always at risk of being prevented from taking advantage of inpatient utilization. Fortunately, the contribution of work status and income to the IOp of inpatient care utilization is decreasing according to our findings. Policy efforts should continue to focus on increasing the income level or providing medical subsidies for middle-aged and elderly people with low socioeconomic status to reduce the inequality of opportunities .The change of medical distance's contribution to IOp of the three types of health service utilization also deserves our attention. Medical distance's contribution to the IOp of hospitalization and self-treatment decreased from 2013 to 2018, which may reflect the recent implementation effect of the new medical reform policy in China, such as timely settlement of medical insurance in non-local treatment , which mOur study also confirms the dramatical influence of residence area on the unequal utilization of health services, which is consistent with existing studies , 51, 52,There are still some limitations that need to be acknowledged. We only used expenditure variables to measure personal health service utilization, without addressing more detailed health care demands or health-service quality variables, which may underestimate health service utilization to some extent. Besides, the expenditure data we used were self-reported by respondents, which may have recall bias and may not reflect the true health service utilization. Furthermore, there may be more circumstance factors leading to the IOp of health service utilization than those listed in this study. However, we could not include all related factors due to questionnaire limitations. Further studies will further explore the inequality caused by price or supply factors of medical services.Our research found that the absolute IOp of outpatient, inpatient, and self-treatment health service utilization among China's middle-aged and elderly people declined from 2013 to 2018, but the contribution of circumstance factors to the total inequality was still significant. The OOP ratio, residence area, work status, and income were the main contributors to the IOp of health service utilization, indicating that policies should focus on further increasing the security level and narrowing the reimbursement gap between different basic medical insurances to alleviate the IOp. In addition, policy efforts should pay more attention to the middle-aged and elderly residents in rural areas, further optimize health resource allocation, improve the social security systems, and narrow the socioeconomic gap between urban and rural areas in China.