Schistosoma mansoni that lives in the vasculature for decades and causes the tropical disease schistosomiasis.The consequences of the earth’s daily rotation have led to 24-h biological rhythms in most organisms. Even some parasites are known to have daily rhythms, which, when in synchrony with host rhythms, can optimise their fitness. Understanding these rhythms may enable the development of control strategies that take advantage of rhythmic vulnerabilities. Recent work on protozoan parasites has revealed 24-h rhythms in gene expression, drug sensitivity and the presence of an intrinsic circadian clock; however, similar studies on metazoan parasites are lacking. To address this, we investigated if a metazoan parasite has daily molecular oscillations, whether they reveal how these longer-lived organisms can survive host daily cycles over a lifespan of many years and if animal circadian clock genes are present and rhythmic. We addressed these questions using the human blood fluke Using round-the-clock transcriptomics of male and female adult worms collected from experimentally infected mice, we discovered that ~ 2% of its genes followed a daily pattern of expression. Rhythmic processes included a stress response during the host’s active phase and a ‘peak in metabolic activity’ during the host’s resting phase. Transcriptional profiles in the female reproductive system were mirrored by daily patterns in egg laying (eggs are the main drivers of the host pathology). Genes cycling with the highest amplitudes include predicted drug targets and a vaccine candidate. These 24-h rhythms may be driven by host rhythms and/or generated by a circadian clock; however, orthologs of core clock genes are missing and secondary clock genes show no 24-h rhythmicity.S. mansoni, but they appear less pronounced than in other organisms. The rhythms reveal temporally compartmentalised internal processes and host interactions relevant to within-host survival and between-host transmission. Our findings suggest that if these daily rhythms are generated by an intrinsic circadian clock then the oscillatory mechanism must be distinct from that in other animals. We have shown which transcripts oscillate at this temporal scale and this will benefit the development and delivery of treatments against schistosomiasis.There are daily rhythms in the transcriptomes of adult The online version contains supplementary material available at 10.1186/s12915-021-01189-9. CLOCK and ARNTL (BMAL1/CYCLE) and the negative regulators TIMELESS, CRYPTOCHROME and PERIOD, and secondary clock genes that modulate the effects of the core feedback loop; together, these drive oscillations in many clock-controlled genes. Circadian and clock-controlled genes are a subset of the genes that show daily patterns of expression (diel genes), and all together they lead to 24-h patterns in physiology and behaviour.Most organisms have biological rhythms that coordinate activities with the consequences of the earth’s daily rotation . These bPlasmodium chabaudi, these rhythms maximise its fitness in terms of within-host survival and between-host transmission  < 0.05) over a 24-h period: 206 in females, 194 in males and 48 in male heads   and between males and male heads  in males that encodes a BPTI/Kunitz protease inhibitor domain protein, as well as hsp70  in male heads and hsp90  in females that each encode heat shock proteins (HSPs) (Table ). In males, Hsp90 has a 4.8-fold change; it falls just outside the strict JTK BH.Q < 0.01 threshold [JTK BH.Q = 0.0103] but we include it in downstream analyses  and FKBP-type peptidylprolyl isomerase (PPIase) (Smp_004780); hsp90 peaks at the start of the dark phase, 4 h before PPIase , that PPIase is expressed in sperm throughout the testes and in all oocytes in the ovary , which has homologues located on the different sex chromosomes (known as gametologues)  Smp_332310; and RGS20 Smp_123050) peaked at night and are markers for muscle and nerve cells, respectively  and gtp-binding Ras-related protein (Smp_084940), a member of the histone co-chaperone pathway, also peaked at night in males and are markers for nerve cell types  and 3-phosphoinositide-dependent protein kinase 1 (Smp_094250) in males and hexokinase (Smp_043030) and protein phosphatase 1 regulatory subunit 3B (Smp_167660) in females   and Venom-allergen-like 12 . SmKI-1 is a tegument localised protein [Val12 is a marker for late tegumental progenitor cells (Sm13+ cells) [Val12 transcripts reach the body wall musculature, with some extending in the tubercles of the dorsal tegument  , and we In males, two additional day-time networks of interacting genes were predicted. The first was formed of four genes involved in DNA repair Fig. : Fig. S6Trematode Eggshell Synthesis domain-containing protein . To investigate whether transcriptional rhythms might reveal patterns in egg laying, we recorded day and night egg counts for individual worm pairs in vitro. For 54 worm pairs in total, we calculated day and night egg counts as the median of three consecutive days and nights, respectively (three replicates of each). Females, on average, laid 13 more eggs over the 12-h night-time, corresponding to a 50% increase compared with the 12-h day-time: for biological replicate 1, n = 12 females/pairs, median difference in night–day egg counts = 12.0, paired Wilcoxon test p = 0.003; biological replicate 2, n = 17, paired Wilcoxon test p = 0.002, median (night-day) = 5.3; replicate 3, n = 17, p = 0.0003, median (night-day) = 17.5; and replicate 4, n = 8, p = 0.008, median (night-day) = 14.3  and transmission. Seventeen of the 21 diel genes that are markers for cell types of the female reproductive system  peaked during the day, between 2.5 and 6.5 ZT. These included a , Smvlg2 ) and a sS. mansoni pairs (and population) inside the mammalian host. Host rhythms may be driving the parasite rhythms, and doing so either directly or by entraining an endogenous time-keeping machinery within the parasite—an intrinsic circadian clock.Of the 33 genes that cycle in both sexes, 17 showed identical phases and the remaining ones peaked within 4 h of the opposite sex , there were no putative hits for Period or any Cryptochromes , but not its paralogue Timeless , 32 with mouse  and only 4 with another lophotrochozoan, the limpet Cellana rota [p = 0.01117; 2 for males p = 0.3675).We identified putative homologues for many secondary clock genes, including orthologues for vrille, slmb/lin23, shaggy/GSK3 and doubletime/Ck1e/KIN-20  that had very high amplitudes in both sexes. By searching the ChEMBL database, we identified existing drugs that are predicted to target the encoded protein of 26 diel genes, 12 of which are phase IV approved drugs (i.e. with the best safety record for humans), including four metabolic chokepoints  has four phase IV compounds that are predicted to target it , and between pooled worms from the three host mice at each time point. The low amplitude of diel genes may also be due to this technical issue. Diel genes in male head samples had a higher median amplitude than the whole-body samples, suggesting that organ-specific cycling might have been dampened in our whole animal samples. It will be interesting to see the extent to which there are differences in diel expression patterns between tissues, as specific cell types and organs are sampled at this temporal scale, facilitated by recent advances in single cell transcriptomic methods [Drosophila, for example [S. mansoni adults live in the mammalian vasculature, a relatively predictable environment with muted oscillations (e.g. temperature), compared with the external environment. Living in a comparatively low amplitude environment may explain why we see fewer diel genes, and lower amplitudes in those genes, compared with the diel genes of free-living (non-parasitic) animals. However, the number of diel genes, and amplitude, is also low compared with the blood-dwelling protozoan parasite Plasmodium chabaudi. Over 80% of the transcriptome in this single-celled organism shows 24-h periodicity, with cycling genes having high amplitudes . TeUnlike other studies of which we are aware, we introduced an initial filtering step in our analysis. We excluded those genes that were not differentially expressed over time, prior to determining whether or not they were cycling. The aim was to reduce false-positive genes that had low amplitude cycling patterns due to chance fluctuations in the data. Additionally, this step reduced false negatives, by reducing the effect of the multiple hypothesis testing correction upon determining which genes were cycling using JTK_Cycle. This approach may therefore be useful in future studies of diel and circadian rhythms in transcriptome data.S. mansoni adults, and we have interpreted these rhythms within the context of daily oscillations in mouse vasculature  are controlled by the circadian clock to regulate protein aggregation and toxicity [S. mansoni, an increased unfolded protein response at night might be a reaction to, or anticipation of, periods of high ER-protein-folding demand that could be triggered by host cyclical rhythms, and this may be an adaptive stress response. One possible trigger is the daily body temperature cycle of the mouse, which increases by ~ 2.5 °C as the mouse becomes active at the onset of the dark phase [S. mansoni form part of the night-time interaction network in male worms  and in anti-phase (at the opposite time of day) to rhythmic processes involved in translation regulation and protein synthesis (e.g. ribosome biogenesis). The nightly peaks in hsp90 and PPIase, and PPIase expression in gametes, suggest possible rhythms in hormone cell signalling related to reproduction. In other animals, these proteins form part of a heterocomplex that chaperones steroid hormones S. mansonS. mansoni interactions with its host  peaked during the day. These included proteins that are secreted or localised to the tegument, e.g. SmKI-1 (Smp_307450) [carbonic anhydrase 12 (Smp_168730) [Val12 (Smp_123540) [SmK1-1 (Smp_307450) reaches its peak at midday (ZT 4.5)  , carboni_168730)  and Val1_123540) . The SmK_123540)  and inteS. mansoni. The lack of most of the animal core circadian clock genes suggests there is either an unusual oscillatory mechanism or a functional endogenous clock has been lost and the S. mansoni rhythms are responding directly to host rhythms. The last common ancestor of bilaterian animals is hypothesised to have had all the core animal circadian clock components—Period, Timeless and Timeout, Clock, Cycle/Bmal1 and Cryptochromes [S. mansoni genome was Timeout, suggesting all other core clock genes have been lost or have diverged beyond recognition. We identified S. mansoni orthologs of the secondary clock genes, vrille, slmb/lin23, shaggy/GSK3 and doubletime/Ck1e/KIN-20. However, neither Timeout nor the secondary clock genes showed 24-h periodicity. This could be a sampling artefact due to sequencing RNA from pooled, whole worm samples as clock gene rhythmicity may be limited to a subset of tissues [S. mansoni has one too. If, in future studies, any of these daily rhythms are discovered to be endogenous circadian rhythms, then our findings suggest that the S. mansoni clockwork must be quite distinct from that in other animals, and novel endogenous oscillators may be discovered within our list of diel genes.A daily program of gene expression clearly exists in ochromes —and combochromes –86, the ochromes . The onl tissues , or the  tissues , 90. How tissues . Neverth tissues , we predDespite the profound global impact of schistosomiasis, there is complete reliance on only a single drug (praziquantel) for treatment, and evidence of reduced susceptibility in some schistosome populations , 94 raisS. mansoni adults have daily rhythms in their transcriptomes is, therefore, not entirely unexpected. These daily rhythms in the parasite may be driven by host rhythms, either directly, and/or generated by an intrinsic circadian clock that is entrained to host cues. What is surprising, however, is that exploration of the genome revealed a lack of core clock genes that are generally conserved across other animals, and this is suggestive of an unusual oscillatory mechanism or loss of a functional endogenous clock. Most importantly, our identification of diel genes and daily processes has revealed fine-scale temporal partitioning of biological processes, some of which may serve the particular time-of-day challenges of life within the host, such as the activity of the host immune and haemostatic systems. These rhythmic genes and processes give us insight into how these parasites can survive for decades in this environment, and highlight the need to incorporate daily oscillations in transcript abundance into functional genomic studies aimed at developing and delivering novel therapeutics against schistosomiasis.This study has advanced our understanding of daily molecular oscillations in organisms to now include a metazoan parasite. Schistosome adults live in the bloodstream of a mammalian host, which is a 24-h rhythmic environment. Our finding that Schistosoma mansoni NMRI (Puerto Rican) strain is maintained at the Wellcome Sanger Institute (WSI) by breeding and infecting susceptible Biomphalaria glabrata snails and mice. Female Balb/c mice were bred at the WSI and maintained on individual air handling units at 19 to 23 °C and 45–65% humidity. Animals were given access to food and water ad libitum, maintained on a 12-h light/dark cycle and housed in groups of no more than 5 adults per cage. Welfare assessments are carried out daily, and abnormal signs of behaviour or clinical signs of concern are reported. All personnel at the WSI performing welfare checks on animals are trained and assessed as competent by a NTCO (Named training competency officer).The life cycle of Schistosoma mansoni cercariae collected from 13 infected snails as described [Thirty-six 6-week-old females were percutaneously infected with 200 mixed-sex escribed . In brievivoPHIXTM  at 4 °C for the dissection of heads the following week. We used male heads only as they are bigger and easier to dissect than female heads. RNA was extracted from each pool and sequenced  0.5) and then 4 h subsequently giving us collection times of ZT: 0.5, 4.5, 8.5, 12.5, 16.5 and 20.5 over two 24-h periods. ZT: 0.5 corresponds to 8 am in the human 24-h clock, so actual collection times were 08:00, 12:00, 16:00, 20:00, 00:00 and 04:00. At each collection time, worms perfused from mice were washed gently in serum-free DMEM media warmed to 37 °C. Mature, paired male and female worms from each mouse were separated from each other by gently pipetting with a glass Pasteur pipette. Six female worms were pooled in an Eppendorf tube, the media removed and replaced with 1 ml of TRIzol, and the same was done for six male worms. The worms were put in Trizol within 60 min of being perfused from the mouse. The worms were left in Trizol for 1 h at room temperature before being stored at −80 °C. A further 10 mature male worms were uncoupled from female worms, pooled from each mouse and fixed and stored in the RNA stabiliser vivoPHIXTM by cutting posterior to the ventral sucker and anterior to the testes. The heads were rinsed in 50% ethanol and pooled in TRIzol and the RNA extracted as for the whole worm samples. RNA quality was assessed using a Pico RNA kit for the BioAnalyzer (Agilent). Good quality RNA was extracted from all but one sample . Total RNA was enriched for mRNA using poly(A) pulldown. The sequencing libraries were prepared using the NEB Ultra II RNA custom kit on an Agilent Bravo WS automation system. All samples had 14 cycles of PCR, which was set up using Kapa HiFi Hot start mix and Eurofins dual indexed tag barcodes on Agilent Bravo WS automation system. Paired-end 75-nucleotide RNA sequencing reads were produced from the pooled worm libraries across six lanes of an Illumina HiSeq2500 v4 sequencing platform. All sequencing data are available through ENA study accession number ERP108923 [RNA was isolated from the pooled whole worm samples in TRIzol reagent according to the manufacturer’s instructions (Life Technologies). For the male head samples, ten additional male worms per mouse per time point were dissected in RP108923 .S. mansoni genome  using Slease 14 . Read colease 14  with defFDR greater than 0.05 were then excluded. Then to identify genes with ~ 24-h periodicity from those that are differentially expressed over 24 h, we used JTK _cycle [FDR < 0.01(JTK BH.Q < 0.01) were identified as cycling. We chose this strict threshold to minimise false positives.The popular JTK_Cycle method for identifying cycling genes does not determine whether a transcript varies significantly over time; it only determines whether it follows a pattern similar to a sine wave. So to identify robustly cycling diel genes, we first excluded genes that were not differentially expressed across the time course using the general linear model (GLM) approach in edgeR v3.24.3 . Only geK _cycle , using tS. mansoni (v7) gene set, Additional file Filtering the genes using edgeR increased the number of genes detected as cycling ; it reduced the input number of genes for JTK_cycle, thereby reducing the effect of the multiple hypothesis testing correction. Without initial edgeR filtering, JTK_cycle and Meta2d  (BH.Q < 0.01) only identified 15 and 35 diel genes in female worms, 40 and 97 in males and 4 and 5 in male heads . The combined number of diel genes from the three datasets without edgeR filtering was 58 (JTK_Cycle) and 133 (Meta2d) diel genes /min (trough), taking the mean of the replicates.For visualisation of cycling gene expression, normalised counts for replicates were averaged and then log-transformed to generate heatmaps using the FDR < 0.05, node_size = 5, method = ‘weight01’ and statistic = ‘Fisher’. GO terms for S. mansoni were downloaded from WormBase ParaSite using BioMart [To better understand the function of genes identified as cycling, we performed GO enrichment analysis of our gene lists using topGO , with FD BioMart  on the 5S. mansoni gene products to the KEGG pathway database was performed on the KAAS server (https://www.genome.jp/kegg/kaas/) using the GHOSTX program and BBH method. The significance of cycling gene enrichment in pathways was assessed using Fisher’s exact test, and resulting p values were adjusted using the Benjamini-Hochberg procedure, where maps in the KEGG categories 1–4 (https://www.genome.jp/kegg/pathway.html) were tested. Pathways with FDR < 0.05 were considered as significant. For visualisation, the R package Pathview [Mapping of Pathview  was usedwww.string-db.org; V 11) [Molecular interactions were predicted using the online search tool STRING  . Proteinp values were corrected using the Benjamini-Hochberg method. A Python script implementing this method is available from our GitHub page (https://github.com/adamjamesreid/schistosoma_daily_rhythms/).We used publicly available single cell transcriptome data from mature adult male and female worms  to deterSeurat uniform manifold approximation and projection (UMAP) plots were used to explore the expression of cycling transcripts across cell types and whether they were ubiquitously expressed, or enriched/specific to one or more cell type . Some ofPPIase) and Smp_123540 . Probes, buffers and hairpins for third-generation in situ hybridisation chain reaction (HCR) experiments were purchased from Molecular Instruments . Molecular Instruments designed probes against Smp_004780 for PPIase (Lot:PRA764) and Smp_123540 for Val12 (Lot: PRA767) from sequences on WormBase ParaSite . The temporal expression profiles of both genes were checked  for an hour at room temperature, incubating in blocking media plus 1:500 anti-acetylated tubulin over night at 4 °C, rinsing in 3 × 30 min in SSCT, adding secondary antibody (1:500) and phalloidin (1:500) in blocking media over night at 4 °C, then rinsing 3 × 30 min in SSCT at room temperature. Samples were mounted using DAPI fluoromount-G (Southern Biotech) and imaged on a confocal laser microscope (Leica Sp8).We carried out fluorescent in situ hybridisation on two diel genes; Smp_004780 (ted from ): anaestted from , 107 and2 in the dark. At 8 am and 8 pm every day for 72 h, eggs were collected from each well and counted, giving 3 day-time counts and 3 night-time counts per worm couple. To collect the eggs , the well plate was taken out of the incubator for 10 min, and the eggs were gently swirled into the centre of the well and collected in 1 ml of media using a stereomicroscope with low light settings. One millilitre of fresh media (warmed to 37 °C) was then added to each well and the plate returned to the incubator. The eggs collected in media were then counted manually using a stereo microscope. The first 12-h period post-perfusion was discounted to allow the worms to acclimate to the in vitro conditions. This experiment was replicated 3 times, each time with worms freshly perfused from one mouse. The median egg number for each worm for day-time and night-time was calculated, and a paired Wilcoxon test was carried out to determine if there was a significant difference in the number of eggs laid between day or night.Worms were perfused from one mouse, and twelve pairs of adult worms (still coupled) were placed into individual wells of a 12-well plate containing 3 ml of ABC169 media  and keptS. mansoni homologues of animal circadian clock genes using two methodologies. The first was a BLASTP sequence similarity search with a cutoff e-value of 1e−10 against the S. mansoni genome (v7) in WormBase ParaSite (WBPS release 14), using previously defined circadian protein sequences from UniProtKB [S. mansoni genome  and the Cryptochromes/ Photolyases, and the secondary clock proteins: Vrille, Slmb, Shaggy and Doubletime  transcripts identified by Schnytzer et al. [S. mansoni (WBPS15) to identify one-to-one orthologues using OrthoFinder [S. mansoni. Sixty-seven limpet cyclers and 96 S. mansoni cyclers were in shared orthogroups. We used the Fisher exact test to determine whether the number of one-to-one orthologues cycling in both species was greater than expected by chance.We identified 2925 cycling genes in se (CGDB ) and 323ParaSite . Of the hoFinder . Here weS. mansoni protein sequences of the cycling genes were used to search the ChEMBL v25 database [S. mansoni gene, we identified its top BLASTP hit amongst all ChEMBL targets, as well as any ChEMBL targets having BLAST hits with E-values within 1e+5 of the top hit’s E-value, and then extracted from ChEMBL the drugs/compounds with bioactivities against those particular ChEMBL targets. To calculate ‘compound scores’ for these drugs/compounds, we prioritised compounds in high clinical development phases, oral/topic administration, crystal structures, properties consistent with oral drugs and lacking toxicity . Brieflity (see  for detaity (see  (Proteinity (see  for detaity (see  and (2),Additional file 1: Supplementary tables 1-17. Table S1. All genes expressed in female Schistosoma mansoni samples over the two 24 hour periods including those with ~ 24 hr rhythmicity. Table S2. All genes expressed in male Schistosoma mansoni samples over the two 24 hour periods including those with ~ 24 hr rhythmicity. Table S3. All genes expressed in male head samples of Schistosoma mansoni over the two 24 hour periods including those with ~ 24 hr rhythmicity. Table S4. - Genes with differential expression over the two 24 hour periods in female Schistosoma mansoni samples including those with ~ 24 hr rhythmicity. Table S5. - Genes with differential expression over the two 24 hour periods in male Schistosoma mansoni samples including those with ~ 24 hr rhythmicity. Table S6. - Genes with differential expression over the two 24 hour periods in male head samples of Schistosoma mansoni including those with ~ 24 hr rhythmicity. Table S7. - Diel genes from the three datasets . Table S8. - Orthologs of Schistosoma mansoni diel genes. Table S9. - GO terms enriched in diel genes. Table S10. - Schistosoma mansoni adult cell types enriched in diel genes. Table S11. Diel genes involved in KEGG pathways. Table S12. Enrichment of Pfam families, InterPro domains, and KEGG pathways with diel genes. Table S13. Putative Schistosoma mansoni core and secondary circadian clock proteins. Table S14. Domain identifiers for main clock proteins were used to query WormBaseParaSite against the Schistosoma mansoni genome to identify putative orthologues. Table S15. -Animal circadian clock genes used in BLASTP searches and phylogenetic analyses to identify orthologues in Schistosoma mansoni.Table S16. One-to-one orthologs that show ~ 24 hr periodicity in Schistosoma mansoni and three other Metazoa; Drosophila melanogaster, Mus musculus and Cellana rota. Table S17. Diel genes with predicted drug inhibitors.Additional file 2: Supplementary Figs. 1-14. Figure S1. - STRING interaction of night-time peaking genes in female worms.Figure S2. – Heatmaps of diel genes involved in heat shock response and recovery. Figure S3-5. – Diel genes in KEGG pathways ‘Protein processing in endoplasmic reticulum’, ‘PI3K-AKT signaling pathway’ and ‘Estrogen signaling pathway’. Figure S6. - STRING interaction of day-time peaking genes in female and male worms. Figure S7. – In situ hybrisation of Val12 gene in males. Figure S8. – Heatmaps of sex-specific 24-hour rhythmic processes. Figure S9. – Day vs night egg counts. Figure S10. – Heatmaps of diel genes common to female and male worms. Figure S11. – Phylogenies of animal core circadian clock genes. Figure S12. – Phylogenies of animal secondary circadian clock genes. Figure S13. – PCA plot of all samples collected for male and male head time series. Figure S14. – Temporal expression profiles of genes validated by in situ hybridisation.Additional file 3: Supplementary information 1. SmKI-1, a Bovine Pancreatic Trypsin Inhibitor/Kunitz protease inhibitor domain protein. Supplementary information 2. Clock gene protein structure.


Mycobacterium tuberculosis complex bacteria, mainly Mycobacterium bovis (M. bovis), is a major threat to public health and economic development. There has been no systematic epidemiological assessment concerning bTB in dairy cattle in China.Bovine tuberculosis (bTB), caused by members of the Literature related to bTB in China was retrieved from China National Knowledge Infrastructure (CNKI), PubMed, ScienceDirect, VIP Chinese Journals Database, and Wan Fang Database to build the first meta-analysis for estimating the prevalence and infection moderators of bTB in dairy cattle in China. A total of 100 relevant studies published from 2010 to 2019 were included. We estimated the overall prevalence of bTB was 2.4% (95% CI: 2.1–2.8) during this decade. In the sampling year subgroup, the prevalence was lowest in 2017 or later at 0.8% (95% CI: 0.3–1.5). The lowest prevalence was 0.7% (95% CI: 0.5–1.0) in Northwestern China. The lowest prevalence was 2.1% (95% CI: 1.8–2.5) using SIT test. Heifer cows had the highest prevalence, which was 27.1% (95% CI: 9.7–49.2). The prevalence in scale farming was 3.7% (95% CI: 3.1–4.3), significantly higher than that in free-range farming . The prevalence of bTB was highest in summer at 4.0% (95% CI: 1.7–7.0). In addition, the influence of different geographical factors  on the prevalence was analyzed.The results showed that bTB was widespread in China but has been gradually reduced through concerted national intervention. It is suggested that different countries should formulate corresponding prevention and control measures according to the epidemic situation in its cattle industry. Enhanced monitoring of warm and humid areas may play an important role in reducing the incidence of bTB. In addition, when large-scale breeding is promoted, attention should be paid to standardizing breeding management and improving animal welfare to reduce the prevalence of bTB in cattle. Mycobacterium tuberculosis complex bacteria , which is also called “TB disease from bovine sources” when it occurs in humans. It is still widespread in China although the prevalence has gradually reduced through national intervention. Because beef and dairy products are important human dietary sources of protein, bTB has a great impact on public health and safety. We constructed the first meta-analysis to assess the epidemic of bTB in dairy cattle in China over the past 10 years and analyzed the potential moderators of bTB. A total of 100 studies were included. The results showed that bTB was common and unevenly distributed in China. The potential moderators affecting the bTB epidemic were region, sampling year, detection methods, age of dairy cattle, feeding mode, season, and certain geographical and climatic factors . These important findings may help in the formulation of policies to control the prevalence of bTB in cattle, thereby reducing the economic losses caused by bTB and reducing the threat of bTB to human health.bTB is a neglected zoonotic disease caused by members of the  Mycobacterium bovis (M. bovis)  OR Tuberculoses OR Kochs Disease OR Koch’s Disease OR Koch Disease OR AND (“China”[Mesh] OR People’s Republic of China OR Mainland China OR Manchuria OR Sinkiang OR Inner Mongolia).In ScienceDirect, the keywords of “Cattle”, “Tuberculosis”, “prevalence”, and “China” and article type of “Research articles” were used to search. The same Chinese search formula with fuzzy search and synonym expansion was used in advanced search of the three Chinese databases :Mycobacterium tuberculosis” AND “cattle”) extracted data from qualified studies. In case of disagreement, the author (QLG) conducted further evaluation. We strictly selected qualified literature according to the following criteria: (1) It was a cross-sectional prevalence study. (2) Data concerned cattle in China. (3) The test was performed using standard bTB testing methods, including single intradermal test (SIT), enzyme-linked immunosorbent assay (ELISA), IFN-γ-ELISA, SIT&IFN-γ-ELISA, and colloidal gold test. (4) The study provided sample totals and prevalence. (5) The subjects were dairy cattle. (6) The full text was available.We used standardized forms in Microsoft Excel 2007 to collect the following information: first author, publication year, sampling year, geographical region, sampling season, age, detection method, feeding mode, total number of samples, and positive numbers.According to the criteria derived from the Grading of Recommendations Assessment, Development and Evaluation method (GRADE) , we asseWe did all analyses using the “meta” package (version 4.12–0) in R software version 3.5.2 . As suggNote: t: transformed prevalence; n = sample size; r = positive number; se = standard error.2, Cochran’s Q, and χ2 tests. A random-effects model was used to perform the meta-analysis due to the expected strong heterogeneity. The publication bias was evaluated by the funnel plot, trim and fill analysis, and Egger’s test. It has been shown that different subgroups may generate different funnel plots because prevalence changes over time [To facilitate reporting, we reconverted the transformed summary proportion and its confidence interval . The varver time . TherefoSubgroup analysis and univariate meta-regression analysis were used to reveal factors that may contribute to heterogeneity among studies. The factors included geographical region (comparison of Northern China with others), sampling year (comparison of 2012 or before with other groups), detection methods (comparison of SIT with other groups), age (comparison of heifer with others), sampling season (comparison of summer with others), quality level (comparison of high with others). In addition, in order to reduce the heterogeneity caused by different detection methods, when a study used multiple detection methods, we pooled the data that came from the SIT method (because most of the studies we included used SIT).2.China has a vast territory and significant climatic differences between different regions. Thus, we also assessed the impact of different geographical factors on the study, including latitude (comparison of 30–35° with other groups), longitude (comparison of >110° with other groups), average annual precipitation (comparison of >1500 mm with other groups), average annual temperature (comparison of 10–15°C with other groups), average annual humidity (comparison of >70% with other groups), altitude (comparison of <1000 m with other groups). All surface meteorological data are from China Integrated Meteorological Information Sharing System (CIMISS). For sampling sites without weather stations, we selected weather station data from nearby counties through high-resolution maps. In case that was unavailable as well, meteorological station data from the nearest city were selected. For sampling sites without sampling years, we extracted data concerning only the altitude, longitude, and latitude. In addition, correlation analysis was conducted for each subgroup with detection methods and provinces respectively to trace the source of heterogeneity. The explained heterogeneity of the covariates is expressed in RAccording to the inclusion and exclusion criteria, there were two low-quality papers (which were excluded), 15 medium-quality papers, and 85 high-quality papers; ultimately, 100 included studies were used for meta-analysis Figs .The forest plot shows great heterogeneity between selected studies . The funM. bovis among dairy cattle in China was 2.4%   .2-methods), and that explained by provinces (the covariate) was 5.51%–41.78% (R2-provinces) . Among them, the prevalence in 2012 or before was much higher than in the other two time periods. Compared with other detection methods, IFN-γ-ELISA yields higher values of the prevalence. The prevalence of bTB in heifers was highest. The prevalence of bTB in cattle with scale breeding was higher than that in free-range breeding. The prevalence was much higher in summer than in other seasons. Medium-quality articles showed the highest prevalence . The hetovinces) .After subgroup analysis of geographical factors, we found that the prevalence in the groups of latitude 30–35°, longitude >110°, average annual precipitation >1500 mm, average annual temperature 10–15°C, average annual humidity 70%, altitude < 100 m was significantly higher than in the corresponding other groups . More intuitively, except for Xinjiang Province, the areas with high prevalence of dairy cow tuberculosis in China were generally concentrated in provinces with large dairy cow populations. Intensive farming has been shown to be a contributing factor to bTB in animals ,27. It i farming . In rece farming . However farming .Understanding age-specific risks is critical to accurately interpreting trends in pooled epidemiological data and to designing and evaluating control strategies such as vaccination . Almost M. bovis does not easily survive in a hot and dry environment [M. bovis to survive or to spread, and that a humid environment is conducive to its spread. It is therefore recommended to strengthen epidemic prevention in moist and warm areas to create a healthy living environment for livestock and reduce the incidence of diseases.In the seasonal subgroup, the prevalence in summer was significantly higher than in winter (P < 0.05). Cattle are more stressed in the hotter and wetter summer months ,31. In aironment , so we sIn terms of regional subgroups, the prevalence was lowest in Northwestern China and highest in Easthern China (P < 0.05). The geographical factors showed that the longitude range was >110° and the latitude was 20–35°, the highest prevalence was consistent with the regional results. This range belongs to subtropical humid monsoon climate and temperate monsoon climate. It is hot and rainy in summer, mild and humid in winter. The results were basically consistent with the above seasonal and geographical factors. We speculated that warm and humid weather may exacerbate the prevalence of bTB ,29,31.Among provinces of China, Shandong had the highest prevalence, while Guangdong and Gansu had the lowest prevalence (P < 0.001). In recent years, a number of modern standardized dairy enterprises that are strong on fundamentals have emerged in Gansu. They have abandoned the extensive model and focused on quantity and quality, so that the dairy farming industry in Gansu has developed well . HoweverThe 100 studies included five methods . Diverse detection methods usually bring heterogeneity to the meta-analysis of prevalence. Therefore, we used detection methods as a covariate to perform joint analysis with other moderators, and the range of heterogeneity explained by the detection method was only 0–4.95%, which implied that the detection methods had a small influence on each subgroup. The SIT is the standard method for detection of bovine tuberculosis. It involves measuring skin thickness, injecting bovine tuberculin intradermally into the measured area, and measuring any subsequent swelling at the site of injection 72 hours later. SIT (n = 89), which has been approved by the OIE and the European Commission as the main screening tool for bTB, is most frequently used in China ,42. The We analyzed the research quality and found that most of the articles of medium quality lacked detailed sampling methods and clear sampling time. It is suggested that personnel record relevant information in detail when conducting epidemiological investigation, to provide scientific data and theoretical support for the follow-up study of bTB.This study’s large sample size and rigorous methods, including comprehensive analysis of moderators, provide a reference for the prevention and control of bTB. However, several limitations may affect this meta-analysis. Firstly, due to the different retrieval strategies of different databases, we used several different retrieval forms and five databases in order to retrieve more qualified studies. Yet there may still be studies that have fallen through the cracks. Secondly, insufficient research on some subgroups  will affect the analysis results to some extent. Thirdly, due to lack of data, we were unable to extract all the potential moderators that are considered important, such as the number of calves produced and cattle breeds .Our study has shown that bTB is widespread among cattle in China. From our findings about factors affecting prevalence in China, we recommend that countries adapt their own prevention and control policies to local conditions, particularly strengthening the screening of cattle in warm and wet areas, strengthen the breeding of animals and management of animal welfare, and slaughter sick cattle in a timely manner. Carrying out bTB epidemiological investigation in more areas can extend the good foundation that has been laid for the prevention and control of bTB in the future.S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.S14 Fig(TIF)Click here for additional data file.S15 Fig(TIF)Click here for additional data file.S16 Fig(TIF)Click here for additional data file.S1 TextTable A. PRISMA Checklist item. Table B. The code in R for this meta-analysis. Table C. Egger’s test for publication bias. Table D. Included studies and quality scores. Table E. Normal distribution test for the normal rate and the different conversion of the normal rate(DOCX)Click here for additional data file.


The Chinese herbal formula TiaoGanYiPi (TGYP) showed effective against chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection. Hence, we aimed to clarify the mechanisms and potential targets between TGYP and CHB. The active compounds and related putative targets of TGYP, and disease targets of CHB were obtained from the public databases. The key targets between TGYP and CHB were identified through the network construction and module analysis. The expression of the key targets was detected in Gene Expression Omnibus (GEO) dataset and normal hepatocyte cell line LO2. We first obtained 11 key targets which were predominantly enriched in the Cancer, Cell cycle and HBV-related pathways. And the expression of the key targets was related to HBV infection and liver inflammation verified in GSE83148 database. Furthermore, the results of real-time quantitative PCR and CCK-8 assay indicated that TGYP could regulate the expression of key targets including CCNA2, ABL1, CDK4, CDKN1A, IGFR and MAP2K1, and promote proliferation of LO2 cells. In coclusion, we identified the active compounds and key targets btween TGYP and CHB, and found that the TGYP might exhibite curative effect on CHB via promoting hepatocyte proliferation and inhibiting the liver inflammatory processes. The worldwide estimates suggested that more than 2 billion people have been infected with HBV, with 248 million of these people are chronic hepatitis B (CHB) patients. And about 15–25% of CHB patients died from cirrhosis or liver cancer2, which caused a substantial global burden3. In China, there are 20 ~ 30 million CHB patients, and the mortality rate of HBV-related complications is higher than that in the world4. The disease process of CHB is affected by both HBV and the host immune system, while the pathogenesis of CHB is complex and still unclear. Meanwhile, existing treatment options including nucleos(t)ide analogs (NAs) and interferons (IFN) are limited in their efficacy and scope of application. At present, the discontinuation of NAs lacked operability in clinc5, and oral antiviral drugs also had limitations in improving complications6. Meanwhile, the contraindications and side effects hindered the clinical application of IFN7.It has been a global public health problem for the hepatitis B virus (HBV) infection for so long9. As for CHM prescriptions which contained a comlex compounds, often function with various multi-targeting and synergistic effects. Among CHM, the TiaoGanYiPi formula (TGYP) was used together with entecavir (ETV), and reported better efficacy for CHB treatment in our previous multi-center, double-blind, randomized placebo-controlled trial10. And in Chinese medicine theory, CHB is a complex-syndrome disease, and the TGYP is targeting at the core syndrome which is stagnation of Gan and deficiency of Pi qi, and damp-heat in the Gan and Dan11. However, the pharmacological mechanisms of TGYP regarding the therapeutic effect of CHB have not been fully explained. Here, we aimed to explore the relationship between CHB and TGYP, identify the key target genes and mechanisms of TGYP treatment on CHB through bioinformatics methods and in vitro experiments.For a long time, Chinese herbal medicine (CHM) plays a crucial role in CHB treatment and shows its superiorityIn this study, we first performed network pharmacology based on system biology and molecular docking, screened out 11 key target genes that exert essential functions during the treatment of TGYP on CHB. And the results of CCK-8 assay and real-time quantitative PCR (RT-qPCR) indicated that curative effect of TGYP could be related to promoting normal hepatocytes proliferation and regulation on the expression of partial key target genes. These results revealed the relationship between TGYP and CHB, further confirmed the effect of TGYP on hepatocytes, and indicated directions and targets for further research.Based on the previous work, which showed the anti-HBV effect of TGYP, we designed the flowchart of this study Fig. . As the To construct the compound-putative targets network of TGYP, we first analyzed the four main herbs in TGYP by CNKI and TCMSP databases and found 26 active components in Chaihu, 25 active compounds in Huangqi, 25 active compounds in Danggui, and 17 active compounds in Kudiding, respectively Figure A. Among To explore the biological function of TGYP target genes, the GO and KEGG pathway enrichment analysis were performed by DAVID. The GO enrichment analysis revealed that the GO-Cellular Component (CC) of TGYP target genes were predominantly involved in the Cytoplasm, Plasma membrane and Nucleus Fig. A; the GOWe analyzed and screened CHB disease targets, where we identified 8,037 targets in the GeneCards database, 415 targets in the DisGeNET database, and 236 targets in the NCBI Gene database, respectively Table . And we Since there was one gene lacked a corresponding protein structure and seven proteins did not interact with others based on PPI network, the remaining 1,192 proteins from STRING data were imported into the Cytoscape to further obtain interaction networks Fig. A. FilterFunctional enrichment analysis of the key target genes was performed after data screening to further understand the biological behaviors Table . The GO-To better understand the interactions between the compounds and target genes, we constructed an interaction network between the key target genes and corresponding active compounds in TGYP Fig. A. Accord12. Thus, we further analyzed the key target genes expressions in CHB liver tissues and their correlations with liver inflammation in the GEO dataset GSE83148. Compared to healthy controls, some key target genes were significantly higher expressed in CHB patients (P < 0.05), including ABL1, CASP8, CCNA2, CCNB1, CDK4, CDKN1A, HIF1A, and IGF1R . The active compounds in TCMSP were mainly filtered by integrating the pharmacokinetic properties comprising oral bioavailability (OB) ≥ 30%42, drug-likeness (DL) ≥ 0.1843, as well as half-life (HL) ≥ 4 h. The corresponding compounds chemical 2D structure SDF and canonical SMILES acquired from PubChem database (https://pubchem.ncbi.nlm.nih.gov). Data formats were transformed with Open Babel GUI software44. The Venn diagram was charted by the Venn tool (http://bioinformatics.psb.ugent.be/webtools/Venn/).Compounds of the main herbs in TGYP, including Chaihu, Huangqi, Danggui, and Kudiding were searched in CNKI and TCMSP database (http://www.swisstargetprediction.ch/) and the STITCH databases (http://stitch.embl.de/), respectively. Putative targets of TGYP were collected under the following conditions: selecting the species is homo sapiens. The probability value of each potential target listed in the SwissTargetPrediction database was used to investigate the accuracy of the current predictions, whose probability value ≥ 0.1 was identified. In addition, the potential target proteins with confidence score ≥ 0.15 in the STITCH database.The canonical SMILES or SDF of the active compounds were imported into the SwissTargetPrediction (https://www.genecards.org/), the DisGeNET database (https://www.disgenet.org/) and the NCBI gene database (https://www.ncbi.nlm.nih.gov/gene/). The search condition was using the keyword “Chronic Hepatitis B” and selecting the organisms “Homo sapiens” from the three platforms. The above gene ID was identified and standardized in the UniProt database (https://www.uniprot.org). We used Retrieve/ID mapping tool of UniProt to convert identifiers, which are of a different type to UniProt identifiers.The target genes related to CHB were gathered from followed three databases. The GeneCards database (https://string-db.org/) with confidence scores ≥ 0.4 and the species limited to “Homo sapiens”, and exported protein–protein interaction (PPI) data. Analyzed the PPI data and constructed network by the Cytoscape 3.8.0 (https://cytoscape.org/), and the non-connection genes were removed. We use the Analyzer plugin to analyze the PPI network and get the degree, through taking the over a double median of degree to get the preliminary hub network45. Then, using the CytoNCA plugin to analyze the preliminary hub network to get network topological parameters: BC, CC, DC, LAC, NC and SC. We took the excess median of BC, CC, DC, LAC, NC, and SC to obtain hub network46. The MCODE application calculated the hub network to get the dense regions47. The genes in dense regions from MCODE is called key targets.The overlapped genes (OGEs) of the compound-putative targets and disease targets were charted by the Venn tool. We inputted the OGEs into the STRING database . The GO function enrichment and KEGG pathway enrichment were analyzed by DAVID database (https://david.ncifcrf.gov/). The filtering of retrieval results is with a threshold value of p < 0.05 and count in descending order. The value of P reflected the significance of protein biological function, and the purpose of filtering is to select the most enriched biological annotation from a given list of genes. Bubble Diagrams were drawn by Sangerbox tools (http://sangerbox.com/AllTools?tool_id=9697991).The screened out genes were analyzed by the GO function analysis included BP, MF and CChttps://www.rcsb.org). Moreover, the 2D structures of the compounds were obtained from the PubChem Database. AutoDock Vina could markedly improve the average accuracy of the combined mode prediction49. Thereby the Molecular docking simulation was performed by AutoDock Vina software, and the result was visualized by PyMOL software50. In this analysis, the value of the vina score indicates the binding activity between a compound and a protein, and the lower the vina score, the more stably the compound binds to the protein.Molecular docking simulation was performed to verify the critical components' binding ability to the key targets and explore their accurate binding modes. The macromolecular protein target receptors were acquired from the RCSB PDB database (http://www.ncbi.nlm.gov/geo/). The keywords were set as "hepatitis B" and "homo sapiens", the tissue type was limited to liver tissue, and the gene expression data of GSE8314851 was downloaded from the GEO database. The platform of dataset GSE83148 is the GPL570 (HG-U133_Plus_2) Affymetrix Human Genome U133 Plus 2.0 Array, which includes 122 patients with CHB and 6 healthy liver samples, and includes 71 patients with abnormal transaminase and 34 patients with normal transaminase, which threshold of ALT was 40 and the threshold of AST was 35.The expression of key genes was verified in the GEO database  supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin and 5% CO2. For the acquisition of Chinese herbal compounds, the granules of TGYP formula were obtained and authenticated by the Nanning Peili Pharmaceutical Co., Ltd. The granules was dissolved in DMSO to 500 mg/mL and stored at -20 °C. The TGYP solution was added into cell culture with 500 µg/mL and use DMSO as control.5 LO2 cells were seeded in 6-well plates. After incubated for 24 h, the cells were treated with TGYP (500 μg/ml) for 48 h. Total RNAs were extracted using the RaPure Total RNA Mini Kit  according the manufacturer’s instructions. The reverse transcription of total RNA to cDNA was performed with qPCR RT Master Mix kit . RT-qPCR was performed using the Real-time PCR Detection System  with the SYBR Green Real-time PCR Master Mix . The primers used in this study are provided in Supplementary Table The 5 × 103 LO2 cells were seeded in 96-well plates with four duplications, and after TGYP treatment, CCK-8 assay kit  was carried out to assess the ability of cell growth through measuring the absorbance at the wavelength of 450 nm by the TECAN infinite M200 Multimode microplate reader .The 5 × 10Data for graphing was processed with GraphPad Prism 8.0 software. Statistical analyses were performed using the SPSS 25.0 statistical software package. For the comparison of quantitative data between groups, the Student’s t-test is used. Differences in means were considered statistically significant at P < 0.05. Significance levels are: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.


To investigate the prevalence of and factors associated with metabolic syndrome (MetS) in patients with psoriatic arthritis (PsA) or ankylosing spondylitis (AS).This cross-sectional study included PsA or AS patients who attended Siriraj Hospital  during March 2014 to October 2017. The Harmonized MetS definition was used to diagnose MetS. Demographic, clinical, and spinal radiographic data were collected. Logistic regression was used to identify factors associated with MetS.p < 0.001). Multiple regression analysis identified body mass index (BMI) > 23 (odds ratio [OR]: 3.7), female gender (OR range: 3.8–3.9), and the number of syndesmophytes or ankylosis [SynAnk] (OR: 1.1) were associated with MetS among PsA patients. For AS patients, BMI > 23 (OR: 9.1) and age > 40 (OR: 4.3) were associated with MetS. Disease activity index was not associated with MetS.Among 319 patients, 153 had AS and 166 had PsA. MetS was present in 43% of PsA and 19% of AS (> 23 or evidence of spinal change should be informed to screen for MetS. AS patients with age > 40 or BMI > 23 should be informed to screen for MetS.MetS was significantly more prevalent in PsA than in AS. Structural change of the spine was associated with MetS in PsA. PsA patients with being female, BMI  Spondyloarthritis (SpA) comprises a group of chronic inflammatory rheumatic diseases, including ankylosing spondylitis (AS) and psoriatic arthritis (PsA) . ChronicPublished systematic reviews and meta-analyses concluded that the prevalence of CVD is increased in PsA patients (odds ratio [OR]: 1.6) , and in Several studies reported the prevalence of MetS to be higher in patients with PsA (38.0 to 59.0%) than in general population , 14–17. This cross-sectional study enrolled patients older than 18 years that were diagnosed with AS according to Modified New York criteria , or diagDemographic data, disease duration, history of comorbidities , occupation, smoking status , current use of non-steroidal anti-inflammatory drugs (NSAIDs) and/or disease modifying anti-rheumatic drugs (DMARDs), current habitual exercise, and scores from the Thai versions of patient self-reported outcome assessments were collected, recorded, and analyzed. The aforementioned outcome assessments included the Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) , Patient> 130 and/or diastolic > 85 mmHg) was identified, participants were asked to rest in a seated position for 10 min, and then the BP measurement was repeated. All physical examinations were performed regardless of questionnaire and blood test results. Blood was drawn after 12 h of fasting to determine fasting glucose (FG), triglycerides (TG), HDL-C, and C-reactive protein (CRP). CRP was tested by particle-enhanced immunoturbidimetric assay  .Physical examination of peripheral joints (66 swollen/68 tender joint counts) and dactylitis were assessed by one rheumatologist (PC or CP). Body weight, height, waist circumference (WC), and blood pressure (BP) were collected. WC was measured at the midpoint between the lower margin of the last rib and the top of the iliac crest while standing, and at the end of normal expiration. If elevated BP (systolic 2 (kg/m2), and 23 kg/m2 (BMI > 23) was determined to be the threshold for overweight status in Asian population  was not associated with MetS (Table p = 0.009) and smoking status (p = 0.041) were also significantly associated with MetS in PsA patients. Factors that were significant in univariate analysis were further analyzed in multiple regression analysis. BMI > 23 (p = 0.008) and female gender (p = 0.007) were the two strongest associations with MetS. PsA patients with a higher number of SynAnk were more likely to be MetS (p = 0.002). DAPSA was not associated with MetS (p > 0.05).Regarding PsA patients, age > 40 and BMI > 23. Conversely, gender, smoking, disease duration, the number of SynAnk, and disease activity were not associated with MetS (Table > 23 (p < 0.001) and age > 40 (p = 0.01) were significantly and independently associated with MetS in AS patients.In univariate analysis, MetS was significantly associated with age p = 0.008] and a lower median [inter quartile range (IQR)] number of SynAnk  than male patients with PsA. Conversely, the prevalence of MetS in male patients with AS (22.3%) was higher than in female patients (13.6%) (p = 0.177). Men with AS also had a significantly higher median (IQR) number of SynAnk  and a longer median (IQR) disease duration  than women with AS. As a result, variables of interest from our analysis of the entire study cohort were further analyzed stratified by gender.Interestingly, female patients with PsA had a significantly higher prevalence of MetS . Moreover, male gender was found to be strongly negatively associated with MetS in PsA patients after adjustment for other factors, including smoking. Therefore, the association between smoking and MetS in PsA patients only in univariate analysis may be influenced by male gender.The association between smoking and MetS is not clear . In our This study has some limitations. First, the cross-sectional design does not permit exploration of changes in disease over time. Our measurements of disease activity could not assess the duration of activity of disease over time. Therefore, our results may not reflect the long-term course of the disease, except to the extent that structural changes of the spine are the result of chronic inflammation. Second, we did not have an age- and gender-matched control group. A control group is needed to better understand the contribution of specific risk factors. Third, our study had a small number of AS patients with MetS, which limited our ability to conduct subgroup analysis. Fourth, our participants came from a single tertiary care hospital, so our results may not be generalizable to other care settings and to a larger population of Thai PsA and AS patients. Fifth, some participants did not perform CRP, so there was no ASDAS-CRP or DAPSA assessment. However, our study included other disease status indexes (cDAPSA), and the results were comparable (data not shown). Sixth and last, radiographic change was determined by one rheumatologist who is a well-trained reader. However, the use of two well-trained interpreters may have yielded more accurate assessments of radiographic change. Syndesmophyte and intervertebral ankylosis which were irreversible lesions were counted as radiographic change in this study. They may be less sensitive to detect structural change than the Modified Ankylosing Spondylitis Spine Score. Diffuse idiopathic skeletal hyperostosis (DISH) which is strongly associated with the metabolic syndrome is characterized by chunky bridging osteophyte . An earl> 23 and structural change of the spine were associated with MetS in PsA. Traditional factors (BMI > 23 and age > 40) were associated with MetS in AS patients. Female patients with PsA were more likely to have MetS than male patients with PsA and female patients with AS. Patients with PsA or AS should be advised to maintain their BMI < 23 kg/m2. In addition to traditional factors of MetS, evidence of structural spinal change should be informed to screen for MetS among PsA patients.MetS was significantly more prevalent in PsA than in AS. Female, BMI


The effects of vitamin C on clinical outcomes in critically ill patients remain controversial due to inconclusive studies. This retrospective observational cohort study evaluated the effects of vitamin C therapy on acute kidney injury (AKI) and mortality among septic patients.n = 212) or Comparison  were reviewed. Propensity score matching was conducted to balance a number of covariates between groups. Multivariate logistic regressions were conducted predicting AKI and in-hospital mortality among the full sample and a sub-sample of patients seen in the ICU.Electronic medical records of 1390 patients from an academic hospital who were categorized as Treatment  and in-hospital mortality (OR = 1.67 95% CI [1.003–2.78]) after adjusting for demographic and clinical covariates. When stratified to examine ICU patients, vitamin C therapy remained a significant risk factor of AKI (OR = 1.61 95% CI [1.09–2.39]) and provided no protective benefit against mortality (OR = 0.79 95% CI [0.48–1.31]).Ongoing use of high dose vitamin C in sepsis should be appraised due to observed associations with AKI and death. Existing research regarding the effect of vitamin C on outcomes in patients with sepsis is inconsistent.This retrospective study of patients hospitalized for sepsis revealed an association between IV vitamin C usage and subsequent AKI and in-hospital mortality.The relationship between early treatment of high dose IV vitamin C and mortality in septic patients might suggest potential nephrotoxicity of vitamin C therapy.Findings, coupled with results from multiple randomized controlled prospective studies of IV vitamin C with mixed results, suggests that wide-spread use of high dose IV vitamin C requires further assessment.The benefits of ascorbic acid (vitamin C) therapy were first noted in the 1970s, related to infections and the common cold , it presSepsis remains a major public health issue as the 10th leading cause of death among non-Hispanic black individuals and 12th among non-Hispanic white individuals in the U.S., and infecting more than 1.7 million U.S. Americans annually . Within Early examinations of vitamin C supplementation, alone or in combination with thiamine (vitamin B1) and corticosteroids, revealed decreased inflammatory markers and end-organ dysfunction , 15. ExtSimilarly, research assessing the role of high dose vitamin C in causing or protected against acute kidney injury (AKI) is also conflicting and lacks validity. Several case reports have identified AKI and oxalate nephropathy in patients who received high doses of vitamin C –25. OtheIn August 2016, a sepsis vitamin order set was formalized but not mandated in the hospital in which the study was completed. The decision to utilize the order set was up to the individual provider. The order panel included IV vitamin C at a dose of 1.5 g, administered every 6 h for a maximum of 16 doses; IV hydrocortisone 50 mg every 6 h up to 28 doses; and IV thiamine 200 mg every 12 h for 8 doses. In December 2018, melatonin 0.5 mg at bedtime was added to the order set with no stop required. The order panel did not include diagnostic studies to determine the severity of sepsis nor did it include treatment option directions based on clinical parameters.This retrospective cohort study Fig.  was condThe following data were collected from the EHR: patient demographics; past medical history; visit characteristics ; primary and secondary diagnoses; hospital and ICU LOS; vasoactive drug use; and in-hospital mortality. Vasoactive drug use was defined as having received Dobutamine, Dopamine, Epinephrine, Norepinephrine, Phenylephrine, or Vasopressin. AKI was defined using ICD-10 code discharge diagnoses. Doses of IV vitamin C received were captured to categorize patients into two groups; comparison group (No or < 1.5 g of IV vitamin C), and treatment group . Individuals who received ≥1.5 g of IV vitamin C more than 12 h after hospital admission were excluded from the study as research suggests that vitamin C treatment after the first 12 h may not be as efficacious . SimilarTo assess bivariate associations, categorical data were compared using chi-square or Fisher’s exact test when appropriate, and continuous data were analyzed using independent-samples t-tests. Preliminary analyses revealed statistically significant demographic differences between individuals in the treatment versus comparison groups Table . Considep-value of less than 0.05 and 95% confidence intervals (CI) not including one were considered significant. All analyses were performed using SAS 9.4 software.Using the matched sample, preliminary bivariate associations with primary outcome variables  were conducted and statistically significant and clinically meaningful variables were entered into two multiple logistic regressions. As the majority of patients (96%) who received high dose IV vitamin C were admitted to the ICU, subsequent analyses were conducted on the sub-sample of ICU patients. Similar to the full population, PSM using the aforementioned variables was performed to balance between treatment and comparison groups who were seen in the ICU, with matching performed using a 2:1 ratio (comparison: treatment) to match the ICU groups’ sample sizes. Preliminary bivariate analyses and multiple logistic regressions were then conducted on the ICU sample predicting AKI and mortality. A Of the 26,346 patient admissions between 1 August 2016 and 31 December 2018, 16,159 were admitted via the ED. Of the ED admissions, 1390 were identified as having a diagnosis of sepsis alone, based on 42 ICD-10 codes and were included for analysis. Overall, diagnosis of AKI and in-hospital mortality rates were 50 and 9%, respectively. Patients were primarily male (54%), identified as black (52%) or white (42%), and had an average age of 59 years. The majority of patients had government insurance (77%), 38 and 9% of patients had a diagnosis of hypertension and diabetes, respectively, and 16% of patients received vasoactive drugs during their visit (see Table p = 0.02), had longer ICU LOS , were less likely to have a history of hypertension , more likely to have AKI , and more likely to have died in the hospital  compared to those in the comparison group  and more likely to have died in the hospital . Results of the multivariate logistic regressions are described below.Bivariate analyses revealed that individuals in the IV vitamin C group were slightly older  revealed that age (AOR: 1.03 95%CI: 1.02–1.04), ICU LOS (AOR: 1.003 95%CI: 1.001–1.004), race , sex , and IV vitamin C (AOR: 2.07 95%CI: 1.46–2.93) were associated with an increased likelihood of AKI. Other covariates including insurance, hypertension, diabetes, and vasoactive drug use were not significant. See Table A multivariate logistic regression predicting mortality revealed that age (AOR: 1.03 95%CI: 1.007–1.05), ICU LOS (AOR: 1.002 95% CI: 1.001–1.003), diagnosis of AKI (AOR: 3.21 95%CI: 1.70–6.03), and IV vitamin C (AOR: 1.67 95% CI: 1.003–2.78) were associated with in-hospital mortality. Insurance, race, hypertension, diabetes, and vasoactive drug use were not significant. See Table A multivariate logistic regression performed on the sub-set of individuals who spent time in the ICU found that age (AOR: 1.02 95% CI: 1.01–1.04), ICU LOS (AOR: 1.003 95% CI: 1.001–1.004), and IV vitamin C (AOR: 1.61 95% CI: 1.09–2.39) were significant predictors of AKI. All other covariates were not significant. See Table A multivariate logistic regression revealed that age (AOR: 1.03 95% CI: 1.01–1.04) and AKI (AOR: 2.01 95% CI: 1.12–3.59) were significant predictors of in-hospital mortality. All other variables in the regression analysis, including IV vitamin C, were not significant. See Table This retrospective study of patients hospitalized for sepsis revealed an association between IV vitamin C usage and subsequent AKI and in-hospital mortality. When examining the association of IV vitamin C therapy and AKI, the odds of experiencing AKI for individuals treated with IV vitamin C were 107% higher than the odds for individuals not treated with IV vitamin C, after controlling for a number of demographic and clinical variables. Findings also revealed that risk of AKI was increased for those who were male and older, which is supported in previous research , 32. FinWhen examining in-hospital mortality, results revealed that being older, having AKI, longer ICU LOS, and receiving IV vitamin C were associated with increased risk of mortality. Specifically, the odds of mortality were 67% higher for the IV vitamin C group compared to those in the comparison group. The association between high dose vitamin C and death was unexpected and not easily explained. Another variable found to be associated with an increased risk of death, which may lend insight into the relationship between vitamin C and mortality, was the diagnosis of AKI. Previous research indicates kidney failure increases the risk of death six to eight times in septic patients . Howeverhigh dose IV vitamin C (≥1.5 g), which may account for outcome differences. One study that examined higher doses (200 mg/kg/day) of vitamin C showed evidence of increasing rates of mortality, with higher mortality (50.6%) among a high dose vitamin C group compared to patients who received lower doses of vitamin C (50 mg/kg/day) (38%), though both treatment groups had lower mortality than the placebo group (63%) [The current finding that IV vitamin C is associated with increased risk of death is inconsistent with another large retrospective study which found IV vitamin C to be associated with reduced mortality among patients with sepsis . One reaup (63%) . NotablySub-analyses of the ICU sample revealed that age, longer ICU LOS, and receiving IV vitamin C remained predictors of AKI, while identifying as black and being male were no longer significant predictors. Older age, and ICU LOS are both known risk factors supported by previous research –38. A se. Others have suggested that similar myocardial tissue damage can occur in secondary forms of hyperoxalosis [The current study observed a moderate relationship between IV vitamin C and mortality in septic patients, which might suggest potential nephrotoxicity of vitamin C therapy. The association between high dose vitamin C and AKI due to oxalate deposition in renal tubules is well established . Since eoxalosis , 50. AcuThis study was limited in the retrospective nature of the design, which precludes causal-effect conclusions and prevents patient follow-up. Relatedly, the EHR data utilized did not include time stamps for diagnoses and treatment, thus temporal order is unknown. However, strict inclusion and exclusion criteria were applied in an effort to reduce selection bias and focus on community-acquired sepsis. Further, as vasoactive drug use and ICU admission were the only available surrogates for illness severity, these factors were considered in the PSM and sample stratification, respectively. Although the current study was limited to one hospital, the large sample size provided adequate power to detect statistically significant associations between patient-level demographic and clinical variables, and specifically, vitamin C usage, AKI, and mortality. Additionally, existing literature has commonly reported SOFA scores, which were unable to be utilized in the current study due to inconsistencies in collection. Many studies have used SOFA scores as a representation of mortality risk, whereas in the current study, we were able to directly examine in-hospital mortality as a primary outcome. Additionally, the usage of serum lactic acid and procalcitonin levels were reviewed as a potential measure of disease severity but could not be included in analyses due to data missing not at random. In other words, these laboratory tests were not performed equally across all groups of patients. Finally, the current study did not assess for other nephrotoxic drugs that could be related to AKI, such as antimicrobials and radiologic contrast dye. Future research should include these as well as other factors to assess the unique relationship between IV vitamin C and AKI.increase the likelihood of AKI and does not serve as a protective factor against mortality. The association of vitamin C with AKI, along with the association of AKI and mortality in patients admitted to the ICU, suggests that high doses of vitamin C may not be beneficial but toxic. This finding, coupled with results from multiple randomized controlled prospective studies of IV vitamin C with mixed results, suggests that wide-spread use of high dose IV vitamin C requires further assessment. Research examining whether there are significant differences in IV vitamin C therapy toxicity among sub-group populations is necessary to further identify appropriate usage of IV Vitamin C therapy. These investigations should also include the different sources of sepsis related to hospital admissions, including those admitted through other routes than the emergency department. Additional research utilizing existing hospital data from across the nation and worldwide are needed to better understand the relationship between high dose IV vitamin C therapy and subsequent outcomes as well as determine the toxic/effective dose.Vitamin C is required for many biological functions including tissue repair, proper immune function, and healing. In sepsis patients, vitamin C levels are depleted thus IV vitamin C therapy is proposed as a mechanism of sepsis treatment. This retrospective review of patients with sepsis revealed though that early use of high dose IV vitamin C may


Background: Butyric acid, a major short-chain fatty acid (SCFA), has an important role in the microbiota–gut–brain axis and brain function. This study investigated the role of butyric acid in treatment response in drug-naïve first episode schizophrenia.Methods: The study recruited 56 Chinese Han schizophrenia inpatients with normal body weight and 35 healthy controls. Serum levels of butyric acid were measured using Gas Chromatography-Mass Spectrometer (GC-MS) analysis at baseline  and 24 weeks after risperidone treatment (for patients). Clinical symptoms were measured using the Positive and Negative Syndrome Scale (PANSS) for patients at both time points.Results: At baseline, there was no significant difference in serum levels of butyric acid between patients and healthy controls (p = 0.206). However, there was a significant increase in serum levels of butyric acid in schizophrenia patients after 24-week risperidone treatment (p = 0.030). The PANSS total and subscale scores were decreased significantly after 24-week risperidone treatment (p's < 0.001). There were positive associations between baseline serum levels of butyric acid and the reduction ratio of the PANSS total and subscale scores after controlling for age, sex, education, and duration of illness (p's < 0.05). Further, there was a positive association between the increase in serum levels of butyric acid and the reduction of the PANSS positive symptoms subscale scores  after controlling for potential confounding factors.Conclusions: Increased serum levels of butyric acid might be associated with a favorable treatment response in drug-naïve, first episode schizophrenia. The clinical implications of our findings were discussed. Schizophrenia is a chronic, severe, and disabling neuropsychiatric disorder that affects ~1% of the world population and imposes an enormous burden on society . The undIn vivo study found that patients with schizophrenia have histone deacetylases dysregulation (Short-chain fatty acids (SCFAs), the main metabolites produced by gut microbiota, are believed to play a key role in microbiota–gut–brain crosstalk . SCFAs agulation . In addigulation , 18. TheFrom July 2017 to December 2018, Chinese Han drug-naïve, first episode schizophrenia patients with normal body weight were recruited from the psychiatric inpatient wards of the First Affiliated Hospital of Zhengzhou University. Healthy controls were recruited from the local community through advertising during the same time period. This study was approved by the Human Ethics Committee of the First Affiliated Hospital of Zhengzhou University, China . Written informed consents were obtained from all subjects after complete descriptions provided.The inclusion criteria for patients were: (1). met the criteria for Diagnostic and Statistical Manual of Mental Disorders fourth version (DSM-IV) schizophrenia; two independent psychiatrists used the Structured Clinical Interview for DSM-IV (SCID) to make the diagnosis , 20; (2)The psychopathology of patients was evaluated using the Positive and Negative Syndrome Scale (PANSS) by two research psychiatrists together. The PANSS is a standardized, semi-structured clinical interview that rates the presence and severity of positive and negative symptoms, as well as general psychopathology for people with schizophrenia within the past week. Of the 30 items, seven are positive symptoms, seven are negative symptoms, and 16 are general psychopathology symptoms. Symptom severity for each item is rated according to which anchoring points in the 7-point scale  best describe the presentation of the symptom . After b* 0.25 mm i.d. * 0.25 μm)  and helium was used as the carrier gas at 1 mL/min. Injection was made in split mode at 10:1 with an injection volume of 1 μL and an injector temperature of 250°C. The temperature of the ion source, interface, and quadrupole were 230, 250, and 150°C, respectively. The column temperature was initially 90°C, then increased to 120°C at 10°C/min, to 150°C at 5°C/min, and finally to 250°C at 25°C/min and kept at this temperature for 2 min . The detector was operated in electron impact ionization mode (electron energy 70 eV) using full scan and selected ion monitoring (SIM) mode. The quantity of each sample was analyzed with a calibration curve using standard chemicals.After fasting overnight, venous blood was collected between 7:00 and 8:00 A.M. for all subjects at baseline; the PANSS was administered to patients on the same day. For patients, fasting blood samples were collected and the PANSS was administered on the same day again after 24-week risperidone treatment. Serum was obtained through centrifugation at 3,000 rpm for 10 min, then transferred into Eppendorf tubes, and stored at −70°C for SCFA assay. SCFAs standards were prepared and stored at 0°C for use. In brief, 100 μL serum samples were vortexed in 50 μL of 15% phosphoric acid with 10 μL of 75 μg/mL isohexanoic acid solution as internal standard and 140 μL ether (30 s). Subsequently, the samples were centrifuged at 4°C for 10 min  and the supernatant was transferred into the vial prior to Gas Chromatography-Mass Spectrometer (GC-MS) analysis. The GC was fitted with a capillary column Agilent HP-INNOWAX /baseline PANSS score. A 50% or greater reduction ratio was defined as “good” response . Normality was tested with the Shapiro-Wilk test, and equal variance was evaluated before analysis of variance analysis. To compare demographic and clinical variables between groups, we used χresponse . The lonp's > 0.05). In addition, there was no significant difference between the two groups in serum levels of butyric acid (p = 0.206) (p's > 0.05).Fifty-six drug-naïve, first-episode schizophrenia patients were screened and 54 were enrolled because they met the inclusion and exclusion criteria. Forty-four patients completed the 24-week assessment . Thirty-= 0.206) . No signp's <0.001). The serum levels of butyric acid were significantly increased after 24-week risperidone treatment (p = 0.030)  .r = 0.34, p = 0.025) . There were no significant correlations between the reduction of PANSS total scores or other subscale scores and the increase in serum levels of butyric acid (p's > 0.05).We further found that there was a positive association between the reduction of the PANSS positive symptoms subscale scores and the increase in serum levels of butyric acid after 24-week treatment in schizophrenia patients (= 0.025) . After cr = 0.44, p = 0.003), positive symptoms subscale scores , negative symptoms subscale scores , and general psychopathology subscale scores  (p's < 0.05).We found that there were significant positive associations between baseline serum levels of butyric acid and the reduction ratio of the PANSS total scores (= 0.016) . After cn = 21) and poor response . Stepwise logistic regression analysis indicated that higher baseline serum levels of butyric acid predict a better treatment response after controlling for baseline age, sex, education, duration of illness .We further defined patients in two groups: good response  schizophrenia patients had increased serum levels of butyric acid after 24-week treatment; (2) the baseline serum levels of butyric acid were positively correlated with the reduction ratio of PANSS total and all subscale scores in schizophrenia patients; and (3) the increase in serum levels of butyric acid was positively correlated with the reduction of PANSS positive symptoms subscale scores after 24-week risperidone treatment in schizophrenia patients.Previous studies indicated that ASD and schizophrenia may share some common biological basis and genetic vulnerability . SeveralRecent studies have shown that butyrate may help normalize the behavioral and physiological abnormalities in ASD , 29. SchIt has been well-established that dopamine is associated with positive symptoms of schizophrenia , 38. SomThe strengths of the present study include the use of a group of drug-naïve, first-episode schizophrenia patients with normal body weight and risperidone monotherapy. The selection of study participants in our study minimized potential confounding factors such as previous exposure to antipsychotic medications, variations in disease state (acute vs. chronic) or duration, differences in obesity and other metabolic disturbances at baseline. In longitudinal analyses, we take both non-completers and completers into account and a linear mixed model was used to calculated them. Several limitations of the present study should be noted. First, this study included a relatively small sample size and a short time duration of treatment. Furthermore, some important factors related to SCFAs, such as food intake and intestinal microbial condition, were not measured.In summary, our study suggests that increased serum levels of butyric acid might be associated with a favorable treatment response in drug-naïve, first episode schizophrenia. Therefore, approaches that affect butyric acid metabolism, for example, a change in gut microbiota, may be considered as an adjunctive treatment for schizophrenia. Future studies further examining the mechanism by which butyric acid may affect the development and treatment of schizophrenia are warranted.The original contributions presented in the study are included in the article/supplementary files, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by the Human Ethics Committee of the First Affiliated Hospital of Zhengzhou University, China . The patients/participants provided their written informed consent to participate in this study.XL and XF designed the study and wrote the manuscript. XL, XY, and LP were in charge of conducting clinical assessment, collecting fasting blood samples, and doing experiments. XL and SH undertook the statistical analysis and wrote the first draft of the manuscript. YW, XH, and XS were responsible for supervision and reviewing. All authors contributed to and have approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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. 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