Berberine Repairs Intestinal Mucosal Barrier by Targeting HSP90AA1 and MAPK14

Sophoricoside improved Crohn's disease-like colitis by inhibiting intestinal epithelial cell apoptosis through PI3K/AKT signaling

The incidence of inflammatory bowel disease (IBD) is increasing in Asian countries. Treatment of IBD is difficult in Asian countries due to low socioeconomic status, lack of medical facilities, and high prevalence of infections such as tuberculosis, hepatitis virus, etc. which have to be excluded before treating IBD. The severity of Crohn’s disease (CD) can be categorized into three levels: mild, moderate and severe. Severe CD has poor prognosis and is characterized by severe lower gastrointestinal bleeding, intestinal stricture or stenosis, intestinal fistula, severe condition, fever, severe ileocolitis appearances on colonoscopy examination and Crohn’s Disease Activity Index of more than 450. The basic treatment for CD includes nutritional, pharmacological, surgical and CD complication treatment. In severe CD, we usually give low-residue diet. In mild CD, we give balanced nutrition, high-protein diet depending on the daily demand, food that does not irritate and food that does not cause lactose intolerance. We suggest avoiding cow’s milk and food containing lactase (lactaid). Iron, calcium, magnesium, zinc, vitamin B12, vitamin D, vitamin K, vitamin E, vitamin C supplementation must be given according to the deficiency. Pharmacological treatment is divided into general supportive therapy (antidiarrheal, antispasmodic, analgesic), anti-inflammatory drugs (aminosalicylates, corticosteroids), immunosuppressive drugs, biological agents, antibiotics, probiotics, complementary alternative medicine/herbal drugs, and other medications. Aminosalicylates include sulphasalazine, 5-aminosalicylates, olsalazine and balsalazide. Corticosteroids include prednisone/prednisolone, methyl prednisolone and budesonide. Immunosuppressive drugs that can be given are 6-mercaptopurine, azathioprine, methotrexate and cyclosporine. Biological agents include infliximab, adalimumab (fully human), certolizumab pegol and natalizumab. Antibiotics that can be given to CD patients are metronidazole, ciprofloxacine, rifaximin, anti-mycobacterial agents. Probiotics can be given to CD patients, but this is still controversial. Herbals that can be given to CD patients include Boswellia serrata gum resin and Curcuma longa extracts. Non-herbal medicine which has already been given in CD includes seal oil, acupuncture and moxibustion. Other medications that can be given to CD are antidiarrheals, laxatives, acetaminophen, teduglutide, helminthic therapy, cannabis-based drugs and stem cell therapy. To predict a severe course or prognosis of CD, the disease activity (severity) index and the endoscopic disease activity score may be used. The endoscopic response to treatment in CD is defined as a significant change in endoscopic disease activity score, such as the Crohn’s Disease Endoscopic Index of Severity (CDEIS) or the Simple Endoscopic Score for Crohn’s Disease (SES-CD). The disease activity (severity) index can be evaluated with some scoring parameters such as the CDEIS, SES-CD, Rutgeerts score, and Crohn’s Disease Activity Index.

Background and aimsPatients with Crohn’s disease (CD) exhibit excessive apoptosis of intestinal epithelial cells (IECs), which contributes to damage to the intestinal barrier structure and function, thereby playing a role in the progression of colitis. Preventing IEC apoptosis and protecting the intestinal barrier are critical to alleviating colitis. Natural plant monomers have been reported to possess multiple pharmacological properties, particularly with the potential to treat CD. This study focuses on Cynaroside (Cyn) to explore its effect on IEC apoptosis and evaluate its pharmacological impact on the intestinal barrier and colitis.MethodsThe 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis mice model was employed in this study. We assessed the therapeutic effect of Cyn on CD-like colitis by evaluating the disease activity index (DAI), body weight changes, intestinal tissue pathological damage, and inflammatory factor levels. Immunofluorescence and Western blotting were used to detect the expression and localization of tight junction (TJ) proteins, allowing us to analyze the intestinal barrier structure. The function of the intestinal barrier was examined using FITC-dextran (FD4), TEER values, and bacterial translocation. Network pharmacology enrichment analysis revealed that Cyn could inhibit cell apoptosis. We also explored the effect and underlying mechanism of Cyn in inhibiting IEC apoptosis on intestinal barrier function and colitis using both the TNF-α-induced colonic organoid model and the TNBS-induced mouse model.ResultsOur findings show that Cyn significantly alleviates TNBS-induced colitis symptoms in mice, as evidenced by reduced body weight loss, colon shortening, DAI score, colon histopathology score, and lower levels of inflammatory factors (IL-1β, TNF-α, and IL-6) compared to the model group. Additionally, the Cyn intervention group showed significant improvements in both the intestinal barrier structure (elevated tight junction protein levels and proper localization) and function (reduced serum FD4 levels, increased intestinal TEER, and decreased bacterial translocation rates in mesenteric lymph nodes [MLNs] and livers). Combining network pharmacology prediction analysis with our validation data from animal models and colonic organoids, we demonstrated that Cyn significantly inhibits IEC apoptosis, as indicated by a decrease in the proportion of TUNEL-positive cells and changes in apoptosis-related protein levels. KEGG enrichment analysis and signaling pathway intervention experiments confirmed that Cyn inhibits the activation of PI3K/AKT signaling.ConclusionCyn inhibits IEC apoptosis by blocking the PI3K/AKT signaling pathway, which is the primary mechanism underlying its protective effects on the intestinal barrier and its ability to improve CD-like colitis. This study also supports the potential of the Chinese medicine monomer Cyn as a promising therapeutic agent for the treatment of CD.

Infliximab treatment of postoperative ulcers in Crohn's disease: to inject or not to inject—that is the question

Context: Maintenance therapy for Crohn disease features the use of immunosuppressive drugs, which are associated with an increased risk of infection. Identification of safe and effective maintenance strategies is a priority. Objective: To determine whether the oral administration of omega‐3 free fatty acids is more effective than placebo for prevention of relapse of Crohn disease. Design, Setting, and Patients: Two randomized, double‐blind, placebo‐controlled studies (Epanova Program in Crohn's Study 1 [EPIC‐1] and EPIC‐2) conducted between January 2003 and February 2007 at 98 centers in Canada, Europe, Israel, and the United States. Data from 363 and 375 patients with quiescent Crohn disease were evaluated in EPIC‐1 and EPIC‐2, respectively. Interventions: Patients with a Crohn's Disease Activity Index (CDAI) score of less than 150 were randomly assigned to receive either 4 g/d of omega‐3 free fatty acids or placebo for up to 58 weeks. No other treatments for Crohn disease were permitted. Main Outcome Measure: Clinical relapse, as defined by a CDAI score of 150 points or greater and an increase of more than 70 points from the baseline value, or initiation of treatment for active Crohn disease. Results: For EPIC‐1, 188 patients were assigned to receive omega‐3 free fatty acids and 186 patients to receive placebo. Corresponding numbers for EPIC‐2 were 189 and 190 patients, respectively. The rate of relapse at 1 year in EPIC‐1 was 31.6% in patients who received omega‐3 free fatty acids and 35.7% in those who received placebo (hazard ratio, 0.82; 95% confidence interval, 0.51‐1.19; P = .30). Corresponding values for EPIC‐2 were 47.8% and 48.8% (hazard ratio, 0.90; 95% confidence interval, 0.67‐1.21; P = .48). Serious adverse events were uncommon and mostly related to Crohn disease. Conclusion: In these trials, treatment with omega‐3 free fatty acids was not effective for the prevention of relapse in Crohn disease. (JAMA. 2008;299:1690‐1697.)Feagan BG, Sandborn WJ, Mittmann U, et al.

Background:The effect and mechanism of Saccharomyces boulardii (Sb) in inflammatory bowel disease are unclear. The objective of the study was to evaluate the impact of Sb on intestinal mucosal barrier and intestinal flora in a colitis mouse model.Methods:Forty C57BL/6J male mice were randomly assigned to five groups: normal control group (A), pathologic control group (B), Sb treatment group (C), mesalazine treatment group (D), and Sb combined with mesalazine treatment group (E). Colitis was induced by the addition of 2.5% (wt/vol) dextran sodium sulfate (DSS) in the drinking water ad libitum for 7 days. The general condition, weight change, stool property, and bloody stool level of mice were observed to evaluate the disease activity index. The expression of zona occludens-1 (ZO-1) and occludin in intestinal tissue were measured by immunohistochemistry. The level of tumor necrosis factor-α (TNF-α) and interleukin (IL)-8 in plasma was measured by enzyme linked immunosorbent assay. Inter-cellular tight junctions were observed by transmission electron microscopy. The feces and intestinal contents were collected sterilely, and intestinal flora was analyzed by 16S rRNA sequencing.Results:Compared with group B, Sb reduced the disease activity index and histological score of group C (disease activity index: group B 2.708 ± 0.628, group C 1.542 ± 0.616, PBC = 0.005; histological score: group B 9.875 ± 3.271, group C 4.750 ± 1.832, PBC = 0.005) in DSS-induced colitis in mice. Sb exerted a protect effect on the expression of ZO-1 (group B 2.075 ± 1.176, group C 4.225 ± 1.316, PBC = 0.019) and occludin (group B 2.200 ± 0.968, group C 3.525 ± 1.047, PBC = 0.023). Compared with group B, Sb decreased the level of TNF-α and IL-8 of group C (TNF-α: group B 716.323 ± 44.691 ng/L, group C 521.740 ± 90.121 ng/L, PBC = 0.001; IL-8: group B 128.992 ± 11.475 pg/mL, group C 106.283 ± 15.906 pg/mL, PBC = 0.012). Treatment with Sb preserved the tight junctions and ameliorated microvilli and inter-cellular space. Treatment with Sb also showed its own characteristics: a higher percentage of Bacteroidetes and a lower percentage of Firmicutes, with significant differences or a significant trend. The proportion of the S24-7 family was increased significantly in the Sb treatment group.Conclusions:Sb shows an anti-inflammatory effect and has a protective effect on the intestinal mucosal mechanical barrier. Sb may up-regulate the abundance of family S24-7 specifically, and maybe a mechanism underlying its function.

Intestinal epithelial barrier dysfunction is intricately linked to the pathogenesis of ulcerative colitis (UC). Dietary interventions that bolster intestinal epithelial barrier function can effectively thwart UC onset. Our prior research revealed that p-Hydroxy benzaldehyde (HD), a phenolic compound from Nostoc commune (an edible cyanobacterium), markedly upregulated the expression of E-cadherin, a pivotal protein in intestinal mucosa, thereby mitigating mucosal damage in mice afflicted with dextran sulfate sodium (DSS)-induced colitis. Nevertheless, the precise molecular mechanisms underpinning HD's ameliorative effects on intestinal epithelial barrier dysfunction remain elusive. Dextran sodium sulfate (DSS)-induced colitis mouse model was established, and the successful establishment of the model was determined by evaluating the changes in body weight, disease activity index (DAI), colonic histopathology, and white blood cell count. Transmission electron microscopy (TEM) observed the ultrastructural changes of intestinal villi. The levels of inflammatory factors ( IFN-γ IL-13 ) and intestinal permeability indicators (FITC-Dextran, DAO, ET, and D-LA ) were detected by Enzyme-linked immunosorbent assay (ELISA). Western blotting (WB) and immunohistochemistry (IHC) were used to detect the expression of intestinal barrier integrity-related factors such as tight junction protein TJs (ZO-1, occludin) and adhesion junction protein AJs (E-cadherin). Furthermore, WB, Pull-down assay, drug affinity reaction target stability (DARTS) assay, molecular docking and molecular dynamics (MD) simulation were used to determine the potential target and molecular mechanism of HD. HD intervention significantly alleviated the symptoms of colitis mice, inhibited the weight loss and colon shortening, reduced DAI score and colon pathological score, maintained the ultrastructure of intestinal villi in colon tissue, and significantly reduced the inflammatory factors IFN-γ, IL-13 and the number of white blood cells in colon tissue of colitis mice. HD could also reduce the levels of FITC-Dextran, DAO, ET, and D-LA and increase the expression of ZO-1, occludin, and E-cadherin in the colonic tissues of colitis mice, thereby maintaining the impaired intestinal barrier function caused by colitis. Mechanically, HD augmented the expression of hepatocyte nuclear factor 1β (HNF-1β) and DRA. Adeno-associated virus (AAV)-HNF-1β shRNA or Lentivirus-mediated HNF-1β knockdown effectively abolished HD-induced intestinal barrier protection, as well as the promotion of solute carrier family 26 member 3 (SLC26A3) expression levels. SLC26A3 siRNA effectively reversed the inhibition of intestinal permeability by HD. Pull-down assay, DARTS analysis, molecular docking, and MD results showed high binding strength, interaction efficiency and remarkable stability between HNF-1β and HD. This study elucidates HD's role in forestalling intestinal epithelial barrier disruption under colitis conditions. Mechanistic investigations revealed that HD fortifies TJs and AJs expression via the HNF-1β/SLC26A3 pathway, thus preserving the lower intestinal epithelial barrier's integrity in UC.

Unique Role of Junctional Adhesion Molecule-A in Maintaining Mucosal Homeostasis in Inflammatory Bowel Disease

An enriched environment (EE) is a promising strategy for protecting the intestinal mucosal barrier and regulating the brain-gut axis, but the optimal EE intervention duration is unknown. Here, different EE intervention durations were applied to assess the optimal intervention duration in rats with colorectal cancer. We used a rat model of 1, 2-dimethylhydrazine-induced colorectal cancer. The rats were housed in an EE for 0, 2, 4, 8 weeks and 8-week blank group. The intestinal mucosa and serum TNF-α, IL-6, IL-10, ATP, CRF, and occludin levels and bacterial translocation (BT) were measured, and the intestinal mucosa morphology was evaluated. In 8 weeks, the effect of tumor on intestinal mucosal barrier was not obvious and the EE had a greater impact on it. Eight weeks of EE was more beneficial to the intestinal mucosal mechanical barrier than 2 or 4 weeks of intervention. A significant difference in BT was found between the 4- and 8-week groups. Overall, the analysis of inflammatory factor regulation revealed that the two blank groups exhibited the worst effect, and the intervention effect at 8 weeks was better than that at 2 and 4 weeks. CRF at 4 weeks was higher than that at 8-week blank group. The effect of 8-week intervention duration on the intestinal mucosal barrier was generally better than that of 2- and 4-week durations and intervention within 4 weeks can help to stabilize and promote the secretion of brain gut peptide, but the effect of different intervention durations on the brain-gut peptide levels was not obvious. In the future, we can further explore the molecular biological mechanism of the effect of different EE intervention durations on the intestinal mucosal barrier and analyze the effect of an EE on other brain-gut peptides.

Intestinal mucosal mechanical barrier is one of the most important structure to maintain the body homeostasis. The occurrences of inflammatory bowel disease, necrotizing enterocolitis and poor prognosis of patients with obstructive jaundice are closely related to the damage of the mucosal barrier function. Long-term high fat diet and obstructive jaundice can cause the abnormality of bile acids metabolism. These pathological conditions are often associated with the destruction of intestinal mucosal barrier function. So the correlations between abnormal bile metabolism and intestinal mucosal mechanical barrier function have aroused interests of many researchers. They found that bile acids the important component of bile are closely related to the intestinal barrier function. The paper reviewed the recent articles and summarized the mechanisms of the deficiency of bile acids, excessive bile acids and abnormal bile acids composition damaging the intestinal mucosal barrier function. It will provide refe-rence for the new fields of study, prevention of the toxic effects of bile acids and the improvement of the prognosis of patients. Key words: Inflammatory bowel disease; Bile acids; Intestinal barrier function; High-fat diet; Obstructive jaundice

An enriched environment (EE) is an animal housing technique where animals are given increased amounts of space, physical activity, and social interaction. Presently, researchers studying EEs focus mainly on their effects within the context of neurological diseases. However, little is known about how EEs affect the intestinal mucosal barrier. This study assessed the effects of an EE on the intestinal mucosal barrier in rats with colorectal cancer.

The present study aimed to identify hub genes associated with the treatment and control of active and inactive Crohn's disease (CD). Differentially expressed genes (DEGs) were identified in normal, active CD, and inactive CD samples from GSE95095 dataset. Intersection genes screened by Venn diagram in DEGs. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted on the intersection genes. The protein-protein interaction (PPI) network was used to screen of hub gene. The expression and mRNA levels of CXCL12 in CD and ROC curves in GSE95095 dataset. Signaling pathways of hub genes and their correlation with immune cells were analyzed by gene set enrichment analysis (GSEA), EPIC, and ESTIMATE, respectively. Finally, immunohistochemistry (IHC) and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were used to detect the expression of the hub gene in normal, inactive, and active CD tissues. In GSE95095 dataset, CXCL12 was identified as the most hub gene by limma analysis, Venn diagram and A protein-protein interaction (PPI) network. CXCL12 expression was highest in active CD (p < 0.001) followed by inactive CD (p < 0.01). Subsequently, it was validated through IHC and RT-PCR in normal intestinal mucosal, active CD, and inactive CD. CXCL12 was overexpressed in active and inactive CD (IHC: p < 0.001 and RT-PCR: p < 0.001, respectively). CXCL12 expression in active CD was determined via analysis with receiver operating characteristic (ROC) curves. The specificity and sensitivity were 0.875 and 0.625, respectively, the accuracy was 72.92%, the area under the curve (AUC) was 0.780, and the 95% confidence interval (CI) was in the range of 0.648-0.912. CXCL12 expression was closely correlated with various immune cells. CXCL12 is overexpressed in active CD and is closely correlated with various immune cells. We propose that CXCL12 as a potential target genes for the treatment and management of both active and inactive CD.

The intestinal mucosal barrier is a complex structure that separates the internal and lumen environments. An impaired intestinal barrier may lead to excessive mucosal immune system activation and further to intestinal diseases, including inflammatory bowel disease (IBD). Therefore, improving the integrity of the intestinal barrier may be a therapeutic approach to prevent or treat IBD. In this study, we aimed to elucidate the effects of the new 1,3,4-oxadiazole derivatives of pyrrolo[3,4-d]pyridazinone, compounds 7b, 10b, and 13b, in intestinal epithelial damage. In this study, we used biobank colon and feces samples collected during our previous original experiment, in which we induced colitis in rats by trinitrobenzenesulfonic acid (TNBS) administration. We assessed the expression of tight junction (TJ) proteins, ie, claudin 1 (CLDN1), occludin (OCLN), zonula occludens 1 (ZO1), and mucus layers proteins, ie, mucin 2 (Muc2) and trefoil factor 3 (TFF3), and the goblet cells and mucus content in colon tissues. We also assessed matrix metalloproteinase 9 (MMP9) and MAP kinases (MAPKs) levels in colon tissues and the level of α1-antitrypsin (α1-AT) in feces samples. We found that compounds 7b and 13b at a dose of 20 mg/kg prevented TNBS-induced loss of goblet cells and mucus layer with normalizing Muc2 and TFF3 expression. Both these compounds prevented TNBS-induced loss of the TJ proteins and normalized the fecal α1-AT level. Compounds 7b and 13b (20 mg/kg) counteracted the TNBS-induced increase of MMP9 concentration and MAPK activation. New pyrrolo[3,4-d]pyridazinone derivatives normalized the colonic expression of TJ and mucus layer proteins and prevented goblet cells and mucus depletion in rats with experimental colitis, exerting a beneficial effect on mucosal epithelial barrier integrity. They protect against intestinal barrier dysfunction, and the potential mechanism may involve the inhibition of MAPKs and MMP9 activation.

Objective: Currently, transcriptome-level investigations into the therapeutic mechanisms of electroacupuncture (EA) on intestinal mucosal barrier dysfunction in diarrhoea-predominant irritable bowel syndrome (IBS-D) models remain scarce. This study was designed to establish a comprehensive competing endogenous RNA (ceRNA) network through integrated RNA sequencing (RNA-seq) and bioinformatics analyses while elucidating the underlying mechanisms through which EA restores intestinal barrier integrity in IBS-D rats via modulation of the long non-coding RNA (lncRNA)-microRNA (miRNA) -messenger RNA (mRNA) regulatory network. Methods: The IBS-D model was established by neonatal maternal separation (NMS), 4% acetic acid enema, and restrain stress (RS). The rats were randomly divided into three groups: control, model and EA groups. After 2 weeks of EA, the morphological changes of the rat colon were observed by hematoxylin-eosin staining (HE) and Transmission electron microscope (TEM), and the expression of substances related to the damage of the intestinal mucosal barrier was detected by Enzyme-linked Immunosorbent Assay (ELISA) and Western blot (WB) to verify the protective effect of EA on the intestinal mucosal barrier of IBS-D rats. Then RNA-seq was used to analyse rat colon differentially expressed RNAs (DE RNAs) and construct relevant ceRNA networks. Subsequently, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the differentially expressed mRNAs (DE mRNAs) altered by EA to elucidate the mechanism of EA in improving the damage of the intestinal mucosal barrier. Finally, Real-Time Quantitative Reverse Transcription PCR (RT-qPCR) was used to verify the RNA-seq results, and WB and immunofluorescence (IF) were used to verify the involvement of mast cells (MCs) in the relevant signalling pathways regulated by EA. Results: Firstly, EA had an alleviating effect on the intestinal mucosal barrier damage in IBS-D rats. Then, RNAseq results showed that 426 DE mRNAs, 342 differentially expressed lncRNAs (DE lncRNAs) and 10 differentially expressed miRNAs (DE miRNAs) were up-regulated and 429 DE mRNAs, 362 DE lncRNAs and 48 DE miRNAs were down-regulated by EA. Meanwhile, the ceRNA networks of 7 DE lncRNAs-miR-139-3p-Bid and 7 DE lncRNAs-miR-378b-Slc4a5 were successfully constructed. GO indicated that EA protected the intestinal mucosal barrier of IBS-D rats mainly by regulating a series of defense responses (e.g., against viruses and bacteria), participating in regulating the secretion and transport of hormones, and affecting the function of cytokines. KEGG indicated that there were key signal pathways such as antigen processing and presentation, neuroactive ligand-receptor interaction, PPAR signaling pathway and glutathione metabolism, which were related to the participation of MC in immune inflammation after degranulation. RT-qPCR results were consistent with RNA-seq. Further experiments confirmed that EA ameliorated the damage to the intestinal mucosal barrier in IBS-D rats by inhibiting MC activation. Conclusion: This study explored the multi-system, multi-level and multi-target mechanism of EA in the treatment of IBS-D through RNA-seq and found that EA could improve the intestinal mucosal barrier damage in IBSD rats. The overall regulatory effect of EA was related to the regulation of the ceRNA networks composed of 7 DE lncRNAs-miR-139-3p-Bid and 7 DE lncRNAs-miR-378b-Slc4a5 by affecting multiple genes in IBS-D rats.

Recent studies have confirmed that increased intestinal permeability and gut-origin lipopolysaccharide (LPS) translocation are important causes of metabolic inflammation in type 2 diabetes (T2D), but there are no recognized therapies for targeting this pathological state. Scutellaria baicalensis and Coptis chinensis are a classic herbal pair often used to treat diabetes and various intestinal diseases, and repair of intestinal barrier damage may be at the core of their therapeutic mechanism. This study investigated the effects of oral administration of Scutellaria-Coptis (SC) on the intestinal mucosal barrier in diabetic rats and explored the underlying mechanism from the perspective of anti-inflammatory and gut microbiota-modulatory effects. The main results showed that, in addition to regulating glycolipid metabolism disorders and inhibiting serum inflammatory factors, SC could also upregulate the expression levels of the tight junction proteins claudin-1, occludin, and zonula occludens (ZO-1), significantly improve intestinal epithelial damage, and inhibit excessive LPS translocation into the blood circulation. Furthermore, it was found that SC could reduce the levels of the inflammatory factors interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α) in intestinal tissue and that the anti-inflammatory effects involved the TLR-4/TRIF and TNFR-1/NF-κB signalling pathways. Moreover, SC had a strong inhibitory effect on some potential enteropathogenic bacteria and LPS-producing bacteria, such as Proteobacteria, Enterobacteriaceae, Enterobacter, Escherichia-Shigella, and Enterococcus, and could also promote the proliferation of butyrate-producing bacteria, such as Lachnospiraceae and Prevotellaceae. Taken together, the hypoglycaemic effects of SC were related to the protection of the intestinal mucosal barrier, and the mechanisms might be related to the inhibition of intestinal inflammation and the regulation of the gut microbiota.