Antiviral activity of Vigna radiata extract against feline coronavirus in vitro.

Feline infectious peritonitis (FIP) is a fatal illness caused by a mutated feline coronavirus (FCoV). This disease is characterized by its complexity, resulting from systemic infection, antibody-dependent enhancement (ADE), and challenges in accessing effective therapeutics. Extract derived from Vigna radiata (L.) R. Wilczek (VRE) exhibits various pharmacological effects, including antiviral activity. This study aimed to investigate the antiviral potential of VRE against FCoV, addressing the urgent need to advance the treatment of FIP. We explored the anti-FCoV activity, antiviral mechanism, and combinational application of VRE by means of in vitro antiviral assays. Our findings reveal that VRE effectively inhibited the cytopathic effect induced by FCoV, reduced viral proliferation, and downregulated spike protein expression. Moreover, VRE blocked FCoV in the early and late infection stages and was effective under in vitro ADE infection. Notably, when combined with VRE, the polymerase inhibitor GS-441524 or protease inhibitor GC376 suppressed FCoV more effectively than monotherapy. In conclusion, this study characterizes the antiviral property of VRE against FCoV in vitro, and VRE possesses therapeutic potential for FCoV treatment.

Inductive line tunneling FET using epitaxial tunnel layer with Ge-source and charge enhancement insulation.

In this paper, we propose an inductive line tunneling FET using Epitaxial Tunnel Layer with Ge-Source and Charge Enhancement Insulation (CEI ETL GS-iTFET). The CEI ETL GS-iTFET allows full overlap between the gate and source regions, thereby enhancing the line tunneling. In addition, a germanium layer is introduced on the source side to form a heterojunction, effectively improving the device's conduction current. An ETL is incorporated to combat point tunneling leakage, resulting in a steeper subthreshold swing. Furthermore, a CEI consisting of Si3N4 is introduced between the germanium source and the Schottky metal, which effectively reduces carrier losses in the inversion layer and improves the overall device performance. This study presents a calibration-based approach to simulations, taking into account practical process considerations. Simulation results show that at VD = 0.2 V, the CEI ETL GS-iTFET achieves an average subthreshold swing (SSavg) of 30.5 mV/dec, an Ion of 3.12 × 10-5 A/µm and an Ion/Ioff ratio of 1.81 × 1010. These results demonstrate a significantly low subthreshold swing and a high current ratio of about 1010. In addition, the proposed device eliminates the need for multiple implantation processes, resulting in significant manufacturing cost reductions. As a result, the CEI ETL GS-iTFET shows remarkable potential in future low-power device competition.

Isolation, full sequence analysis, and in situ hybridization of pigeon paramyxovirus-1 genotype VI.2.1.1.2.2 from oriental turtle doves (Streptopelia orientalis).

Pigeon paramyxovirus-1 (PPMV-1), a genetic variant of avian paramyxovirus-1 (APMV-1), has been identified in Columbiformes and is the primary cause of diseases in captive and free-ranging pigeons. However, it has also been reported that PPMV-1 can infect chickens naturally and experimentally, thus posing a potential threat to the poultry industry. This study investigated a lethal outbreak of paramyxovirus infection that occurred among 16 oriental turtle doves (Streptopelia orientalis) in a walk-in aviary at a zoo from March to April 2021. Necropsies were performed, and histopathological findings revealed mild to moderate lymphoplasmacytic infiltration in several organs, such as the pancreas, liver, kidneys, and lungs. Reverse transcription polymerase chain reaction (RT-PCR) using formalin-fixed paraffin-embedded tissue blocks, virus isolation from fresh tissue, and in situ hybridization against the fusion (F) protein confirmed the diagnosis for PPMV-1 infection. The isolated strain NTU/C239/21 was fully sequenced by next-generation sequencing, and the results of phylogenetic analyses revealed that the F protein of NTU/C239/21 shared 98.8% nucleotide sequence identity with Pigeon/Taiwan/AHRI121/2017, which was isolated from a feral pigeon in Taiwan. The present study is the first to identify PPMV-1 infection in Streptopelia orientalis and suggests that Streptopelia orientalis may also play an important role in spreading the infection, similar to pigeons in APMV-1 spreading.

Effects of selective cyclooxygenase-2 inhibitor robenacoxib on primary cells derived from feline injection-site sarcoma.

Feline injection-site sarcomas (FISSs) are highly invasive malignant mesenchymal neoplasms that arise from injection sites in cats. Although the tumorigenesis of FISSs is still uncertain, there is a consensus that FISS is associated with chronic inflammation caused by irritation of injection-related trauma and foreign chemical substances. Chronic inflammation can provide a proper microenvironment for tumour development, which has been known as one of the risk factors of tumorigenesis in many tumours. To investigate the tumorigenesis of FISS and screen for its potential therapeutic targets, cyclooxygenase-2 (COX-2), an inflammation-enhancing enzyme, was selected as a target for this study. In vitro experiments using FISS- and normal tissue-derived primary cells and robenacoxib, a highly selective COX-2 inhibitor, were performed. The results demonstrated that expression of COX-2 could be detected in formalin-fixed and paraffin-embedded FISS tissues and FISS-derived primary cells. Cell viability, migration and colony formation of FISS-derived primary cells were inhibited, and cell apoptosis was enhanced by robenacoxib in a dose-dependent manner. However, susceptibility to robenacoxib varied in different lines of FISS primary cells and was not completely correlated with COX-2 expression. Our results suggest that COX-2 inhibitors could be potential adjuvant therapeutics against FISSs.

Cross-reactivities and cross-neutralization of different envelope glycoproteins E2 antibodies against different genotypes of classical swine fever virus.

Classical swine fever (CSF) is a highly contagious swine disease caused by the classical swine fever virus (CSFV), wreaking havoc on global swine production. The virus is divided into three genotypes, each comprising 4-7 sub-genotypes. The major envelope glycoprotein E2 of CSFV plays an essential role in cell attachment, eliciting immune responses, and vaccine development. In this study, to study the cross-reaction and cross-neutralizing activities of antibodies against different genotypes (G) of E2 glycoproteins, ectodomains of G1.1, G2.1, G2.1d, and G3.4 CSFV E2 glycoproteins from a mammalian cell expression system were generated. The cross-reactivities of a panel of immunofluorescence assay-characterized serum derived from pigs with/without a commercial live attenuated G1.1 vaccination against different genotypes of E2 glycoproteins were detected by ELISA. Our result showed that serum against the LPCV cross-reacted with all genotypes of E2 glycoproteins. To evaluate cross-neutralizing activities, hyperimmune serum from different CSFV E2 glycoprotein-immunized mice was also generated. The result showed that mice anti-E2 hyperimmune serum exhibited better neutralizing abilities against homologous CSFV than heterogeneous viruses. In conclusion, the results provide information on the cross-reactivity of antibodies against different genogroups of CSFV E2 glycoproteins and suggest the importance of developing multi-covalent subunit vaccines for the complete protection of CSF.

Use of Leukotriene-Receptor Antagonists During Pregnancy and Risk of Neuropsychiatric Events in Offspring.

This cohort study examines the association of the use of leukotriene-receptor antagonists during pregnancy with the risk of neuropsychiatric events in offspring.

Identification and genomic characterization of Baculovirus penaei in Litopenaeus vannamei in Taiwan.

Baculovirus penaei (BP), the causative agent of tetrahedral baculovirosis, causes the death of penaeid genera at the larval and post-larval stages. BP has been reported in the Western Pacific, South-East Atlantic, and the State of Hawaii, but never in Asia. The clinical features of BP infection are non-specific, and diagnosis relies on histological and molecular methods. In the present study, we report the first identification of BP infection in a shrimp farm in Northern Taiwan in 2022. Histopathologically, several tetrahedral eosinophilic intranuclear occlusion bodies were observed in or budding out of the nuclei of the degenerative hepatopancreatic cells. In situ hybridization and polymerase chain reaction confirmed tetrahedral baculovirosis infection caused by BP. Sequence alignment of the TW BP-1 with the USA BP strain reported in 1995 revealed 94.81% identity in the partial gene. The possibility of the emergence of USA-like BP in Taiwan highlights the importance of further epidemiological investigations on the prevalence and impact of BP in Asia.

Associations between parental psychiatric disorders and autism spectrum disorder in the offspring.

Whether parental psychiatric disorders are associated with autism spectrum disorder (ASD) in offspring has remained inconclusive. We examined the associations of parental psychiatric disorders with ASD in offspring. This population-based case-control study used Taiwan's National Health Insurance Research Database to identify a cohort of children born from 2004 to 2017 and their parents. A total of 24,279 children with ASD (diagnostic ICD-9-CM code: 299.x or ICD-10 code F84.x) and 97,715 matched controls were included. Parental psychiatric disorders, including depressive disorders, bipolar spectrum disorders, anxiety disorders, obsessive-compulsive disorder, schizophrenia, substance use disorders, autism spectrum disorder, attention-deficit hyperactivity disorder (ADHD), and adjustment disorders were identified. Conditional logistic regressions with covariate adjustment were performed. The results suggest that parental diagnosis with any of the psychiatric disorders is associated with ASD in offspring (adjusted odds ratio [AOR] = 1.45, 95%CI: 1.40-1.51 for mothers; and AOR = 1.12, 95%CI: 1.08-1.17 for fathers). ASD in offspring was associated with schizophrenia, depressive disorders, obsessive-compulsive disorder, adjustment disorders, ADHD and ASD in both parents. The relationship between parental psychiatric disorders and the timing of the child's birth and ASD diagnosis varied across the different psychiatric disorders. The present study provides supportive evidence that parental psychiatric disorders are associated with autistic children. Furthermore, because the associations between parental psychiatric disorders and the timing of child's birth and ASD diagnosis varied across psychiatric disorders, the observed relationships may be affected by both genetic and environmental factors. Future studies are needed to disentangle the potential influence of genetic and environmental factors on the observed associations.

The Effects of Bacillus licheniformis-Fermented Products on the Microbiota and Clinical Presentation of Cats with Chronic Diarrhea.

Bacillus licheniformis-fermented products (BLFP) are probiotics with antibacterial, antiviral, and anti-inflammatory properties that can improve growth performance. This study aimed to compare the fecal microbiota of diarrheal cats with chronic diarrhea (n = 8) with that of healthy cats (n = 4) from the same household using next-generation sequencing, and evaluate the effectiveness of oral administration of BLFP in relieving clinical signs and altering the intestinal microbiota in diarrheal cats. Six out of eight diarrheal cats showed clinical improvement after BLFP administration for 7 days, and the stool condition of the other two was normal. A higher Firmicutes/Bacteroidetes ratio was noted in the feces of diarrheal cats without clinical improvement as compared with those in the healthy cats and in the diarrheal cats with clinical improvement after receiving BLFP. The phylum Bacteroidetes and class Bacteroidia decreased significantly in diarrheal cats regardless of BLFP administration. Blautia spp., Ruminococcus torques, and Ruminococcus gnavus, which belong to the Clostridium cluster XIVa and have been reported as beneficial to intestinal health, increased significantly in feces after treatment. Furthermore, Clostridium perfringens also significantly decreased in diarrheal cats after BLFP administration. Overall, BLFP could be a potential probiotic to relieve gastrointestinal symptoms and improve fecal microbiota in cats with chronic diarrhea.

In situ structure and dynamics of an alphacoronavirus spike protein by cryo-ET and cryo-EM.

Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, bringing substantial economic losses in the pork industry. The trimeric spike (S) glycoprotein of PEDV is responsible for virus-host recognition, membrane fusion, and is the main target for vaccine development and antigenic analysis. The atomic structures of the recombinant PEDV S proteins of two different strains have been reported, but they reveal distinct N-terminal domain 0 (D0) architectures that may correspond to different functional states. The existence of the D0 is a unique feature of alphacoronavirus. Here we combined cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) to demonstrate in situ the asynchronous S protein D0 motions on intact viral particles of a highly virulent PEDV Pintung 52 strain. We further determined the cryo-EM structure of the recombinant S protein derived from a porcine cell line, which revealed additional domain motions likely associated with receptor binding. By integrating mass spectrometry and cryo-EM, we delineated the complex compositions and spatial distribution of the PEDV S protein N-glycans, and demonstrated the functional role of a key N-glycan in modulating the D0 conformation.

Investigating the role of signal transducer and activator of transcription 3 in feline injection site sarcoma.

BACKGROUND: Feline injection-site sarcomas (FISSs) are malignant mesenchymal tumors of different histotypes. The pathogenesis of FISS has been correlated with chronic inflammation, resulting in neoplastic transformation. Activation of the Janus kinase-signal transducer and activator of transcription 3 (STAT3) have been demonstrated to play a critical role in tumor development by regulating signaling pathways involved in cell proliferation, survival, metastasis, and angiogenesis in human medicine. To characterize the role of STAT3 in FISS, we first detected STAT3 and phosphorylated STAT3 in formalin-fixed and paraffin-embedded (FFPE) FISS tissues using immunohistochemical staining. RESULTS: STAT3 was detected in 88.9% (40/45) of FISS cases, and phosphorylated STAT3 was detected in 53.3% (24/45) of cases. However, the expression levels of both forms of STAT3 were not correlated with tumor grade. To study the role of STAT3 in tumor survival, two primary cells derived from FISSs of two cats exhibiting consistent immunophenotypes with their parental FFPE tissues were established. A dose-dependent inhibitory effect on cell proliferation was observed in both primary FISS cells treated with the STAT3 inhibitor, 5-hydroxy-9,10-dioxo-9,10-dihydroanthracene-1-sulfonamide. CONCLUSIONS: The STAT 3 may play an important role in the tumorigenesis of FISS and be a potential molecular therapeutic target for FISS.

Molecular and serological detection of Toxoplasma gondii infection in mammals in the Taipei Zoo.

Toxoplasmosis caused by Toxoplasma gondii affects both conservation and public health efforts. In the Taipei Zoo, toxoplasmosis was diagnosed in ring-tailed lemurs and a meerkat in 2019 while a freeze-thaw meat strategy had been applied to carnivores before the event. To investigate the possible risk factors associated with T. gondii infection in the Taipei Zoo, 179 veterinary visiting mammals from 2019-2021 and six stray cats were included to detect anti-T. gondii IgG and IgM in their serum via ELISA, and T. gondii in their faeces and blood via PCR. Although the overall T. gondii IgG seroprevalence was 33.5% and PCR positivity was 16.2% in the zoo mammals, the correlation between T. gondii PCR and systemic IgG results was low. An omnivorous diet (adjusted OR = 0.4; 95% CI: 0.2-1.0), a herbivorous diet (adjusted OR = 3.2; 95% CI: 1.1-9.6), and animals in the Conservation Area where stray cats appeared (adjusted OR = 18.3; 95% CI: 3.9-85.9) were independent risk factors for T. gondii infection. The low T. gondii-specific IgM positivity (0.6%) suggests that most animals did not have acute T. gondii infection. In conclusion, our findings indirectly support that feeding frozen meat to carnivores, cleaning fresh food, and restricting access to stray cats to prevent faecal contaminants could prevent animals from T. gondii exposure.

Concurrent infection of a novel genotype of hepatopancreatic parvovirus and Enterocytozoon hepatopenaei in Penaeus vannamei in Taiwan.

Hepatopancreatic parvovirus (HPV) and Enterocytozoon hepatopenaei (EHP) are emerging and reemerging pathogens in shrimps. In the present study, a novel genotype of HPV concurrently infected with EHP in Penaeus vannamei in Taiwan leading to severe atrophy and damage of hepatopancreas were confirmed by histopathology, in situ hybridization, and PCR. The novel genotype of HPV exhibited 66%-69.5% sequence identities with all known HPVs and carried unique amino acid deletions and insertions in the VP gene. According to phylogenetic analysis, the Taiwan HPV isolates were classified as the genotype IV. The present study not only provided the histopathological and molecular proof of HPV and EHP co-infection in Taiwan, but also revealed the importance of investigating the geographical expansion of novel HPV genotypes.

Oxidative Stress-Induced Alterations of Cellular Localization and Expression of Aquaporin 1 Lead to Defected Water Transport upon Peritoneal Fibrosis.

Being one of the renal replacement therapies, peritoneal dialysis (PD) maintains around 15% of end-stage kidney disease patients' lives; however, complications such as peritoneal fibrosis and ultrafiltration failure during long-term PD compromise its application. Previously, we established a sodium hypochlorite (NaClO)-induced peritoneal fibrosis porcine model, which helped to bridge the rodent model toward pre-clinical human peritoneal fibrosis research. In this study, the peritoneal equilibration test (PET) was established to evaluate instant functional changes in the peritoneum in the pig model. Similar to observations from long-term PD patients, increasing small solutes transport and loss of sodium sieving were observed. Mechanistic investigation from both in vivo and in vitro data suggested that disruption of cytoskeleton induced by excessive reactive oxygen species defected intracellular transport of aquaporin 1, this likely resulted in the disappearance of sodium sieving upon PET. Functional interference of aquaporin 1 on free water transport would result in PD failure in patients.

Generation and Characterization of a Spike Glycoprotein Domain A-Specific Neutralizing Single-Chain Variable Fragment against Porcine Epidemic Diarrhea Virus.

The emergence of the genotype (G) 2 and re-emergence of the G1 porcine epidemic diarrhea virus (PEDV) has caused severe economic impacts in the past decade. Developments of efficient vaccines against new variants of PEDV have been challenging, not least because of the difficulties in eliciting mucosal and lactogenic immunity. A single-chain fragment variable (scFv) capable of efficient antigen recognition is an alternative to vaccination and treatment of a viral infection. In the present study, the variable regions of the light chain and the heavy chain of a G2b PEDV spike domain A (S1A)-specific neutralizing monoclonal antibody (mAb) were sequenced, constructed with a (G4S) x3 linker, and produced by a mammalian protein expression system. Our results demonstrated that the PEDV S1A domain scFv was able to bind to S proteins of both G1 and G2b PEDVs. Nevertheless, the scFv was only capable of neutralizing the homologous G2b PEDV but not the G1 PEDV. The binding ability of the G2b-specific neutralizing scFv was not able to predict the neutralizing ability toward heterologous PEDV. The anti-PEDV S1A scFv presented herein serves as a potential therapeutic candidate against the virulent G2b PEDV.

Investigation of Lethal Concurrent Outbreak of Chlamydiosis and Pigeon Circovirus in a Zoo.

During the spring, an outbreak of sudden death involving 58 birds occurred in a zoo. Histopathological examinations revealed variable numbers of intracytoplasmic basophilic microorganisms in the macrophages, hepatocytes, and renal epithelium of most birds, along with occasional botryoid intracytoplasmic inclusion bodies within histiocytes in the bursa of Fabricius. Based on the results of histopathological examinations, immunohistochemical staining, transmission electron microscopy, and polymerase chain reactions, genotype B Chlamydia psittaci infection concurrent with pigeon circovirus (PiCV) was diagnosed. A retrospective survey, including two years before the outbreak and the outbreak year, of C. psittaci and PiCV infections of dead birds in the aviaries, revealed that the outbreak was an independent episode. The findings of this study indicate that concurrent infection with C. psittaci and PiCV might lead to lethal outbreaks of chlamydiosis, particularly Streptopelia orientalis. In addition, persistently monitoring both pathogens and identifying potential PiCV carriers or transmitters might also help prevent lethal disease outbreaks.

Association of Oral Corticosteroid Bursts With Severe Adverse Events in Children.

Importance: The adverse effects from the long-term use of oral corticosteroids are known, but, to our knowledge, few studies have reported the risk of corticosteroid bursts, particularly among children. Objective: To quantify the associations of corticosteroid bursts with severe adverse events, including gastrointestinal (GI) bleeding, sepsis, pneumonia, and glaucoma, in children. Design, Setting, and Participants: This study used data derived from the National Health Insurance Research Database in Taiwan from January 1, 2013, to December 31, 2017, on children younger than 18 years of age and used a self-controlled case series design. Data were analyzed from January 1 to July 30, 2020. Exposure: Oral corticosteroid bursts (defined as oral corticosteroid use for ≤14 days). Main Outcomes and Measures: Incidence rates were calculated of 4 severe adverse events (GI bleeding, sepsis, pneumonia, and glaucoma) in children who did or did not receive corticosteroid bursts. Conditional fixed-effect Poisson regression was used to estimate incidence rate ratios (IRRs) of severe adverse events within 5 to 30 days and 31 to 90 days after initiation of corticosteroid bursts. Results: Among 4 542 623 children, 23% (1 064 587; 544 268 boys [51.1%]; mean [SD] age, 9.7 [5.8] years) were prescribed a single corticosteroid burst. The most common indications were acute respiratory tract infections and allergic diseases. The incidence rate differences per 1000 person-years between children administered a single corticosteroid burst and those not prescribed corticosteroids were 0.60 (95% CI, 0.55-0.64) for GI bleeding, 0.03 (95% CI, 0.02-0.05) for sepsis, 9.35 (95% CI, 9.19-9.51) for pneumonia, and 0.01 (95% CI, 0.01-0.03) for glaucoma. The IRRs within 5 to 30 days after initiating corticosteroid bursts were 1.41 (95% CI, 1.27-1.57) for GI bleeding, 2.02 (95% CI, 1.55-2.64) for sepsis, 2.19 (95% CI, 2.13-2.25) for pneumonia, and 0.98 (95% CI, 0.85-1.13) for glaucoma; the IRRs within the subsequent 31 to 90 days were 1.10 (95% CI, 1.02-1.19) for GI bleeding, 1.08 (95% CI, 0.88-1.32) for sepsis, 1.09 (95% CI, 1.07-1.11) for pneumonia, and 0.95 (95% CI, 0.85-1.06) for glaucoma. Conclusions and Relevance: This study suggests that corticosteroid bursts, which are commonly prescribed for children with respiratory and allergic conditions, are associated with a 1.4- to 2.2-fold increased risk of GI bleeding, sepsis, and pneumonia within the first month after initiation of corticosteroid therapy that is attenuated during the subsequent 31 to 90 days.

Parenterally Administered Porcine Epidemic Diarrhea Virus-Like Particle-Based Vaccine Formulated with CCL25/28 Chemokines Induces Systemic and Mucosal Immune Protectivity in Pigs.

Generation of a safe, economical, and effective vaccine capable of inducing mucosal immunity is critical for the development of vaccines against enteric viral diseases. In the current study, virus-like particles (VLPs) containing the spike (S), membrane (M), and envelope (E) structural proteins of porcine epidemic diarrhea virus (PEDV) expressed by the novel polycistronic baculovirus expression vector were generated. The immunogenicity and protective efficacy of the PEDV VLPs formulated with or without mucosal adjuvants of CCL25 and CCL28 (CCL25/28) were evaluated in post-weaning pigs. While pigs intramuscularly immunized with VLPs alone were capable of eliciting systemic anti-PEDV S-specific IgG and cellular immunity, co-administration of PEDV VLPs with CCL25/28 could further modulate the immune responses by enhancing systemic anti-PEDV S-specific IgG, mucosal IgA, and cellular immunity. Upon challenge with PEDV, both VLP-immunized groups showed milder clinical signs with reduced fecal viral shedding as compared to the control group. Furthermore, pigs immunized with VLPs adjuvanted with CCL25/28 showed superior immune protection against PEDV. Our results suggest that VLPs formulated with CCL25/28 may serve as a potential PEDV vaccine candidate and the same strategy may serve as a platform for the development of other enteric viral vaccines.

Fermented Soy Paste Alleviates Lipid Accumulation in the Liver by Regulating the AMPK Pathway and Modulating Gut Microbiota in High-Fat-Diet-Fed Rats.

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of liver disease due to lipid accumulation in the hepatocyte. Diet, especially a high-fat diet, is one risk factor that leads to NAFLD. Many natural compounds such as isoflavones have antiobesity effects. Therefore, intake of these functional compounds through daily dietary choices is a method of improving health. Miso is a kind of fermented soy paste, which is rich in isoflavones and has a different biological activity. In this study, we investigated the effects of different concentrations of fermented soy paste on NAFLD in high-fat-diet (HFD)-fed Sprague-Dawley (SD) rats. The results showed that 2% fermented soy paste decreased serum triacylglycerol (TG) and alanine aminotransferase (ALT) and reduced lipid accumulation in the liver through induced fatty acid oxidation by activating the adenosine 5'-monophosphate -activated protein kinase (AMPK) pathway and increasing PGC1α and CPT1α protein expression. Furthermore, we found that 2% fermented soy paste increased the abundance of Prevotellaceae NK3B31 and Desulfovibrio. Taken together, fermented soy paste improved HFD-induced lipid accumulation in the liver by activating fatty acid oxidation and modulating gut microbiota.