Metabolomic profile of plasma approach to investigate the mechanism of Poria cocos oligosaccharides attenuated LPS-induced acute lung injury in mice.

Poria cocos (Schw.) Wolf (PCW) are the dried sclerotia of Poaceae fungus Poria cocos that contain many biological activity ingredients such as polysaccharides and triterpenoids. The carbohydrates from Poria cocos have been proven to possess anti-inflammatory and antioxidant effects. This study aimed to investigate the impact and mechanism of Poria cocos oligosaccharides (PCO) protecting mice against acute lung injury (ALI). We examined the histopathological analysis of lung injury, inflammatory, and edema levels to evaluate the benefits of PCO during ALI. As a result, PCO improved the lipopolysaccharide (LPS) induced lung injury and decreased the inflammatory cytokines of lung tissue. Simultaneously, PCO alleviated lung edema by regulating the expression of aquaporin5 (AQP5) and epithelial Na+ channel protein (ENaC-α). Additionally, untargeted metabolomics was performed on the plasma of ALI mice via HUPLC-Triple-TOF/MS. The results indicated that linoleic acid, linolenic acid, arachidonic acid, carnosine, glutamic acid, and 1-methylhistamine were the biomarkers in ALI mice. Besides, metabolic pathway analysis suggested PCO affected the histidine and fatty acid metabolism, which were closely associated with inflammation and oxidative reaction of the host. Consequently, the effects of PCO inhibiting inflammation and edema might relate to the reducing pro-inflammatory mediators and the reverse of abnormal metabolic pathways.

Association between oral microbiome and five types of respiratory infections: a two-sample Mendelian randomization study in east Asian population.

Objective: To explore the causal relationship between the oral microbiome and specific respiratory infections including tonsillitis, chronic sinusitis, bronchiectasis, bronchitis, and pneumonia, assessing the impact of genetic variations associated with the oral microbiome. Methods: Mendelian randomization was used to analyze genetic variations, leveraging data from genome-wide association studies in an East Asian cohort to identify connections between specific oral microbiota and respiratory infections. Results: Our analysis revealed that Prevotella, Streptococcus, Fusobacterium, Pauljensenia, and Capnocytophaga play crucial roles in influencing respiratory infections. Prevotella is associated with both promoting bronchitis and inhibiting pneumonia and tonsillitis, with a mixed effect on chronic sinusitis. Streptococcus and Fusobacterium show varied impacts on respiratory diseases, with Fusobacterium promoting chronic sinusitis, bronchiectasis, and bronchitis. Conversely, Pauljensenia and Capnocytophaga are linked to reduced bronchitis and tonsillitis, and inhibited pneumonia and bronchitis, respectively. Discussion: These findings underscore the significant impact of the oral microbiome on respiratory health, suggesting potential strategies for disease prevention and management through microbiome targeting. The study highlights the complexity of microbial influences on respiratory infections and the importance of further research to elucidate these relationships.

Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation.

Tissue regeneration therapy based on human dental pulp cells (hDPCs) faces the distinct challenge of cellular senescence during massive expansion in vitro. To further explore the regulatory mechanism of cellular senescence in hDPCs, we conduct experiments on young cells (Passage 5, P5) and replicative senescent (Passage 12, P12) hDPCs. The results confirm that hDPCs undergo replicative senescence with passaging, during which their ability to proliferate and osteogenic differentiation decreases. Notably, during replicative senescence, phosphoglycerate dehydrogenase (PHGDH), the key enzyme of the serine synthesis pathway (SSP), is significantly downregulated, as well as S-adenosylmethionine (SAM) levels, resulting in reduced H3K36me3 modification on Sirtuin 1 (SIRT1)and Runt-related transcription factor 2 (RUNX2) promoters. Inhibition of PHGDH leads to the same phenotype as replicative senescence. Serine supplementation fails to rescue the senescence phenotype caused by replicative senescence and inhibitors, in which folate metabolism-related genes, including serine hydroxymethyl transferase 2 (SHMT2), methylenetetrahydrofolate dehydrogenase 1(MTHFD1), methylenetetrahydrofolate dehydrogenase 2(MTHFD2), are notably decreased. Our research raised a possibility that PHGDH may be involved in cellular senescence by affecting folate metabolism and histone methylation in addition to serine biosynthesis, providing potential targets to prevent senescence.

Sex-specific differences in the associations between adiposity indices and incident hyperuricemia among middle-aged and older adults: a nationwide longitudinal study.

Objective: Although obesity is a known risk for hyperuricemia (HUA), the associations between adiposity indices and incident HUA and whether sex-specific differences exist is still unknown. We aimed to investigate the associations between adiposity indices and incident HUA in a longitudinal study. Methods: Data from the China Health and Retirement Longitudinal Study (CHARLS) in 2011-2012 and 2015-2016 were used to conduct a cohort study. Participants aged ≥45 years without HUA at baseline were included in this study. Adiposity indices, including body mass index (BMI), waist circumference (WC), waist-to-height ratio body roundness index (BRI), conicity index (CI), lipid accumulation product (LAP) index, waist-to-height ratio (WHtR), visceral adiposity index (VAI), and Chinese visceral adiposity index (CVAI), were calculated. Logistic analysis was used to analyze the association between adiposity indices and incident HUA risk stratified by gender. Receiver operating characteristic curve analysis was performed to evaluate the power of predictions for incident HUA. Results: Of 5,873 participants aged 59.0 ± 8.7 years enrolled in this study, 578 (9.8%) participants developed HUA during the 4-year follow-up period. After adjusting for confounding variables, LAP, VAI, and CVAI showed significant association with incident HUA. BMI, WC, WHtR, BRI, and CI were significantly associated with incident HUA in women but not in men. LAP had the highest area under the curve (AUC) (0.612) followed by CVAI (0.596) in men, while CVAI had the highest AUC (0.707) followed by LAP (0.691) in women. All indices showed better predictive ability in women than in men. Conclusion: Our findings indicated that adiposity indices were effective predictors of incident HUA and showed better predictive power in women than men. In clinical practice, adiposity indices could be used to assess and prevent incident HUA among Chinese middle-aged and older adults.

Tannic Acid Suppresses HBV Replication via the Regulation of NF-κB, MAPKs, and Autophagy in HepG2.2.15 Cells.

Hepatitis B virus (HBV) infection is a serious global health problem that threatens the health of human. Tannic acid (TA), a natural polyphenol in foods, fruits, and plants, exhibits a variety of bioactive functions. In our research, we decide to explore the pharmacological mechanism of TA against HBV replication. Our results showed that TA effectively reduced the content of HBV DNA and viral antigens (HBsAg and HBeAg) in HepG2.2.15 cells. Meanwhile, TA significantly decreased the mRNA expression of HBV RNA, which include total HBV RNA, HBV pregenomic RNA, and HBV precore mRNA. Besides, TA evidently downregulated the activity of HBV promoters in HepG2.2.15 cells. Furthermore, we found that TA upregulated the expression of IL-8, TNF-α, IFN-α, and IFN-α-mediated antiviral effectors in HepG2.2.15 cells. On the contrary, TA downregulated the expression of IL-10 and hepatic nuclear factor 4 (HNF4α). In addition, TA activated the NF-κB and MAPK pathways that contributed to the inhibition of HBV replication. Finally, TA treatment led to the occurrence of autophagy, which accelerated the elimination of HBV components in HepG2.2.15 cells. Taken together, our results elucidated the suppressive effect of TA on HBV replication and provided inspiration for its clinical application in HBV treatment.

LC-Q-TOF/MS-based Fragmentation Behavior Study and In vitro Metabolites Identification of Nine Benzodiazepines.

BACKGROUND: Benzodiazepines (BZDs) are compounds that contain one diazepine ring and two benzene rings, and are widely used to treat central nervous system diseases. However, drug abuse and BZDs' illegal addition may affect normal life and even lead to grave social harm. As BZDs may be metabolized and eliminated quickly, it is of great theoretical and practical significance to clarify their metabolic profile. OBJECTIVE: In this paper, LC-Q-TOF/MS-based fragmentation behavior has been investigated for nine benzodiazepine drugs available and widely used in clinical treatment (diazepam, nitrazepam, clonazepam, oxazepam, lorazepam, alprazolam, estazolam, triazolam, and midazolam), and their metabolic profile has been studied by using in vitro human liver microsomal incubation. METHODS: A regular human liver microsomal system was used to investigate the potential biotransformation of the nine benzodiazepines in vitro, and an LC-Q/TOF-MS was used to perform fragmentation behavior studies and metabolite identification. RESULTS: As a result, characteristic fragmentation pathway and diagnostic fragment ions of the nine BZDs were analyzed, and 19 metabolites of the 9 benzodiazepines were found and identified, with glucuronidation and hydroxylation considered as their most important metabolic pathways. CONCLUSION: These experimental data add to our knowledge of the nine benzodiazepine drugs and their metabolism study, which could provide useful information and evidence of their in vivo metabolic profile prediction and help promote their monitoring in both clinical use and social/illegal abuse.

Discovering the Intrinsic Causes of Dendrite Formation in Zinc Metal Anodes: Lattice Defects and Residual Stress.

Developing a highly stable and dendrite-free zinc anode is essential to the commercial application of zinc metal batteries. However, the understanding of zinc dendrites formation mechanism is still insufficient. Herein, for the first time, we discover that the interfacial heterogeneous deposition induced by lattice defects and epitaxial growth limited by residual stress are intrinsic and critical causes for zinc dendrite formation. Therefore, an annealing reconstruction strategy was proposed to eliminate lattice defects and stresses in zinc crystals, which achieve dense epitaxial electrodeposition of zinc anode. The as-prepared annealed zinc anodes exhibit dendrite-free morphology and enhanced electrochemical cycling stability. This work first proves that lattice defects and residual stresses are also very important factors for epitaxial electrodeposition of zinc in addition to crystal orientation, which can provide a new mechanism for future researches on zinc anode modification.

Experimental Investigation on Ablation Behaviors of CFRP Laminates in an Atmospheric Environment Irradiated by Continuous Wave Laser.

In order to understand the ablation behaviors of CFRP laminates in an atmospheric environment irradiated by continuous wave laser, CFRP laminates were subjected to a 1080-nm continuous wave laser (6-mm laser spot diameter), with different laser power densities carried out in this paper. The internal delamination damage in CFRP laminates was investigated by C-Scan. The rear- and front-face temperature of CFRP laminates were monitored using the FLIR A 655 sc infrared camera, and the rear-face temperature was monitored by K type thermocouples. The morphology of ablation damage, the area size of the damaged heat affected zone (HAZ), crater depth, thermal ablation rate, mass ablation rate, line ablation rate, etc., of CFRP laminates were determined and correlated to the irradiation parameters. It is found that the area size of the damage HAZ, mass ablation rate, line ablation rate, etc., increased as the laser power densities. The dimensionless area size of the damaged HAZ decreased gradually along the thickness direction of the laser irradiation surface.

Application of Diffusion Kurtosis Imaging in Evaluating Acute Xerostomia in Nasopharyngeal Carcinoma Treated With Induction Chemotherapy Plus Concurrent Chemoradiotherapy.

Purpose: The aim of this study was to identify the efficacy of diffusion kurtosis imaging (DKI) in tracking and monitoring the dynamic change of parotid glands (PGs), submandibular glands (SMGs), sublingual glands (SLGs), and acute xerostomia in nasopharyngeal carcinoma (NPC) patients treated with induction chemotherapy (IC) plus concurrent chemoradiotherapy (CCRT). Methods: The prospective study recruited 42 participants treated with IC+CCRT. All patients underwent DKI scanning six times: before IC, before RT, in the middle of the RT course, immediately after RT, and 1 and 3 months post-RT. Mean diffusion coefficient (MD) and mean kurtosis (MK) of PG, SMG, SLG, saliva flow rate measured under resting (uSFR) and stimulated condition (sSFR), and xerostomia questionnaire (XQ) scores were recorded. Results: At each time point, sSFR was significantly higher than uSFR (p < 0.05 for all). MD of the salivary glands and XQ scores increased over time while MK, uSFR, and sSFR decreased. After IC, the significant differences were detected in MD and MK of bilateral SMG and MK of the left SLG (p < 0.05 for all), but not in MD and MK of PG, uSFR, sSFR, and XQ scores. After RT, sSFR at 1m-RT decreased significantly (p = 0.03) while no significant differences were detected in uSFR and XQ scores. Moderate-strong correlations were detected in ΔMD-PG-R%, ΔMK-PG-R%, ΔMD-PG-L%, ΔMK-PG-L%, ΔMD-SMG-R%, ΔMK-SMG-R%, ΔMD-SMG-L%, ΔMK-SMG-L%, and ΔMD-SLG-R%, with correlation coefficients (p < 0.05 for all) ranging from 0.401 to 0.714. ΔuSFR% was correlated with ΔMD-SMG% (p = 0.01, r = -0.39), ΔMD-SLG% (p < 0.001, r = -0.532), and ΔMK-SMG% (p < 0.001, r = -0.493). ΔsSFR% correlated with ΔMD-PG% (p = 0.001, r = -0.509), ΔMD-SMG% (p = 0.015, r = -0.221), and ΔMK-PG% (p < 0.001, r = 0.524). ΔXQ% was only correlated with ΔMK-PG% (p = 0.004, r = 0.433). Conclusion: DKI is a promising tool for tracking and monitoring the acute damage of PG, SMG, and SLG induced by IC+CCRT in NPC patients.

Regulation of gut microbiota and intestinal metabolites by Poria cocos oligosaccharides improves glycolipid metabolism disturbance in high-fat diet-fed mice.

In this study, we aimed to explore the effect of Poria cocos oligosaccharides (PCO) on glucolipid metabolism disorder. Based on a high-fat diet (HFD)-induced obese mouse model, we demonstrated that PCO ameliorated glucose intolerance and insulin resistance, decreased the levels of blood glucose (187.8±19.8 mg/dL) and insulin (566.3±53.34 ng/L) in HFD-fed mice compared to the Ctrl group (140.4±7.942 mg/dL for glucose, 423.2±19.56 ng/L for insulin). Moreover, PCO treatment suppressed the mRNA expressions of fatty acid synthesis regulators (decreases of 68.8%, 62.8%, and 32.0% for G6Pase, FASN, and DGAT, respectively, vs. HFD group) and pro-inflammatory cytokines in epididymal fat (decreases of 71.9%, 81.5%, 76.0%, 29.3%, and 63.9% for TNF-α, IL-1ß, IL-6, COX-5b, and MCP-1, respectively, vs. HFD group). Also, PCO treatment alleviated damage to the intestinal barrier of HFD-fed mice. By 16S rDNA gene sequencing, PCO partly restored the imbalance of gut microbiota in HFD-fed mice, accompanied by the reversal of several intestinal metabolites, including bile acids (BAs), short-chain fatty acids (SCFAs), and tryptophan metabolites. By Spearman's correlation analysis, we found that the changed gut microbiota and their metabolites were significantly correlated with the alteration of metabolic markers. Finally, the significance of gut microbiota in PCO-mediated improvement on glucolipid metabolism disorder was confirmed by an antibiotic depletion experiment and fecal microbiota transplantation. In summary, PCO may be used as a novel prebiotic in the treatment of glucolipid disorders by reshaping intestinal bacteria structure. Our studies also point towards the potential of Poria cocos as a healthy food in the clinical application to metabolic diseases in the future.

An Intensive Intervention to Reduce Readmissions for Frequently Hospitalized Patients: the CHAMP Randomized Controlled Trial.

BACKGROUND: A small number of patients are disproportionally readmitted to hospitals. The Complex High Admission Management Program (CHAMP) was established as a multidisciplinary program to improve continuity of care and reduce readmissions for frequently hospitalized patients. OBJECTIVE: To compare hospital utilization metrics among patients enrolled in CHAMP and usual care. DESIGN: Pragmatic randomized controlled trial. PARTICIPANTS: Inclusion criteria were as follows: 3 or more, 30-day inpatient readmissions in the previous year; or 2 inpatient readmissions plus either a referral or 3 observation admissions in previous 6 months. INTERVENTIONS: Patients randomized to CHAMP were managed by an interdisciplinary team including social work, physicians, and pharmacists. The CHAMP team used comprehensive care planning and inpatient, outpatient, and community visits to address both medical and social needs. Control patients were randomized to usual care and contacted 18 months after initial identification if still eligible. MAIN MEASURES: Primary outcome was number of 30-day inpatient readmissions 180 days following enrollment. Secondary outcomes were number of hospital admissions, total hospital days, emergency department visits, and outpatient clinic visits 180 days after enrollment. KEY RESULTS: There were 75 patients enrolled in CHAMP, 76 in control. Groups were similar in demographic characteristics and baseline readmissions. At 180 days following enrollment, CHAMP patients had more inpatient 30-day readmissions [CHAMP incidence rate 1.3 (95% CI 0.9-1.8) vs. control 0.8 (95% CI 0.5-1.1), p=0.04], though both groups had fewer readmissions compared to 180 days prior to enrollment. We found no differences in secondary outcomes. CONCLUSIONS: Frequently hospitalized patients experienced reductions in utilization over time. Though most outcomes showed no difference, CHAMP was associated with higher readmissions compared to a control group, possibly due to consolidation of care at a single hospital. Future research should seek to identify subsets of patients with persistently high utilization for whom tailored interventions may be beneficial. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT03097640; https://clinicaltrials.gov/ct2/show/NCT03097640.

The Effect of Reducing Intravenous Push Opioid Use on Hospital Medicine Patients' Pain Experience During Hospitalization.

OBJECTIVE: To determine the efficacy of a program to limit the use of the intravenous (IV) push route for opioids on the experience of pain by inpatients and on associated safety events. DESIGN: Retrospective cohort study. SETTING: Two inpatient general medicine floor units at an urban tertiary care academic medical center. SUBJECTS: 4,752 inpatient opioid recipients. METHODS: Patients in one unit were exposed to a multidisciplinary intervention to limit the prescription of opioids via the IV push route, with the other unit used as a control unit. The primary study outcome was the mean numeric pain score per patient during the hospital stay. Secondary measures included the hospital length of stay and postdischarge patient satisfaction. Fidelity measures included the percentage of the patient population exposed to each opioid administration route and the amount of opioid administered per route. Safety measures included patient disposition, transfer to intensive care, and incidence of naloxone administration. RESULTS: The intervention was successful in decreasing both the percentage of patients exposed to IV push opioids and the amount of opioid administered via the IV push route, but no associated changes in other study outcomes were identified. CONCLUSIONS: For the treatment of acute pain in medical inpatients, no evidence of benefit or harm was identified in relation to an increase or decrease in the use of the IV push opioid route.

Unveiling the Mechanism of Principal Drugs of Lianpu Drink on Chronic Gastritis by Network Pharmacology.

Lianpu drink (LPD) is a traditional Chinese medicine (TCM) formula for the treatment of chronic gastritis (CG), and its clinical effects have been effectively verified. However, due to the complexity of the chemical composition of TCM formulas, its mechanism of action has not yet been clearly explained. Many studies have shown that the principal drugs in the TCM formula play a major therapeutic role. Therefore, in this study, the principal drugs Coptidis Rhizoma (CR) and Magnolia officinalis Rehd. et Wils. (MOR) in LPD were used as the main research objects to predict the mechanism of LPD on CG. We contrasted a "compounds-targets-diseases" network and screened the putative targets of CR and MOR in LPD related to CG, respectively. Furthermore, common targets of CR and MOR related to CG were selected as candidate targets. In this study, the specific target proteins of CR, MOR, and CG were combined by protein-protein interaction (PPI) to construct a pharmacological network of "components-targets-diseases." In addition, we investigated the effects of CR and MOR on the TNF signaling pathway, which mediated the remission of CG. This study preliminarily revealed that CR and MOR play a key role in the treatment of CG. Animal experiments also showed that CR and MOR could significantly improve CG by inhibiting MKK6/p38 and RIP/p38 pathway.

Study on the Mechanism of Lianpu Drink for the Treatment of Chronic Gastritis Based on Network Pharmacology.

Chronic gastritis (CG) places a considerable burden on the healthcare system worldwide. Traditional Chinese Medicine (TCM) formulas characterized by multicompounds and multitargets have been acknowledged with striking effects in the treatment of CG in China's history. Nevertheless, their accurate mechanisms of action are still ambiguous. In this study, we analyzed the effective compounds, potential targets, and related biological pathway of Lianpu Drink (LPD), a TCM formula which has been reported to have a therapeutic effect on CG, by contrasting a "compound-target-disease" network. According to the results, 92 compounds and 5762 putative targets of LPD were screened; among them, 8 compounds derived from different herbs in LPD and 30 common targets related to LPD and CG were selected as candidate compounds and precision targets, respectively. Meanwhile, the predicted common targets were verified by Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway analysis and pharmacological experiments. The results demonstrated that quercetin, ephedrine, trigonelline, crocetin, and ß-sitosterol were major effective compounds of LPD responsible for the CG treatment by inhibiting the activation of the JAK 2-STAT 3 signaling pathway to reduce the expressions of cyclin D1 and Bcl-2 proteins. The study provides evidence for the mechanism of understanding of LPD for the treatment of CG.

Neutralizing Antibodies and Cellular Immune Responses Against SARS-CoV-2 Sustained One and a Half Years After Natural Infection.

Background: COVID-19 has caused more than 2.6 billion infections and several million deaths since its outbreak 2 years ago. We know very little about the long-term cellular immune responses and the kinetics of neutralizing antibodies (NAbs) to SARS-CoV-2 because it has emerged only recently in the human population. Methods: We collected blood samples from individuals who were from the first wave of the COVID-19 epidemic in Wuhan between December 30, 2019, and February 24, 2020. We analyzed NAbs to SARS-CoV-2 using pseudoviruses and IgG antibodies to SARS-CoV-2 spike (S) and nucleocapsid (N) protein using enzyme-linked immunosorbent assay in patients' sera and determined SARS-CoV-2-specific T-cell responses of patients with ELISpot assays. Results: We found that 91.9% (57/62) and 88.9% (40/45) of COVID-19 patients had NAbs against SARS-CoV-2 in a year (10-11 months) and one and a half years (17-18 months), respectively, after the onset of illness, indicating that NAbs against SARS-CoV-2 waned slowly and possibly persisted over a long period time. Over 80% of patients had IgG antibodies to SARS-CoV-2 S and N protein one and a half years after illness onset. Most patients also had robust memory T-cell responses against SARS-CoV-2 one and a half years after the illness. Among the patients, 95.6% (43/45) had an IFN-γ-secreting T-cell response and 93.8% (15/16) had an IL-2-secreting T-cell response. The T-cell responses to SARS-CoV-2 were positively correlated with antibodies (including neutralizing antibodies and IgG antibodies to S and N protein) in COVID-19 patients. Eighty percent (4/5) of neutralizing antibody-negative patients also had SARS-CoV-2-specific T-cell response. After long-term infection, protective immunity was independent of disease severity, sex, and age. Conclusions: We concluded that SARS-CoV-2 infection elicited a robust and persistent neutralizing antibody and memory T-cell response in COVID-19 patients, indicating that these sustained immune responses, among most SARS-CoV-2-infected people, may play a crucial role in protection against reinfection.

Analyzing Active Constituents and Optimal Steaming Conditions Related to the Hematopoietic Effect of Steamed Panax notoginseng by Network Pharmacology Coupled with Response Surface Methodology.

During hundreds of years of medication, it is believed that the steamed Panax notoginseng (SPN) can enrich and regulate the blood, replenish the body, and improve the health. The aim of this study was to optimize the steaming conditions of SPN which are related to the hematopoietic effect. In the study, network pharmacology and pharmacological experiments were used to predict and verify the potential hematopoietic active ingredients of SPN. Three variables including the steaming time (2-10 h), steaming temperature (90-130°C), and different producing areas of PN were investigated by using single-factor analysis. Box-Behnken design response surface methodology (BBD-RSM) was performed to explore the optimized steaming conditions which are responsible for the hematopoietic effect of SPN. Furthermore, the hematopoietic effect of the optimized SPN was evaluated. Results demonstrated that ginsenoside Rd, Rh1, Rh4, Rk3, and 20(S)-Rg3 can significantly increase blood routine parameters and expressions of hematopoietic factors in anemia mice. The total contents of the five ginsenosides were selected as evaluation indexes of the response surface method. We found that the PN from Wenshan steamed at 120°C for 5 h could significantly increase the levels of blood routine parameters and hematopoietic factor expression compared with the model group. The study not only provides data support for the determination of hematinic effect-related markers for SPN but also gives a scientific reference for the processing of SPN which has a better hematopoietic effect. The underlying mechanisms require further research.

[Screening and identification of GABA-producing microbes in fermentation process of Sojae Semen Praeparatum].

A high-content GABA was found in Sojae Semen Praeparatum(SSP), which is a famous traditional Chinese medicine and officially listed in Chinese Pharmacopoeia. To screen out and identify GABA-producing microbes from samples at different time points during the fermenting process of SSP, traditional microbiological methods combined with molecular biological methods were used to study the predominant GABA-producing microorganisms existing in the fermenting process of SSP. This study would lay a foundation for further studying the processing mechanism of SSP. The fermenting process of SSP was based on Chinese Pharmacopoeia(2010 edition), and samples were taken at different time points during the fermenting process of SSP. The bacteria and fungi from samples at different time points in the fermenting process of SSP were cultured, isolated and purified by selective medium, and dominant strains were selected. The dominant bacteria were cultured in the designated liquid medium to prepare the fermentation broths, and GABA in the fermentation broth was qualitatively screened out by thin-layer chromatography. The microbial fermentation broth with GABA spots in the primary screening was quantitatively detected by online pre-column derivatization and high performance liquid chromatography established in our laboratory. GABA-producing microorganisms were screened out from predominant strains, and their GABA contents in fermentation broth were determined. The DNA sequences of GABA-producing bacteria and fungi were amplified using 16S rDNA and 18S rDNA sequences by PCR respectively. The amplified products were sequenced, and the sequencing results were identified through NCBI homology comparison. Molecular biological identification was made by phylogenetic tree constructed by MEGA 7.0 software. Through the homology comparison of NCBI and the construction of phylogenetic tree by MEGA 7.0 software, nine GABA-producing microorganisms were screened out and identified in this study. They were Bacillus subtilis, Enterococcus faecium, E. avium, Aspergillus tamarii, A. flavus, A. niger, Cladosporium tenuissimum, Penicillium citrinum and Phanerochaete sordida respectively. For the first time, nine GABA-producing microorganisms were screened out and identified in the samples at different time points during the fermenting process of SSP in this study. The results indicated that multiple predominant GABA-producing microorganisms exist in the fermenting process of SSP and may play an important role in the formation of GABA.

First record of Leptospira and Blastocystis infections in captive flying squirrels (Trogopterus xanthipes) from Enshi County, China.

In traditional Chinese medicine, the feces of flying squirrels have long been used to promote blood circulation and relieve bodily stasis. However, the excrement of flying squirrels may harbor zoonotic agents that could be hazardous to public health. To understand the occurrence of bacterial and parasitic infections in this species, we investigated selected zoonotic pathogens including Leptospira and Blastocystis in the urine and feces of flying squirrels in China. Urine and fecal samples from flying squirrels were collected from a family-owned flying squirrel farm located in Enshi County, Hubei Province in China. Leptospira and Blastocystis DNA was extracted from the urine and feces of flying squirrels, and used as targets for PCR amplification, using different specific primers. PCR amplification and DNA sequencing showed that 4.4% (3/69) of flying squirrels were positive for Leptospira, while 30.4% (21/69) of the animals were positive for Blastocystis. Notably, 1.4% (1/69) of flying squirrels were found to be co-infected with Leptospira and Blastocystis. Sequence analyses allowed for the detection of 3 Blastocystis subtypes (ST1, ST3 and ST13), and mixed infections of Blastocystis subtype 1 and subtype 3 were found in 4.4% (3/69) of flying squirrels. Phylogenetic analysis of the 16S ribosomal RNA gene (rrs2), the flagellin B gene (flaB), and outer membrane lipoprotein lipL32 gene (LipL32) sequences indicated that the Leptospira species detected in the study was L. interrogans. We concluded that flying squirrels from central China were infected with Leptospira and Blastocystis, suggesting that these animals can be a source of infection for their owners, and using fresh excrement from this animal as traditional medicine could be risky to human health. To the best of our knowledge, this is the first report of Leptospira and Blastocystis infection in flying squirrels from Enshi County, China. Our findings provide new data on the epidemiology of these pathogens in this region.