Exploring of gut microbiota features in dyslipidemia and chronic coronary syndrome patients undergoing coronary angiography.

Chronic coronary syndrome (CCS) has a high mortality rate, and dyslipidemia is a major risk factor. Atherosclerosis, a cause of CCS, is influenced by gut microbiota dysbiosis and its metabolites. The objective of this study was to study the diversity and composition of gut microbiota and related clinical parameters among CCS patients undergoing coronary angiography and dyslipidemia patients in comparison to healthy volunteers in Thailand. CCS patients had more risk factors and higher inflammatory markers, high-sensitivity C-reactive protein (hs-CRP) than others. The alpha diversity was lower in dyslipidemia and CCS patients than in the healthy group. A significant difference in the composition of gut microbiota was observed among the three groups. The relative abundance of Proteobacteria, Fusobacteria, Enterobacteriaceae, Prevotella, and Streptococcus was significantly increased while Roseburia, Ruminococcus, and Faecalibacterium were lower in CCS patients. In CCS patients, Lachnospiraceae, Peptostreptococcaceae, and Pediococcus were positively correlated with hs-CRP. In dyslipidemia patients, Megasphaera was strongly positively correlated with triglyceride (TG) level and negatively correlated with high-density lipoprotein cholesterol (HDL-C). The modification of gut microbiota was associated with changes in clinical parameters involved in the development of coronary artery disease (CAD) in CCS patients.

Roles of qseC mutation in bacterial resistance against anti-lipopolysaccharide factor isoform 3 (ALFPm3).

Propelled by global climate changes, the shrimp industry has been facing tremendous losses in production due to various disease outbreaks, particularly early mortality syndrome (EMS), a disease caused by Vibrio parahaemolyticus AHPND. Not only is the use of antibiotics as EMS control agents not yet been proven successful, but the overuse and misuse of antibiotics could also worsen one of the most challenging global health issues-antimicrobial resistance. To circumvent antibiotic usage, anti-lipopolysaccharide factor isoform 3 (ALFPm3), an antimicrobial peptide (AMP) derived from the shrimp innate immune system, was proposed as an antibiotic alternative for EMS control. However, prolonged use of AMPs could also lead to bacterial cross resistance with life-saving antibiotics used in human diseases. Here, we showed that ALFPm3-resistant strains of E. coli could be induced in vitro. Genome analysis of the resistant mutants revealed multiple mutations, with the most interesting being a qseC(L299R). A study of antibiotic susceptibility profile showed that the resistant strains harboring the qseC(L299R) not only exhibited higher degree of resistance towards polymyxin antibiotics, but also produced higher biofilm under ALFPm3 stress. Lastly, a single cell death analysis revealed that, at early-log phase when biofilm is scarce, the resistant strains were less affected by ALFPm3 treatment, suggesting additional mechanisms by which qseC orchestrates to protect the bacteria from ALFPm3. Altogether, this study uncovers involvement of qseC mutation in mechanism of resistance of the bacteria against ALFPm3 paving a way for future studies on sustainable use of ALFPm3 as an EMS control agent.

Comparative genome features and secondary metabolite biosynthetic potential of Kutzneria chonburiensis and other species of the genus Kutzneria.

Actinobacteria are well known as a rich source of diversity of bioactive secondary metabolites. Kutzneria, a rare actinobacteria belonging to the family Pseudonocardiaceae has abundance of secondary metabolite biosynthetic gene clusters (BGCs) and is one of important source of natural products and worthy of priority investigation. Currently, Kutzneria chonburiensis SMC256T has been the latest type-strain of the genus and its genome sequence has not been reported yet. Therefore, we present the first report of new complete genome sequence of SMC256T (genome size of 10.4 Mbp) with genome annotation and feature comparison between SMC256T and other publicly available Kutzneria species. The results from comparative and functional genomic analyses regarding the phylogenomic and the clusters of orthologous groups of proteins (COGs) analyses indicated that SMC256T is most closely related to Kutzneria sp. 744, Kutzneria kofuensis, Kutzneria sp. CA-103260 and Kutzneria buriramensis. Furthermore, a total of 322 BGCs were also detected and showed diversity among the Kutzneria genomes. Out of which, 38 clusters showing the best hit to the most known BGCs were predicted in the SMC256Tgenome. We observed that six clusters responsible for biosynthesis of antimicrobials/antitumor metabolites were strain-specific in Kutzneria chonburiensis. These putative metabolites include virginiamycin S1, lysolipin I, esmeraldin, rakicidin, aclacinomycin and streptoseomycin. Based on these findings, the genome of Kutzneria chonburiensis contains distinct and unidentified BGCs different from other members of the genus, and the use of integrative genomic-based approach would be a useful alternative effort to target, isolate and identify putative and undiscovered secondary metabolites suspected to have new and/or specific bioactivity in the Kutzneria.

Metatranscriptomics Reveals Sequential Expression of Genes Involved in the Production of Melanogenesis Inhibitors by the Defined Microbial Species in Fermented Unpolished Black Rice.

Fermented products require metabolic enzymes from the microbial community for desired final products. Using a metatranscriptomic approach, the role of microorganisms in fermented products on producing compounds with a melanogenesis inhibition activity has not yet been reported. Previously, unpolished black rice (UBR) fermented with the E11 starter containing Saccharomyces cerevisiae, Saccharomycopsis fibuligera, Rhizopus oryzae, and Pediococcus pentosaceus (FUBR) showed potent melanogenesis inhibition activity. This study aimed to investigate the function of these defined microbial species in producing melanogenesis inhibitors in the FUBR using a metatranscriptomic approach. The melanogenesis inhibition activity increased in a fermentation time-dependent manner. Genes related to melanogenesis inhibitors synthesis such as carbohydrate metabolism, amino acids synthesis, fatty acids/unsaturated fatty acids synthesis, and carbohydrate transporters were analyzed. Most genes from R. oryzae and P. pentosaceus were upregulated in the early stage of the fermentation process, while those of S. cerevisiae and S. fibuligera were upregulated in the late stage. FUBR production using different combinations of the four microbial species shows that all species were required to produce the highest activity. The FUBR containing at least R. oryzae and/or P. pentosaceus exhibited a certain level of activity. These findings were in agreement with the metatranscriptomic results. Overall, the results suggested that all four species sequentially and/or coordinately synthesized metabolites during the fermentation that led to a FUBR with maximum melanogenesis inhibition activity. This study not only sheds light on crucial functions of certain microbial community on producing the melanogenesis inhibitors, but also paves the way to initiate quality improvement of melanogenesis inhibition activity in the FUBR. IMPORTANCE Fermentation of food is a metabolic process through the action of enzymes from certain microorganisms. Although roles of the microbial community in the fermented food were investigated using metatranscriptomic approach in terms of flavors, but no study has been reported so far on the function of the microorganisms on producing compounds with a melanogenesis inhibition activity. Therefore, this study explained the roles of the defined microorganisms from the selected starter in the fermented unpolished black rice (FUBR) that can produce melanogenesis inhibitor(s) using metatranscriptomic analysis. Genes from different species were upregulated at different fermentation time. All four microbial species in the FUBR sequentially and/or coordinately synthesized metabolites during fermentation that led to a FUBR with maximal melanogenesis inhibition activity. This finding contributes to a deeper understanding of the roles of certain microbial community during fermentation and led to the knowledge-based improvement for the fermented rice with potent melanogenesis inhibition activity.

Role of Dietary Defatted Rice Bran in the Modulation of Gut Microbiota in AOM/DSS-Induced Colitis-Associated Colorectal Cancer Rat Model.

Defatted rice bran (DRB) is a by-product of rice bran derived after the oil extraction. DRB contains several bioactive compounds, including dietary fiber and phytochemicals. The supplementation with DRB manifests chemopreventive effects in terms of anti-chronic inflammation, anti-cell proliferation, and anti-tumorigenesis in the azoxymethane (AOM) and dextran sodium sulfate (DSS)-induced colitis-associated colorectal cancer (CRC) model in rats. However, little is known about its effect on gut microbiota. Herein, we investigated the effect of DRB on gut microbiota and short chain fatty acid (SCFA) production, colonic goblet cell loss, and mucus layer thickness in the AOM/DSS-induced colitis-associated CRC rat model. The results suggested that DRB enhanced the production of beneficial bacteria (Alloprevotella, Prevotellaceae UCG-001, Ruminococcus, Roseburia, Butyricicoccus) and lessened the production of harmful bacteria (Turicibacter, Clostridium sensu stricto 1, Escherichia-Shigella, Citrobacter) present in colonic feces, mucosa, and tumors. In addition, DRB also assisted the cecal SCFAs (acetate, propionate, butyrate) production. Furthermore, DRB restored goblet cell loss and improved the thickness of the mucus layer in colonic tissue. These findings suggested that DRB could be used as a prebiotic supplement to modulate gut microbiota dysbiosis, which decreases the risks of CRC, therefore encouraging further research on the utilization of DRB in various nutritional health products to promote the health-beneficial bacteria in the colon.

Distinct skin microbiome modulation following different topical acne treatments in mild acne vulgaris patients: A randomized, investigator-blinded exploratory study.

The effects of topical non-antibiotic acne treatment on skin microbiota have rarely been demonstrated. In the study, we randomized 45 mild acne vulgaris participants into three treatment groups, including a cream-gel dermocosmetic containing Aqua Posae Filiformis, lipohydroxy acid, salicylic acid, linoleic acid, niacinamide and piroctone olamine (DC), retinoic acid 0.025% cream (VAA) and benzoyl peroxide 2.5% gel (BP). At months 0, 1 and 3, skin specimens were swabbed from the cheek and forehead and sequenced by targeting V3-V4 regions of the 16 S rRNA gene. QIIME2 was used to characterize bacterial communities. Acne severity, sebum level and tolerability were assessed concomitantly in each visit. We found that both VAA and BP could significantly reduce the bacterial diversity at month 1 (p-value = 0.010 and 0.004 respectively), while no significant reduction was observed in DC group. The microbiota compositions also significantly altered for beta diversity in all treatments (all p-value = 0.001). An increased Cutibacterium with decreased Staphylococcus relative abundance was observed at months 1 and 3 in DC group, while an opposite trend was demonstrated in VAA and BP groups. These findings suggest a potential impact of DC, VAA and BP on the diversity and composition profiles of the skin microbiota in mild acne participants.

Inhibitory Effect of Dietary Defatted Rice Bran in an AOM/DSS-Induced Colitis-Associated Colorectal Cancer Experimental Animal Model.

Defatted rice bran (DRB) is gaining immense popularity worldwide because of its nutritional and functional aspects. Emerging evidence suggests that DRB is a potential source of dietary fiber and phenolic compounds with numerous purported health benefits. However, less is known about its chemoprotective efficacy. In the present study, we determined and examined the nutrient composition of DRB and its chemopreventive effect on azoxymethane and dextran sulphate sodium (AOM/DSS)-induced colitis-associated colorectal cancer (CRC) in rats. The results showed the presence of several bioactive compounds, such as dietary fiber, phytic acid, and phenolic acids, in DRB. In addition, DRB supplementation reduced the progression of CRC symptoms, such as colonic shortening, disease activity index (DAI), and histopathological changes. Interestingly, a significant decrease was observed in total numbers of aberrant crypt foci (ACFs) and tumors with DRB supplementation. Furthermore, DRB supplementation suppressed the expression of pro-inflammatory cytokines (IL-6) and inflammatory mediators (NF-κB and COX-2) through the inactivation of the NF-κB signaling pathway. The administration of DRB revealed a negative effect on cancer cell proliferation by repressing the expression of nuclear ß-catenin, cyclin D1, and c-Myc. These findings suggest that DRB supplementation mitigates chronic inflammation and cancer cell proliferation and delays tumorigenesis in rat AOM/DSS-induced colitis-associated CRC. Therefore, the establishment of DRB as a natural dietary food-derived chemopreventive agent has the potential to have a significant impact on cancer prevention in the global population.

Host cell transcriptomic response to the multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

The upsurge of multidrug-resistant infections has rendered tuberculosis the principal cause of death among infectious diseases. A clonal outbreak multidrug-resistant triggering strain of Mycobacterium tuberculosis was identified in Kanchanaburi Province, labelled "MKR superspreader," which was found to subsequently spread to other regions, as revealed by prior epidemiological reports in Thailand. Herein, we showed that the MKR displayed a higher growth rate upon infection into host macrophages in comparison with the H37Rv reference strain. To further elucidate MKR's biology, we utilized RNA-Seq and differential gene expression analyses to identify host factors involved in the intracellular viability of the MKR. A set of host genes function in the cellular response to lipid pathway was found to be uniquely up-regulated in host macrophages infected with the MKR, but not those infected with H37Rv. Within this set of genes, the IL-36 cytokines which regulate host cell cholesterol metabolism and resistance against mycobacteria attracted our interest, as our previous study revealed that the MKR elevated genes associated with cholesterol breakdown during its growth inside host macrophages. Indeed, when comparing macrophages infected with the MKR to H37Rv-infected cells, our RNA-Seq data showed that the expression ratio of IL-36RN, the negative regulator of the IL-36 pathway, to that of IL-36G was greater in macrophages infected with the MKR. Furthermore, the MKR's intracellular survival and increased intracellular cholesterol level in the MKR-infected macrophages were diminished with decreased IL-36RN expression. Overall, our results indicated that IL-36RN could serve as a new target against this emerging multidrug-resistant M. tuberculosis strain.

New Normal Mask-Wearing and Its Impact on Underneath Skin Microbiome: A Cross-Sectional Study in Mild Acne Vulgaris Patients.

Introduction: Prolonged mask-wearing could modulate the skin microenvironment resulting in several facial dermatoses. Microbial dysbiosis is proposed to be linked with these changes; however, data regarding the association is still limited. Accordingly, we aimed to explore the impact of face masks on the skin's bacterial microbiota. Methods: We classified participants into short (<4 h/day) and long (≥4 h/day) mask-wearing time (SMWT and LMWT) groups according to mask-wearing time per day in the previous 2 weeks. Specimens were swabbed from the cheek and forehead of 45 mild acne vulgaris patients, representing mask-covered area (MCA) and mask-uncovered area (MUA), respectively. The 16S rRNA gene sequencing and QIIME2 were used to characterize bacterial communities. Results: There were 12 (26.7%) and 33 (73.3%) participants in SMWT and LMWT, respectively. There were no significant differences in beta diversity across MCA/MUA or LMWT/SMWT groups. In alpha-diversity, the evenness on MCA was significantly lower in LMWT than in SMWT (p value = 0.049). Among all groups, the relative abundance of bacterial taxa was similar, showing Actinobacteriota and Firmicutes, and Cutibacterium and Staphylococcus as the most predominant phyla and genera, respectively. Conclusion: Our results showed no significant impact of mask-wearing on the skin microbiota in mild acne vulgaris participants.

Association between gut microbiota and prediabetes in people living with HIV.

The prevalence of prediabetes is rapidly increasing in general population and in people living with HIV (PLWH). Gut microbiota play an important role in human health, and dysbiosis is associated with metabolic disorders and HIV infection. Here, we aimed to evaluate the association between gut microbiota and prediabetes in PLWH. A cross-sectional study enrolled 40 PLWH who were receiving antiretroviral therapy and had an undetectable plasma viral load. Twenty participants had prediabetes, and 20 were normoglycemic. Fecal samples were collected from all participants. The gut microbiome profiles were analyzed using 16S rRNA sequencing. Alpha-diversity was significantly lower in PLWH with prediabetes than in those with normoglycemia (p<0.05). A significant difference in beta-diversity was observed between PLWH with prediabetes and PLWH with normoglycemia (p<0.05). Relative abundances of two genera in Firmicutes (Streptococcus and Anaerostignum) were significantly higher in the prediabetes group. In contrast, relative abundances of 13 genera (e.g., Akkermansia spp., Christensenellaceae R7 group) were significantly higher in the normoglycemic group. In conclusion, the diversity of gut microbiota composition decreased in PLWH with prediabetes. The abundances of 15 bacterial taxa in the genus level differed between PLWH with prediabetes and those with normoglycemia. Further studies on the effect of these taxa on glucose metabolism are warranted.

The autophagy-resistant Mycobacterium tuberculosis Beijing strain upregulates KatG to evade starvation-induced autophagic restriction.

Mycobacterium tuberculosis utilizes several mechanisms to block phagosome-lysosome fusion to evade host cell restriction. However, induction of host cell autophagy by starvation was shown to overcome this block, resulting in enhanced lysosomal delivery to mycobacterial phagosomes and the killing of the M. tuberculosis reference strain H37Rv. Nevertheless, our previous studies found that strains belonging to the M. tuberculosis Beijing genotype can resist starvation-induced autophagic elimination, though the mycobacterial factors involved remain unclear. In this study, we showed that KatG expression is upregulated in the autophagy-resistant M. tuberculosis Beijing strain (BJN) during autophagy induction by the starvation of host macrophages, while such increase was not observed in the H37Rv. KatG depletion using the CRISPR-dCas9 interference system in the BJN resulted in increased lysosomal delivery to its phagosome and decreased its survival upon autophagy induction by starvation. As KatG functions by catabolizing ROS, we determined the source of ROS contributing to the starvation-induced autophagic elimination of mycobacteria. Using siRNA-mediated knockdown, we found that Superoxide dismutase 2, which generates mitochondrial ROS but not NADPH oxidase 2, is important for the starvation-induced lysosomal delivery to mycobacterial phagosomes. Taken together, these findings showed that KatG is vital for the BJN to evade starvation-induced autophagic restriction.

Identification of candidate regulators of mandibular bone loss in FcγRIIB-/- Mice.

Patients with systemic lupus erythematosus (SLE) have increased inflammatory cytokines, leading to periodontitis and alveolar bone loss. However, the mechanisms driving this phenomenon are still unknown. Here, we have identified novel therapeutic targets for and mediators of lupus-mediated bone loss using RNA-sequencing (RNA-seq) in a FcγRIIB-/- mouse model of lupus associated osteopenia. A total of 2,710 upregulated and 3,252 downregulated DEGs were identified. The GO and KEGG annotations revealed that osteoclast differentiation, bone mineralization, ossification, and myeloid cell development were downregulated. WikiPathways indicated that Hedgehog, TNFα NF-κB and Notch signaling pathway were also decreased. We identified downregulated targets, Sufu and Serpina12, that have important roles in bone homeostasis. Sufu and Serpina12 were related to Hedgehog signaling proteins, including Gli1, Gli2, Gli3, Ptch1, and Ptch2. Gene knockdown analysis demonstrated that Sufu, and Serpina12 contributed to osteoclastogenesis and osteoblastogenesis, respectively. Osteoclast and osteoblast marker genes were significantly decreased in Sufu-deficient and Serpina12-deficient cells, respectively. Our results suggest that alterations in Hedgehog signaling play an important role in the pathogenesis of osteopenia in FcγRIIB-/- mice. The novel DEGs and pathways identified in this study provide new insight into the underlying mechanisms of mandibular bone loss during lupus development.

Bioponic system for nitrogen and phosphorus recovery from chicken manure: Evaluation of manure loading and microbial communities.

Bioponics integrates the biological treatment of nutrient-rich waste streams with hydroponics. However, there are several challenges of bioponics, especially nutrient availability and qualities, which affect plant yield. In this study, chicken manure based-nutrient film technique bioponics was examined at manure loadings of 200, 300, and 400 g dry wt. per bioponic system (total of 18 plants). Bioponics effectively released nitrogen and phosphorus (total ammonia nitrogen of 5.8-8.0 mgN/L, nitrate of 7.0-11.2 mgN/L, and phosphate of 48.7-74.2 mgP/L) for efficient growth of lettuce (Lactuca sativa; total yield of 1208-2030 g wet wt. per 18 plants). Nitrogen and phosphorus use efficiencies were 35.1-41.8% and 6.8-8.0%, respectively, and were comparable to aquaponics. Next-generation sequencing was used to examine the microbial communities in digested chicken manure and plant roots in bioponics. Results showed that several microbial genera were associated with organic degradation (e.g., Nocardiopsis spp., Cellvibrio spp.), nitrification (Nitrospira spp.), phosphorus solubilization, and plant growth promotion (e.g., WD2101_soil_group, and Bacillus spp.). Nocardiopsis spp., Romboutsia spp. and Saccharomonospora spp. were found at high abundances and a high degree of co-occurrences among the microbiota, suggesting that the microbial organic decomposition to nitrogen and phosphorus release could be the key factors to achieve better nutrient recovery in bioponics.

Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain.

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

Transcriptional response to the host cell environment of a multidrug-resistant Mycobacterium tuberculosis clonal outbreak Beijing strain reveals its pathogenic features.

Tuberculosis is a global public health problem with emergence of multidrug-resistant infections. Previous epidemiological studies of tuberculosis in Thailand have identified a clonal outbreak multidrug-resistant strain of Mycobacterium tuberculosis in the Kanchanaburi province, designated "MKR superspreader", and this particular strain later was found to also spread to other regions. In this study, we elucidated its biology through RNA-Seq analyses and identified a set of genes involved in cholesterol degradation to be up-regulated in the MKR during the macrophage cell infection, but not in the H37Rv reference strain. We also found that the bacterium up-regulated genes associated with the ESX-1 secretion system during its intracellular growth phase, while the H37Rv did not. All results were confirmed by qRT-PCR. Moreover, we showed that compounds previously shown to inhibit the mycobacterial ESX-1 secretion system and cholesterol utilisation, and FDA-approved drugs known to interfere with the host cholesterol transportation were able to decrease the intracellular survival of the MKR when compared to the untreated control, while not that of the H37Rv. Altogether, our findings suggested that such pathways are important for the MKR's intracellular growth, and potentially could be targets for the discovery of new drugs against this emerging multidrug-resistant strain of M. tuberculosis.

First reported quantitative microbiota in different livestock manures used as organic fertilizers in the Northeast of Thailand.

Northeastern Thailand relies on agriculture as a major economic activity, and has used high levels of agrochemicals due to low facility, and salty sandy soil. To support soil recovery and sustainable agriculture, local farmers have used organic fertilizers from farmed animal feces. However, knowledge about these animal fecal manures remains minimal restricting their optimal use. Specifically, while bacteria are important for soil and plant growth, an abundance and a diversity of bacterial composition in these animal fecal manures have not been reported to allow selection and adjustment for a more effective organic fertilizer. This study thereby utilized metagenomics combined with 16S rRNA gene quantitative PCR (qPCR) and sequencing to analyze quantitative microbiota profiles in association with nutrients (N, P, K), organic matters, and the other physiochemical properties, of the commonly used earthworm manure and other manures from livestock animals (including breed and feeding diet variations) in the region. Unlike the other manures, the earthworm manure demonstrated more favorable nutrient profiles and physiochemical properties for forming fertile soil. Despite low total microbial biomass, the microbiota were enriched with maximal OTUs and Chao richness, and no plant pathogenic bacteria were found based on the VFDB database. The microbial metabolic potentials supported functions to promote crop growth, such as C, N and P cyclings, xenobiotic degradation, and synthesis of bioactive compounds. Pearson's correlation analyses indicated that the quantitative microbiota of the earthworm manure were clustered in the same direction as N, and conductivity, salinity, and water content were essential to control the microbiota of animal manures.

DNA metabarcoding to unravel plant species composition in selected herbal medicines on the National List of Essential Medicines (NLEM) of Thailand.

Traditional medicines are widely traded across the globe and have received considerable attention in the recent past, with expectations of heightened demand in the future. However, there are increasing global concerns over admixture, which can affect the quality, safety, and efficacy of herbal medicinal products. In this study, we aimed to use DNA metabarcoding to identify 39 Thai herbal products on the Thai National List of Essential Medicines (NLEM) and assess species composition and admixture. Among the products, 24 samples were in-house-prepared formulations, and 15 samples were registered formulations. In our study, DNA metabarcoding analysis using ITS2 and rbcL barcode regions were employed to identify herbal ingredients mentioned in the products. The nuclear region, ITS2, was able to identify herbal ingredients in the products at the genus- and family-levels in 55% and 63% of cases, respectively. The chloroplast gene, rbcL, enabled genus- and family-level identifications in 58% and 73% of cases, respectively. In addition, plant species were detected in larger numbers (Family identified, absolute %) in registered herbal products than in in-house-prepared formulations. The level of fidelity increases concerns about the reliability of the products. This study highlights that DNA metabarcoding is a useful analytical tool when combined with advanced chemical techniques for the identification of plant species in highly processed, multi-ingredient herbal products.

A novel diterpene agent isolated from Microbispora hainanensis strain CSR-4 and its in vitro and in silico inhibition effects on acetylcholine esterase enzyme.

An actinomycete strain CSR-4 was isolated from the rhizosphere soil of Zingiber montanum. Taxonomic characterization revealed strain CSR-4 was a member of the genus Microbispora. Whole-genome sequence analysis exhibited the highest average nucleotide identity (ANI) value (95.34%) and digital DNA-DNA hybridization (DDH) value (74.7%) between strain CSR-4 and the closest relative M. hainanensis DSM 45428T, which was in line with the assignment to same species. In addition, a new diterpene compound, 2α-hydroxy-8(14), 15-pimaradien-17, 18-dioic acid, and nine known compounds were isolated from the ethyl acetate crude extract of fermentation broth. Interestingly, a new diterpene displayed the suppressive effect on the recombinant human acetylcholinesterase (rhAChE) enzymes (IC50 96.87 ± 2.31 µg/ml). In silico studies based on molecular docking and molecular dynamics (MD) simulations were performed to predict a binding mode of the new compound into the binding pocket of the rhAChE enzyme and revealed that some amino acids in the peripheral anions site (PAS), anionic subsite, oxyanion site and catalytic active site (CAS) of the rhAChE have interacted with the compound. Therefore, our new compound could be proposed as a potential active human AChE inhibitor. Moreover, the new compound can protect significantly the neuron cells (% neuron viability = 88.56 ± 5.19%) from oxidative stress induced by serum deprivation method at 1 ng/ml without both neurotoxicities on murine P19-derived neuron cells and cytotoxicity against Vero cells.

Intestinal parasites in rural communities in Nan Province, Thailand: changes in bacterial gut microbiota associated with minute intestinal fluke infection.

Gastrointestinal helminth infection likely affects the gut microbiome, in turn affecting host health. To investigate the effect of intestinal parasite status on the gut microbiome, parasitic infection surveys were conducted in communities in Nan Province, Thailand. In total, 1047 participants submitted stool samples for intestinal parasite examination, and 391 parasite-positive cases were identified, equating to an infection prevalence of 37.3%. Intestinal protozoan species were less prevalent (4.6%) than helminth species. The most prevalent parasite was the minute intestinal fluke Haplorchis taichui (35.9%). Amplicon sequencing of 16S rRNA was conducted to investigate the gut microbiome profiles of H. taichui-infected participants compared with those of parasite-free participants. Prevotella copri was the dominant bacterial operational taxonomic unit (OTU) in the study population. The relative abundance of three bacterial taxa, Ruminococcus, Roseburia faecis and Veillonella parvula, was significantly increased in the H. taichui-infected group. Parasite-negative group had higher bacterial diversity (α diversity) than the H. taichui-positive group. In addition, a significant difference in bacterial community composition (ß diversity) was found between the two groups. The results suggest that H. taichui infection impacts the gut microbiome profile by reducing bacterial diversity and altering bacterial community structure in the gastrointestinal tract.

Transcriptome analysis of Pueraria candollei var. mirifica for gene discovery in the biosyntheses of isoflavones and miroestrol.

BACKGROUND: Pueraria candollei var. mirifica, a Thai medicinal plant used traditionally as a rejuvenating herb, is known as a rich source of phytoestrogens, including isoflavonoids and the highly estrogenic miroestrol and deoxymiroestrol. Although these active constituents in P. candollei var. mirifica have been known for some time, actual knowledge regarding their biosynthetic genes remains unknown. RESULTS: Miroestrol biosynthesis was reconsidered and the most plausible mechanism starting from the isoflavonoid daidzein was proposed. A de novo transcriptome analysis was conducted using combined P. candollei var. mirifica tissues of young leaves, mature leaves, tuberous cortices, and cortex-excised tubers. A total of 166,923 contigs was assembled for functional annotation using protein databases and as a library for identification of genes that are potentially involved in the biosynthesis of isoflavonoids and miroestrol. Twenty-one differentially expressed genes from four separate libraries were identified as candidates involved in these biosynthetic pathways, and their respective expressions were validated by quantitative real-time reverse transcription polymerase chain reaction. Notably, isoflavonoid and miroestrol profiling generated by LC-MS/MS was positively correlated with expression levels of isoflavonoid biosynthetic genes across the four types of tissues. Moreover, we identified R2R3 MYB transcription factors that may be involved in the regulation of isoflavonoid biosynthesis in P. candollei var. mirifica. To confirm the function of a key-isoflavone biosynthetic gene, P. candollei var. mirifica isoflavone synthase identified in our library was transiently co-expressed with an Arabidopsis MYB12 transcription factor (AtMYB12) in Nicotiana benthamiana leaves. Remarkably, the combined expression of these proteins led to the production of the isoflavone genistein. CONCLUSIONS: Our results provide compelling evidence regarding the integration of transcriptome and metabolome as a powerful tool for identifying biosynthetic genes and transcription factors possibly involved in the isoflavonoid and miroestrol biosyntheses in P. candollei var. mirifica.