Lacticaseibacillus rhamnosus Fmb14 ameliorates hyperuricemia-induced hepatocyte pyroptosis via NLRP3 inflammasome cascade inhibition

Hyperuricemia is a high-risk factor for the development of gout and renal fibrosis,but the adverse effects of hyperuricemia on the liver have been seriously neglected.This research investigated the ameliorating effect of Lacticaseibacillus rhamnosus Fmb14 on hyperuricemia induced liver dysfunction both in vitro and in vivo.Cell free extracts of high dose L.rhamnosus Fmb14 treatment reduced the death rate of HepG2 cell lines from 24.1%to 14.9%by inhibiting NLRP3 recruitment,which was mainly activated by reactive oxygen species release and mitochondrial membrane potential disorder.In purine dietary induced hyperuricemia(PDIH)mice model,liver oedema and pyroptosis were ameliorated after L.rhamnosus Fmb14 administration through downregulating the expression levels of NLRP3,caspase-1 and gasdermin-D from 1.61 to 0.86,3.15 to 1.01 and 5.63 to 2.02,respectively.L.rhamnosus Fmb14 administration restored mitochondrial inner membrane protein(MPV17)and connexin 43 from 2.83 and 0.73 to 0.80 and 0.98 respectively in PDIH mice,indicating that dysbiosis of mitochondrial membrane potential was restored in liver.Intriguingly,PDIH pyroptosis stimulates the process of apoptosis,which leads to severe leakage of hepatocytes,and both of pyroptosis and apoptosis were decreased after L.rhamnosus Fmb14 treatment.Therefore,L.rhamnosus Fmb14 is a promising biological resource to maintain homeostasis of the liver in hyperuricemia and the prevention of subsequent complications.

Ameliorative effect of Lacticaseibacillus rhamnosus Fmb14 from Chinese yogurt on hyperuricemia

Hyperuricemia is a critical threat to human health,and a high inosine diet can increase the prevalence of it.Lacticaseibacillus rhamnosus Fmb14 was isolated from traditional fermented Chinese yogurt,and its inosine degradation rate reached 36.3%at 109 CFU/mL for 24 h.LC-MS analysis revealed that high concentrations of inosine could activate compensatory metabolic pathways of L.rhamnosus Fmb14 to catalyse inosine as an energy source and produce intracellular folic acid and riboflavin.The contents of folic acid and riboflavin were 6.0 and 4.3 fold increased after inosine treatment in the cell-free extracts(CFE).L.rhamnosus Fmb14 CFE treatment ameliorates hyperuricemia through xanthine oxidase(XOD)inhibition and ATP-binding cassette subfamily G member 2(ABCG2)promotion,both of which are responsible for uric acid(UA)synthesis and secretion in HepG2 and Caco2 cells,respectively.The in vivo results showed that the serum UA level decreased from 236.28 to 149.28μmol/L after 8 weeks of oral administration of L.rhamnosus Fmb14 in inosine-induced hyperuricemia model mice.Our results revealed that L.rhamnosus Fmb14 has a potential as a biological therapeutic agent in hyperuricemia prevention.

Effects of dietary Bacillus amyloliquefaciens supplementation on growth performance,intestinal morphology, inflammatory response, and microbiota of intra-uterine growth retarded weanling piglets

Background: The focus of recent research has been directed toward the probiotic potential of Bacillus amyloliquefaciens(BA) on the gut health of animals. However, little is known about BA's effects on piglets with intra-uterine growth retardation(IUGR). Therefore, this study investigated the effects of BA supplementation on the growth performance,intestinal morphology, inflammatory response, and microbiota of IUGR piglets.Methods: Eighteen litters of newborn piglets were selected at birth, with one normal birth weight(NBW) and two IUGR piglets in each litter(i.e., 18 NBW and 36 IUGR piglets in total). At weaning, the NBW piglet and one of the IUGR piglets were assigned to groups fed a control diet(i.e., the NBW-CON and IUGR-CON groups). The other IUGR piglet was assigned to a group fed the control diet supplemented with 2.0 g BA per kg of diet(i.e., IUGR-BA group). The piglets were thus distributed across three groups for a four-week period.Results: IUGR reduced the growth performance of the IUGR-CON piglets compared with the NBW-CON piglets. It was also associated with decreased vil us sizes, increased apoptosis rates, reduced goblet cel numbers, and an imbalance between pro-and anti-inflammatory cytokines in the smal intestine. Supplementation with BA improved the average daily weight gain and the feed efficiency of the IUGR-BA group compared with the IUGR-CON group(P < 0.05). The IUGR-BA group exhibited increases in the ratio of jejunal vil us height to crypt depth, in ileal vil us height, and in ileal goblet cel density. They also exhibited decreases in the numbers of jejunal and ileal apoptotic cel s and ileal proliferative cel s(P < 0.05). Supplementation with BA increased interleukin 10 content, but it decreased tumor necrosis factor alpha level in the smal intestines of the IUGR-BA piglets(P < 0.05). Furthermore, compared with the IUGR-CON piglets, the IUGR-BA piglets had less Escherichia coli in their jejunal digesta, but more Lactobacil us and Bifidobacterium in their ileal digesta(P < 0.05).Conclusions: Dietary supplementation with BA improves morphology, decreases inflammatory response, and regulates microbiota in the smal intestines of IUGR piglets, which may contribute to improved growth performance during early life.

Yogurt-derived Lactobacillus plantarum Q16 alleviated high-fat diet-induced non-alcoholic fatty liver disease in mice

Accumulating evidence revealed that some probiotics regulated lipid metabolism and alleviated diet-induced non-alcoholic fatty liver disease(NAFLD).This study mainly explored whether yogurt-derived Lactobacillus plantarum Q16 modulated lipid and energy metabolism,and suppressed microbial dysbiosis in high-fat diet(HFD)-fed mice.Results showed that oral administration of L.plantarum Q16 improved serum and hepatic lipid profile.Protein analysis showed that L.plantarum Q16 could reduce hepatic lipid content by reducing the expression of FAS,ACC,SCD-1,Srebp-lc and ATGL,but increasing expression levels of CPT-1α,PPAR-αand ATGL.Meanwhile,L.plantarum Q16 also improved hepatic energy metabolism by regulating FGF21/adiponectin/AMPKα/PGC-1αsignaling pathway.Metagenomic analysis also discovered that L.plantarum Q16 increased species diversity and richness of intestinal microbiota,promoted proliferation of beneficial commensals and suppressed the growth of endotoxin-producing microorganisms in the colon of HFD-fed mice.Overall,L.plantarum Q16 protected against HFD-induced NAFLD by improving hepatic profile and regulating colonic microbiota composition.

Surfactin alleviated hyperglycaemia in mice with type 2 diabetes induced by a high-fat diet and streptozotocin

Type 2 diabetes mellitus(T2DM)is associated with liver dysfunction and intestinal dysbiosis.Bioactive peptides(BAPs)have been reported to ameliorate T2DM by preventing oxidative damage to the liver.Bacillus amyloliquefaciens fmb50 produces the lipopeptide surfactin with a wide range of biological activities.The effects of surfactin on T2DM,on the other hand,have not been studied.In the present study,80 mg/kg body weight surfactin supplementation lowered fasting blood glucose(FBG)levels by 21.05%and insulin resistance(IR)by 18.18%compared with those in the T2DM group,reduced inflammation,and increased antioxidant activity in mice with T2DM induced by a high-fat diet(HFD)and streptozotocin(STZ).According to further research,surfactin administration reduced Firmicutes-to-Bacteroidetes ratios while increasing Bifi dobacterium abundance by 20 times and the level of the tight junction protein Occludin by 18.38%and ZO-1 by 66.60%.Furthermore,surfactin also improved hepatic glucose metabolism by activating the adenosine monophosphate-activated protein kinase(AMPK)signalling pathway,increasing glycogen synthesis and glucose transporter 2(GLUT2)protein expression while reducing glucose-6-phosphatase(G6Pase)protein expression.In addition,the increased Bifi dobacterium abundance indirectly reduced the liver burden of the metabolic products indole,cresol and amine produced by saprophytic bacteria.All of these findings revealed that surfactin not only ameliorated HFD/STZ-induced gut dysbiosis and preserved intestinal barrier integrity but also enhanced hepatic glucose metabolism and detoxifi cation function in T2DM mice.The gut microbiota appeared to be important in controlling glucose metabolism,IR,fat accumulation,inflammation and antioxidation,according to Spearman’s correlation coeffi cients.All data indicated that surfactin alleviated hyperglycaemia in mice with T2DM induced by HFD/STZ.

Substitution of the communication‑mediating domain and the interaction between modules in bacillomycin D produced by Bacillus amyloliquefaciens

Bacillomycin D is a nonribosomal peptide produced by Bacillus amyloliquefaciens fmbJ.In this study,the biosynthesis of bacillomycin D was mediated by replacing the intermodule donor,acceptor,and communication-mediating(COM)domain pairs and introducing the COM domain between the surfactin subunits.Both the homologous donor and the COM domain pair replacement strains eliminated the selective barrier of the original COM domain pair to a certain extent,resulting in a more flexible hybrid biosynthesis system that provides simultaneous biosynthesis of different lipopeptide products.The synthesis of bacillomycin D in the homologous acceptor replacement strains was barely affected.The COM domains between the surfactin subunits cannot establish efficient communication between the bacillomycin D modules.In conclusion,the COM domains compatibility between the bacillomycin D modules are extremely strong,and the conserved amino acid residues in the acceptor domain are an important part of module–module interactions and efficient communication during bacillomycin D synthesis.

Screening and performance of L-14, a novel, highly efficient and low temperature-resistant cellulose-degrading strain

In view of the low bioconversion efficiency of agricultural biomass waste in low-temperature environments in winter,a low-temperature-resistant cellulose-degrading strain,L-14,was successfully screened by restrictive cultures from humus-rich soil in the Daqing Zhalong wetland region.According to morphological observations and 18S rDNA sequence analysis,the cellulose-degrading strain L-14 was identified as a Neurospora sp,belonging to fungus.Different parameters,such as temperature,initial pH,carbon,nitrogen and lecithin,were optimized using a single-factor experiment and a response surface methodology(RSM).When the temperature was 16°C,the optimal conditions for enzyme production were an initial pH 8.20,10.45 g/L of bran,5.28 g/L of yeast powder,and 4.25 g/L of lecithin.The carboxymethyl cellulase(CMCase)activity of strain L-14 was 63.598 IU/mL.Strain L-14 had a high level of cellulose degradation activity,excellent resistance to low temperatures and environmental adaptability,indicating its good application prospects in substrates pretreatment of biogas engineering.

Deletion of COM donor and acceptor domains and the interaction between modules in bacillomycin D produced by Bacillus amyloliquefaciens

Bacillomycin D is a cyclic lipopeptide produced by Bacillus amyloliquefaciens fmbJ.At present,no relevant report has described the combinatorial biosynthesis of bacillomycin D.Due to the strong biosynthetic potential of the communication-mediating(COM)domains,its crosstalk between NRPS subunits has been studied to some extent,but the interaction of COM domain between modules is rarely reported.Therefore,in this study,we conducted the combinatorial biosynthesis of bacillomycin D through the deletion of the COM donor and acceptor domains between the modules and elucidated the interaction between the NRPS modules.The results showed that the deletion of the donor domain between modules 2 and 3 did not affect catalysis by upstream modules,but prevented downstream modules from catalysing the extension of the lipopeptide product,ultimately resulting in mutant complexes that could form linear dipeptides with the sequenceβ-NH_(2)FA-Asn-Tyr.However,the engineered hybrid bacillomycin D NRPSs lacking the donor domains between modules 3 and 4 and modules 6 and 7 could form multiple assembly lines that produced bacillomycin D and its analogs(linear tripeptides,cyclic hexapeptides and linear hexapeptides).In addition,all the acceptor domain deletion strains failed to produce bacillomycin D,only truncated peptides produced by module interruption(except for the acceptor domain deletion strains between modules 3 and 4,which also produced cyclic hexapeptides).In conclusion,deletion of the inter-module donor domains led to a more flexible hybrid biosynthetic system for the production of diverse peptide products;compared with the inter-subunit donor domain deletion strains that could only produce truncated peptides,the former had a greater biosynthetic capacity.Meanwhile,the acceptor domains between modules were an important part of module-module interactions and efficient communication within bacillomycin D synthetase.

抗人脂蛋白相关磷脂酶A2单抗制备及应用

本研究旨在制备抗人脂蛋白相关磷脂酶A2 (Lp-PLA2)单克隆抗体,对单抗进行初步评价,采用免疫层析方法学,建立一种可在社区医疗机构应用的Lp-PLA2快速检测方法。首先从NCBI获得人Lp-PLA2全长基因序列构建表达载体,在CHO-K1细胞中表达Lp-PLA2重组蛋白。以获得的重组蛋白免疫BALB/c小鼠,利用细胞融合技术筛选杂交瘤细胞,小鼠腹水诱生单克隆抗体。通过SDS-PAGE、ELISA等方法评价抗体性能。利用双抗夹心法制备Lp-PLA2量子点荧光免疫层析检测试剂,并使用便携式检测仪器评估试剂性能。制备并筛选得到亲和力达到1×10^(–8)的配对单克隆抗体PLA1与PLA5,抗体亚类为IgG1,能特异性识别血液中Lp-PLA2蛋白。自制Lp-PLA2量子点荧光免疫检测试剂可检测线性范围为20–2000ng/mL,与进口试剂的检测相关系数R达到0.99。综上,本研究制备得到一对高亲和力、高特异性的抗人Lp-PLA2单克隆抗体,并建立了Lp-PLA2免疫层析检测方法,该方法检测准确、操作简便,适合Lp-PLA2检测应用于社区医疗机构,为居民心血管健康管理提供了新途径。

Neomycin biosynthesis is regulated positively by AfsA-g and NeoR in Streptomyces fradiae CGMCC 4.7387

Neomycins are a group of aminoglycoside antibiotics with both clinical and agricultural applications.To elucidate the regulatory mechanism of neomycin biosynthesis,we completed draft genome sequencing of a neomycin producer Streptomyces fradiae CGMCC 4.7387 from marine sediments,and the neomycin biosynthesis gene cluster was identified.Inactivation of the afsA-g gene encoding a γ-butyrolactone(GBL) synthase in S.fradiae CGMCC 4.7387 resulted in a significant decrease of neomycin production.Quantitative RT-PCR analysis revealed that the transcriptional level of neoR and the aphA-neoGH operon were reduced in the afsA-g::aac(3)Ⅳ mutant.Interestingly,a conserved binding site of AdpA,a key activator in the GBL regulatory cascade,was discovered upstream of neoR,a putative regulatory gene encoding a protein with an ATPase domain and a tetratricopeptide repeat domain.When neoR was inactivated,the neomycin production was reduced about 40%in comparison with the WT strain.Quantitative RT-PCR analysis revealed that the transcriptional levels of genes in the aphA-neoGH operon were reduced clearly in the neoR::aac(3)Ⅳ mutant.Finally,the titers of neomycin were improved considerably by overexpression of qfsA-gand neoR in S.fradiae CGMCC 4.7387.

Optimization of fermentation factors to enhance rice straw degradation ability using a microbial consortium LZF-12

Biological pretreatment has broad application prospects in agricultural waste treatment because of its economic benefits,environmental protection and energy saving characteristics.In this study,a microbial consortium LZF-12 was applied for a biological pretreatment to degrade rice straw.Batch experiments were performed under hydrolysis conditions by using the method of Box-Behnken factorial design(BBD).The results showed that the model multiple correlation coefficient R2 was 0.9816,and the effects of three factors on the degradation of rice straw of LZF-12 as descending order were the initial rice content,chicken manure content,and initial pH value.The interaction between straw concentration,chicken manure concentration,and initial pH value had significant effects on the degradation of a microbial consortium.Under the optimum conditions of 0.86%rice straw,0.5%chicken manure and the initial pH value of 7.0,the degradation rate of rice straw reached 72.4%.There is only a small difference of 0.55%between the experimental value and predicted value from BBD model.Therefore,it is feasible for the established model due to the consistent results between the prediction and experimental value.The microbial consortium LZF-12 has high cellulase enzyme activities and degradation ability over a wide range of temperature and pH value,indicating that it has a good development potential and application prospects in waste biodegradation and biomass energy production.

Development of environmentally friendly biological algicide and biochemical analysis of inhibitory effect of diatom Skeletonema costatum

Skeletonema costatum is a diatom widely distributed in red tide microalgae blooms and as one of the main algae causing harmful algal blooms,because of their rapid reproduction and production of toxic and harmful substances,often play a negative role in aquatic ecosystems,and human health and wellbeing.Bacillomycin D is a nonribosomal cyclic antifungal lipopeptide in the iturins family.In this study,Bacillomycin D was tested for its ability to inhibit the growth of S.costatum.The EC_(50 24h) of Bacillomycin D on S.costatum was 24.70μg/mL.The chlorophyll fluorescence parameters F_(v)/F_(m),F_(v)/F_(o),and yield of the diatoms decreased significantly with increasing concentrations of Bacillomycin D.Study of the mechanism showed that Bacillomycin D induced cell death by changing cell membrane permeability,promoting the release of cellular contents.In this study,transcriptomic analysis showed Bacillomycin D significantly inhibited the photosynthesis and metabolism of S.costatum.These findings investigated the inhibitory effect of Bacillomycin D on the growth of S.costatum and provided a theoretical foundation for the development of new environmentally friendly biological algicide.

Translocation of subunit PPSE in plipastatin synthase and synthesis of novel lipopeptides

Nonribosomal peptide synthase(NRPS)is a unique molecular assembly mechanism with high hybridity.Its recombination is conducive to the development of novel lipopeptides.However,there are few reports on NRPS subunit recombination of plipastatin at present.In this paper,plipastatin synthase was modified by the forward movement of subunit PPSE and the replacement of the communication-mediating(COM)domain.The results showed that ppsABE,a new assembly line,could synthesize novel lipopeptides such as cycle pentapeptide(C16-18β-OHFA-E-O-cyclo(Y-T-I),and its antimicrobial activity against Rhizopus stolonifer and Staphylococcus aureus was better than that of plipastatin.However,the reactivity of ppsABCE disappeared,but the substitution of COM^(D)_(ppsC)/COM^(A)_(ppsD) or COM^(D)_(ppsD)/COM^(A)_(ppsE) for COM^(D)_(ppsC)/COM^(A)_(ppsE) could restore its activity and conduct the biosynthesis of linear hexapeptide(C16-17β-OHFA-E-O-Y-T-E-A/V)and heptapeptide(C17-18β-OHFA-E-O-Y-T-E-A-I).Collectively,these findings indicated that the COM donor domain at the C-terminus of PPSB could communicate with the COM acceptor domain at the N-terminus of PPSE and that the compatible COM domain is an important tool for communication between nonpartner subunits.Moreover,the integrity and selective compatibility of the COM acceptor domain of subunit PPSE are essential to promote the interaction between PPSE and other subunits.This work further complemented the rules of NRPS subunit recombination and provided a theoretical basis for the development of novel high-efficiency lipopeptides.