Fibroblast growth factor 21 improves insulin sensitivity by modulating the bile acid-gut microbiota axis in type â ¡ diabetic mice.

BACKGROUND: Fibroblast growth factor 21 (FGF21) is an important regulator of glycolipid metabolism. However, whether the gut microbiota is related to the anti-diabetic and obesity effects of FGF21 remains unclear. METHODS: Our research used KO/KO db/db male mice and streptozotocin (STZ)-induced to simulate the construction of two type II diabetic mellitus (T2DM) models, and detected impaired glucose tolerance in the model by using the ipGTT and ITT assays, and collected feces from the model mice for sequencing of the intestinal flora and the content of short-chain fatty acids. H＆E staining was used to detect changes in intestinal tissue, the serum levels of LPS and GLP-1 were detected by ELISA. RESULTS: In this study, we found that FGF21 significantly improved insulin sensitivity, attenuated intestinal lesions, and decreased serum lipopolysaccharide (LPS) concentrations in T2DM mice. Moreover, FGF21 reshaped the gut microbiota and altered their metabolic pathways in T2DM mice, promoting the production of short-chain fatty acids (SCFAs) and the secretion of glucagon-like peptide 1 (GLP-1). Fecal transplantation experiments further confirmed that feces from FGF21-treated diabetic mice demonstrated similar effects as FGF21 in terms of anti-diabetic activity and regulation of gut microbiota dysbiosis. Additionally, the antibiotic depletion of gut microbiota abolished the beneficial effects of FGF21, including increased GLP-1 secretion and fecal SCFA concentration. Additionally, the FGF21 effects of ameliorating intestinal damage and suppressing plasma LPS secretion were suppressed. All these findings suggest that FGF21 prevents intestinal lesions by modifying the gut microbiota composition. Furthermore, FGF21 affected bile acid synthesis by inhibiting CYP7A1, the key enzyme of bile acid synthesis. CONCLUSSION: Therefore, FGF21 enriched beneficial bacteria by preventing bile acid synthesis and stimulating the secretion of the intestinal hormone GLP-1 via the increased production of gut microbiota metabolites, thereby exerting its anti-diabetic effects.

Enhanced algin oligosaccharide production through selective breeding and optimization of growth and degradation conditions in Cobetia sp. cqz5-12-M1.

Algin oligosaccharides have been applied in diverse industries and could be innovative synthesized by alginate-degrading bacteria. For enhance the alginate degradation efficiency to produce more algin oligosaccharides, a mutant strain (Cobetia sp. cqz5-12-M1) was obtained through the complex mutagenesis using UV and the alkylating agent 1-methyl-3-nitro-1-nitrosoguanidine. The enzyme activity of the fermentation supernatant of mutant exhibited a significant 38.09% (53.98 ± 0.69 U/mL) increase, and its optimal growth conditions were determined as: 5 g/L sodium alginate, 5 g/L yeast powder, 30 g/L NaCl, 2 g/L K2HPO4, 2 g/L KH2PO4, 1 g/L MgSO4•7H2O, 0.01 g/L FeSO4•7H2O, pH 6.5, and 34 ℃. Moreover, its optimal degradation conditions were identified as: 5 g/L sodium alginate, 5 g/L yeast powder, 30 g/L NaCl, 2 g/L K2HPO4, 2 g/L KH2PO4, 1 g/L MgSO4•7H2O, 0.01 g/L FeSO4•7H2O, pH 6.5, 31 ℃ and 72 h, yielding an enzyme activity of 120.98 ± 1.40 U/mL in the fermentation supernatant. Conclusive experiments on reagent tolerance revealed the growth of the mutant strain was significantly inhibited by 3% hydrogen peroxide, 5% carbolic acid, and 10 mg/mL gatifloxacin. Additionally, the alginate degradation capacity of mutant strain was highly significantly inhibited by 75% ethanol and all tested antibiotics.

Polygonatum cyrtonema polysaccharides reshape the gut microbiota to ameliorate dextran sodium sulfate-induced ulcerative colitis in mice.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized inflammatory imbalance, intestinal epithelial mucosal damage, and dysbiosis of the gut microbiota. Polygonatum cyrtonema polysaccharides (PCPs) can regulate gut microbiota and inflammation. Here, the different doses of PCPs were administered to dextran sodium sulfate-induced UC mice, and the effects of the whole PCPs were compared with those of the fractionated fractions PCP-1 (19.9 kDa) and PCP-2 (71.6 and 4.2 kDa). Additionally, an antibiotic cocktail was administered to UC mice to deplete the gut microbiota, and PCPs were subsequently administered to elucidate the potential role of the gut microbiota in these mice. The results revealed that PCP treatment significantly optimized the lost weight and shortened colon, restored the balance of inflammation, mitigated oxidative stress, and restored intestinal epithelial mucosal damage. And, the PCPs exhibited superior efficacy in ameliorating these symptoms compared with PCP-1 and PCP-2. However, depletion of the gut microbiota diminished the therapeutic effects of PCPs in UC mice. Furthermore, fecal transplantation from PCP-treated UC mice to new UC-afflicted mice produced therapeutic effects similar to PCP treatment. So, PCPs significantly ameliorated the symptoms, inflammation, oxidative stress, and intestinal mucosal damage in UC mice, and gut microbiota partially mediated these effects.

Preliminary study on the effect of catabolite repression gene knockout on p-nitrophenol degradation in Pseudomonas putida DLL-E4.

P-nitrophenol (PNP) is a carcinogenic, teratogenic, and mutagenic compound that can cause serious harm to the environment. A strain of Pseudomonas putida DLL-E4, can efficiently degrade PNP in a complex process that is influenced by many factors. Previous studies showed that the expression level of pnpA, a key gene involved in PNP degradation, was upregulated significantly and the degradation of PNP was obviously accelerated in the presence of glucose. In addition, the expression of crc, crcY, and crcZ, key genes involved in catabolite repression, was downregulated, upregulated, and upregulated, respectively. To investigate the effect of the carbon catabolite repression (CCR) system on PNP degradation, the crc, crcY, and crcZ genes were successfully knocked out by conjugation experiments. Our results showed that the knockout of crc accelerated PNP degradation but slowed down the cell growth. However, the knockout of crcY or crcZ alone accelerated PNP degradation when PNP as the sole carbon source, but that knockout slowed down PNP degradation when glucose was added. The results indicate that the CCR system is involved in the regulation of PNP degradation, and further work is required to determine the details of the specific regulatory mechanism.

Isolation, identification, and whole genome sequence analysis of the alginate-degrading bacterium Cobetia sp. cqz5-12.

Alginate-degrading bacteria or alginate lyases can be used to oligomerize alginate. In this study, an alginate-degrading bacterium with high alginolytic activity was successfully screened by using Sargassum fusiforme sludge. When the strain was grown on a plate containing sodium alginate, the transparent ring diameter (D) was 2.2 cm and the ratio (D/d) of transparent ring diameter to colony diameter (d) was 8.8. After 36 h in culture at a temperature of 28 °C shaken at 150 r/min, the enzymatic activity of the fermentation supernatant reached 160 U/mL, and the enzymatic activity of the bacterial precipitate harvested was 2,645 U/mL. The strain was named Cobetia sp. cqz5-12. Its genome is circular in shape, 4,209,007 bp in size, with a 62.36% GC content. It contains 3,498 predicted coding genes, 72 tRNA genes, and 21 rRNA genes. The functional annotations for the coding genes demonstrated that there were 181 coding genes in the genome related to carbohydrate transport and metabolism and 699 coding genes with unknown functions. Three putative coding genes, alg2107, alg2108 and alg2112, related to alginate degradation were identified by analyzing the carbohydrate active enzyme (CAZy) database. Moreover, proteins Alg2107 and Alg2112 were successfully expressed and exhibited alginate lyase activity.

Prdx1 promotes the loss of primary cilia in esophageal squamous cell carcinoma.

BACKGROUND: Loss of primary cilia is frequently observed in tumor cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction, the inability to exit the cell cycle, and promotion of tumor cell invasion. Primary cilia loss also occurs in esophageal squamous cell carcinoma (ESCC) cells, but the molecular mechanisms that explain how ESCC cells lose primary cilia remain poorly understood. METHODS: Inhibiting the expression of Prdx1 in the ESCC cells to detect the up-regulated genes related to cilium regeneration and down-regulated genes related to cilium disassembly by Gene chip. And, mice and cell experiments were carried to confirm the role of the HEF1-Aurora A-HDAC6 signaling axis in ESCC. RESULTS: In this study, we found that silencing Peroxiredoxin 1 (Prdx1) restores primary cilia formation, and over-expressing Prdx1 induces primary cilia loss in ESCC cells. We also showed that the expression of Prdx1 regulates the action of the HEF1-Aurora A-HDAC6 signaling axis to promote the disassembly of primary cilia, and suppression of Prdx1 results in decreased tumor formation and tumor mass volume in vivo. CONCLUSIONS: These results suggest that Prdx1 is a novel regulator of primary cilia formation in ESCC cells.

Fibroblast growth factor 21 alleviates acute pancreatitis via activation of the Sirt1-autophagy signalling pathway.

Fibroblast growth factor 21 (FGF21), a metabolic hormone with pleiotropic effects on glucose and lipid metabolism and insulin sensitivity, alleviates the process of acute pancreatitis (AP). However, its mechanism remains elusive. The pathological and physiological characteristics of FGF21 are observed in both patients with AP and cerulein-induced AP models, and the mechanisms of FGF21 in response to AP are investigated by evaluating the impact of autophagy in FGF21-treated mice and cultured pancreatic cells. Circulating levels of FGF21 significantly increase in both AP patients and cerulein-induced AP mice, which is accompanied by the change of pathology in pancreatic injury. Replenishment of FGF21 distinctly reverses cerulein-induced pancreatic injury and improves cerulein-induced autophagy damage in vivo and in vitro. Mechanically, FGF21 acts on pancreatic acinar cells to up-regulate Sirtuin-1 (Sirt1) expression, which in turn repairs impaired autophagy and removes damaged organs. In addition, blockage of Sirt1 accelerates cerulein-induced pancreatic injury and weakens the regulative effect in FGF21-activated autophagy in mice. These results showed that FGF21 protects against cerulein-induced AP by activation of Sirtuin-1-autophagy axis.

SIRT1 Mediates Effects of FGF21 to Ameliorate Cisplatin-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is a common complication in cancer patients. Kidney function is closely related to patients' quality of life and tumor prognosis. Cisplatin is a highly effective anti-tumor drug. However, the use of cisplatin is limited by its nephrotoxicity. It has been reported that FGF21 has a renal-protective function, but the mechanisms by which it does so remain unclear. In this study, we show that the expression of FGF21 is significantly upregulated in both in vitro and in vivo cisplatin-induced AKI models. Administration of recombinant FGF21 to cisplatin-induced AKI mice resulted in significantly decreased blood urea nitrogen (BUN) and serum creatinine levels, as well as significantly reduced protein levels of kidney injury molecule-1 (TIM-1), C-caspase 3, and Bax. H&E-stained kidney sections from cisplatin-induced AKI mice treated with recombinant FGF21 showed a relatively normal renal tissue structure, a reduced number of necrotic sites and vacuolar changes, and decreased casts, suggesting alleviated renal tubular injury. Experiments with an AKI cell model (cisplatin-treated HK-2 cells) yielded similar results as the mouse model; recombinant FGF21 significantly downregulated protein expression levels of TIM-1, C-caspase 3, and Bax. Furthermore, administration of recombinant FGF21 to cisplatin-treated AKI models significantly increased SIRT1 expression, and the beneficial effects of FGF21 on kidney injury were reversed by SIRT1 knockdown. Collectively, our results suggest that SIRT1 mediates the protective effect of FGF21 on cisplatin-induced kidney injury.

Relationship of Red Blood Cell Distribution Width with Cancer Mortality in Hospital.

BACKGROUND: Red blood cell distribution width (RDW) is a clinical index used to make early diagnosis and to monitor treatment effects in iron deficiency anemia. Recently, several studies have suggested that RDW was associated with mortality from various cancers; however, there has been little evidence regarding RDW and cancer as a whole. Therefore, the purpose of our study was to investigate the relationship of RDW and overall cancer mortality in hospital. METHODS: We extracted patient data from the Multiparameter Intelligent Monitoring in Intensive Care Database III version 1.3 (MIMICIII.1.3). RDW was measured prior to hospital admission. Patients older than 18 who were diagnosed with malignant tumors were included. The primary outcome was cancer mortality in hospital. Logistic regression and multivariate analysis were used to assess the association between the RDW and hospital mortality. RESULT: A total of 3384 eligible patients were enrolled. A positive correlation was observed between RDW and overall cancer mortality. Patients with higher RDW (14.4-16.3%, 16.4-30.5%) were at greater risk of death than the patients with RDW in the reference range (11.5-14.3%). On multivariate analysis, when adjusted for age and gender, the adjusted OR (95% CIs) in the mid-RDW group and high-RDW group were 1.61 (1.28, 2.03) and 2.52 (2.03, 3.13), respectively, with the low-RDW group set as the baseline. Similar trends were also observed in the model adjusted for other clinical characteristics. This suggested that elevated RDW was related to increased risk of cancer mortality, and RDW may play an important role in the prediction of short-term mortality after hospitalization in cancer patients. CONCLUSION: Elevated RDW was associated with overall cancer mortality. To a certain extent, RDW may predict the risk of mortality in patients with cancers; it was an independent prognostic indicator of short-term mortality after hospitalization in cancer patients.

Crystal structure of p-nitrophenol 4-monooxygenase PnpA from Pseudomonas putida DLL-E4: The key enzyme involved in p-nitrophenol degradation.

p-Nitrophenol 4-monooxygenase PnpA, the key enzyme in the hydroquinone pathway of p-nitrophenol (PNP) degradation, catalyzes the monooxygenase reaction of PNP to p-benzoquinone in the presence of FAD and NADH. Here, we determined the first crystal structure of PnpA from Pseudomonas putida DLL-E4 in its apo and FAD-complex forms to a resolution of 2.04 Šand 2.48 Å, respectively. The PnpA structure shares a common fold with hydroxybenzoate hydroxylases, despite a low amino sequence identity of 14-18%, confirming it to be a member of the Class A flavoprotein monooxygenases. However, substrate docking studies of PnpA indicated that the residues stabilizing the substrate in an orientation suitable for catalysis are not observed in other homologous hydroxybenzoate hydroxylases, suggesting PnpA employs a unique catalytic mechanism. This work expands our understanding on the reaction mode for this enzyme class.

Cloning, heterologous expression, and enzymatic characterization of a novel glucoamylase GlucaM from Corallococcus sp. strain EGB.

The gene encoding a novel glucoamylase (GlucaM) from the Corallococcus sp. strain EGB was cloned and heterologous expressed in Escherichia coli BL21(DE3), and the enzymatic characterization of recombinant GlucaM (rGlucaM) was determined in the study. The glucaM had an open reading frame of 1938 bp encoding GlucaM of 645 amino acids with no signal peptide. GlucaM belongs to glycosyl hydrolase family 15 and shares the highest identity 96% with the GH15 glucoamylase of Corallococcus coralloides DSM 2259. The rGlucaM with His-tag was purified by the Ni2+-NTA resin, with a specific activity from 3.4 U/mg up to 180 U/mg, and the molecular weight of rGlucaM was approximately 73 kDa on SDS-PAGE. The Km and Vmax of rGlucaM for soluble starch were 1.2 mg/mL and 46 U/mg, respectively. rGlucaM was optimally active at pH 7.0 and 50 °C and had highly tolerance to high concentrations of salts, detergents, and various organic solvents. rGlucaM hydrolyzed soluble starch to glucose, and hydrolytic activities were also detected with amylopectin, amylase, glycogen, starch (potato), α-cyclodextrin, starch (corn and potato). The analysis of hydrolysis products shown that rGlucaM with α-(1-4),(1-6)-D-glucan glucohydrolase toward substrates. These characteristics indicated that the GlucaM was a new member of glucoamylase family and a potential candidate for industrial application.

Impact of pnpR, a LysR-type regulator-encoding gene, on the cellular processes of Pseudomonas putida DLL-E4.

LysR-type transcriptional regulators (LTTRs) regulate various cellular processes in bacteria. pnpR is an LTTR-encoding gene involved in the regulation of hydroquinone (HQ) degradation, and its effects on the cellular processes of Pseudomonas putida DLL-E4 were investigated at the physiological, biochemical and molecular levels. Reverse transcription polymerase chain reaction revealed that pnpR positively regulated its own expression and that of the pnpC1C2DECX1X2 operon; additionally, pnpR partially regulated the expression of pnpA when P. putida was grown on para-nitrophenol (PNP) or HQ. Strains DLL-E4 and DLL-ΔpnpR exhibited similar cellular morphologies and growth rates. Transcriptome analysis revealed that pnpR regulated the expression of genes in addition to those involved in PNP degradation. A total of 20 genes were upregulated and 19 genes were downregulated by at least 2-fold in strain DLL-ΔpnpR relative to strain DLL-E4. Bioinformatic analysis revealed putative PnpR-binding sites located in the upstream regions of genes involved in PNP degradation, carbon catabolite repression and other cellular processes. The utilization of L-aspartic acid, L-histidine, L-pyroglutamic acid, L-serine, γ-aminobutyric acid, D,L-lactic acid, D-saccharic acid, succinic acid and L-alaninamide was increased at least 1.3-fold in strain DLL-ΔpnpR as shown by BIOLOG assays, indicating that pnpR plays a potential negative regulation role in the utilization of carbon sources.

The Regulation of para-Nitrophenol Degradation in Pseudomonas putida DLL-E4.

Pseudomonas putida DLL-E4 can efficiently degrade para-nitrophenol and its intermediate metabolite hydroquinone. The regulation of para-nitrophenol degradation was studied, and PNP induced a global change in the transcriptome of P. putida DLL-E4. When grown on PNP, the wild-type strain exhibited significant downregulation of 2912 genes and upregulation of 845 genes, whereas 2927 genes were downregulated and 891 genes upregulated in a pnpR-deleted strain. Genes related to two non-coding RNAs (ins1 and ins2), para-nitrophenol metabolism, the tricarboxylic acid cycle, the outer membrane porin OprB, glucose dehydrogenase Gcd, and carbon catabolite repression were significantly upregulated when cells were grown on para-nitrophenol plus glucose. pnpA, pnpR, pnpC1C2DECX1X2, and pnpR1 are key genes in para-nitrophenol degradation, whereas pnpAb and pnpC1bC2bDbEbCbX1bX2b have lost the ability to degrade para-nitrophenol. Multiple components including transcriptional regulators and other unknown factors regulate para-nitrophenol degradation, and the transcriptional regulation of para-nitrophenol degradation is complex. Glucose utilization was enhanced at early stages of para-nitrophenol supplementation. However, it was inhibited after the total consumption of para-nitrophenol. The addition of glucose led to a significant enhancement in para-nitrophenol degradation and up-regulation in the expression of genes involved in para-nitrophenol degradation and carbon catabolite repression (CCR). It seemed that para-nitrophenol degradation can be regulated by CCR, and relief of CCR might contribute to enhanced para-nitrophenol degradation. In brief, the regulation of para-nitrophenol degradation seems to be controlled by multiple factors and requires further study.

Metabolic pathway involved in 2-methyl-6-ethylaniline degradation by Sphingobium sp. strain MEA3-1 and cloning of the novel flavin-dependent monooxygenase system meaBA.

2-Methyl-6-ethylaniline (MEA) is the main microbial degradation intermediate of the chloroacetanilide herbicides acetochlor and metolachlor. Sphingobium sp. strain MEA3-1 can utilize MEA and various alkyl-substituted aniline and phenol compounds as sole carbon and energy sources for growth. We isolated the mutant strain MEA3-1Mut, which converts MEA only to 2-methyl-6-ethyl-hydroquinone (MEHQ) and 2-methyl-6-ethyl-benzoquinone (MEBQ). MEA may be oxidized by the P450 monooxygenase system to 4-hydroxy-2-methyl-6-ethylaniline (4-OH-MEA), which can be hydrolytically spontaneously deaminated to MEBQ or MEHQ. The MEA microbial metabolic pathway was reconstituted based on the substrate spectra and identification of the intermediate metabolites in both the wild-type and mutant strains. Plasmidome sequencing indicated that both strains harbored 7 plasmids with sizes ranging from 6,108 bp to 287,745 bp. Among the 7 plasmids, 6 were identical, and pMEA02' in strain MEA3-1Mut lost a 37,000-bp fragment compared to pMEA02 in strain MEA3-1. Two-dimensional electrophoresis (2-DE) and protein mass fingerprinting (PMF) showed that MEA3-1Mut lost the two-component flavin-dependent monooxygenase (TC-FDM) MeaBA, which was encoded by a gene in the lost fragment of pMEA02. MeaA shared 22% to 25% amino acid sequence identity with oxygenase components of some TC-FDMs, whereas MeaB showed no sequence identity with the reductase components of those TC-FDMs. Complementation with meaBA in MEA3-1Mut and heterologous expression in Pseudomonas putida strain KT2440 resulted in the production of an active MEHQ monooxygenase.

Purification and Characterization of a Novel Extracellular Thermostable Alkaline Protease from Streptomyces sp. M30.

A novel alkaline protease from Streptomyces sp. M30, SapHM, was purified by ammonium sulfate precipitation, hydrophobic interaction chromatography, and DEAE-Sepharose chromatography, with a yield of 15.5% and a specific activity of 29,070 U/mg. Tryptic fragments of the purified SapHM were obtained by electrospray ionization quadrupole time-of- flight mass spectrometry. Nucleotide sequence analysis revealed that the gene sapHM contained 1,179 bp, corresponding to 392 amino acids with conserved Asp156, His187, and Ser339 residues of alkaline protease. The first 24 amino acid residues were predicted to be a signal peptide, and the molecular mass of the mature peptide was 37.1 kDa based on amino acid sequences and mass spectrometry. Pure SapHM was optimally active at 80°C in 50 mM glycine-NaOH buffer (pH 9.0), and was broadly stable at 0-50 °C and pH 4.0-9.0. The protease relative activity was increased in the presence of Ni(2+), Mn(2+), and Cu(2+) to 112%, 113%, and 147% of control, respectively. Pure SapHM was also activated by dimethylformamide, dimethyl sulfoxide, Tween 80, and urea. The activity of the purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, indicating that it is a serine-type protease. The Km and Vmax values were estimated to be 35.7 mg/ml, and 5 × 10(4) U/mg for casein. Substrate specificity analysis showed that SapH was active on casein, bovine serum albumin, and bovine serum fibrin.

Complete Genome Sequence of the p-Nitrophenol-Degrading Bacterium Pseudomonas putida DLL-E4.

The first complete genome sequence of a p-nitrophenol (PNP)-degrading bacterium is reported here. Pseudomonas putida DLL-E4, a Gram-negative bacterium isolated from methyl-parathion-polluted soil, can utilize PNP as the sole carbon and nitrogen source. P. putida DLL-E4 has a 6,484,062 bp circular chromosome that contains 5,894 genes, with a G+C content of 62.46%.