Gut microbial communities are likely remodeled in tandem with accumulated physiological decline during aging, yet there is limited understanding of gut microbiome variation in advanced age. Here, we performed a metagenomics-based enterotype analysis in a geographically homogeneous cohort of 367 enrolled Chinese individuals between the ages of 60 and 94 years, with the goal of characterizing the gut microbiome of elderly individuals and identifying factors linked to enterotype variations. In addition to two adult-like enterotypes dominated by Bacteroides (ET-Bacteroides) and Prevotella (ET-Prevotella), we identified a novel enterotype dominated by Escherichia (ET-Escherichia), whose prevalence increased in advanced age. Our data demonstrated that age explained more of the variance in the gut microbiome than previously identified factors such as type 2 diabetes mellitus (T2DM) or diet. We characterized the distinct taxonomic and functional profiles of ET-Escherichia, and found the strongest cohesion and highest robustness of the microbial co-occurrence network in this enterotype, as well as the lowest species diversity. In addition, we carried out a series of correlation analyses and co-abundance network analyses, which showed that several factors were likely linked to the overabundance of Escherichia members, including advanced age, vegetable intake, and fruit intake. Overall, our data revealed an enterotype variation characterized by Escherichia enrichment in the elderly population. Considering the different age distribution of each enterotype, these findings provide new insights into the changes that occur in the gut microbiome with age and highlight the importance of microbiome-based stratification of elderly individuals.
Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Bacteroides ; China ; Diabetes Mellitus, Type 2/microbiology* ; Escherichia coli/classification* ; Gastrointestinal Microbiome/genetics* ; Metagenomics ; East Asian People

Flavonoid 3-O-glucosyltransferase (3GT) is a key enzyme in the glucosidation of anthocyanins. To investigate the 3GT gene in rhododendron, we cloned an open reading frame (ORF) of 3GT gene (named Rh3GT) from Rhododendron hybridum Hort (Red cultivar) and then characterized this gene and the deduced protein in terms of the biochemical characteristics, expression level, and enzymatic function. The results showed that Rh3GT had a full length of 993 bp and encoded 330 amino acid residues. The deduced protein was hydrophilic, stable, weak acid, belonging to the glycosyltransferase family (GT-B type), with glutamine (Q) at position 44 in the PSPG box. The phylogenetic analysis showed that Rh3GT was most closely related to Vc3GT from Vaccinium corymbosum and Vm3GT from Vaccinium myrtillus. Rh3GT was expressed in the stems, leaves, and flowers and almost not expressed in the roots, with the highest expression level in petals during full blooming stage. Introduction of pCAMBIAL1302-Rh3GT into petals significantly up-regulated the expression level of Rh3GT and increased the total anthocyanin accumulation. Rh3GT was successfully expressed in Escherichia coli BL21 in the form of inclusion bodies with a size of about 36 kDa. The results of HPLC showed that the recombinant Rh3GT after denaturation, purification, and dilution could catalyze the synthesis of cyanidin and UDP-glucose to synthesize cyanidin 3-O-glucoside, indicating that the expressed protein had 3GT activity. This study provides basic data for further studying the molecular regulation mechanism of anthocyanin biosynthesis and theoretical support for molecular breeding of rhododendron.
Rhododendron/classification* ; Glucosyltransferases/metabolism* ; Cloning, Molecular ; Escherichia coli/metabolism* ; Recombinant Proteins/biosynthesis* ; Anthocyanins/biosynthesis* ; Phylogeny ; Plant Proteins/metabolism* ; Amino Acid Sequence

OBJECTIVE: To investigate the phylogenetics and prevalence of bloodstream infections with ST131, the antimicrobial resistance profiles of the pathogens, and the clinical features. METHODS: Non-duplicate isolates were collected from 144 patients with bloodstream infections in our hospital between January and December, 2016.The phylogenetic groups of the isolates were analyzed using multiplex PCR, and O serotyping of ST131 strains was performed by allele-specific PCR.The clinical characteristics of the 144 patients were analyzed to define the differences in the clinical features between patients with ST131 infection and those with non-ST131 infection.Antibiotic susceptibility of the isolates was determined using the Vitek 2 compact system. RESULTS: The phylogenetic group analysis showed a domination by group B2 (41.0%[59/144]), followed by group F, group B1 and group E, which accounted for 16.7%(24/144), 13.9%(20/144), and 13.2% (19/144), respectively.Nine strains (6.3%) of were identified to be ST131 strains, among which 8 were O25b-B2-ST131 strains and 1 was O16-B2-ST131 strain.Of the 9 cases of ST131 infection, 7(77.8%) were found to occur in a nosocomial setting.The demographic characteristics and clinical features of the ST131-infected patients were similar to those of non-ST131-infected patients.ST131 strains were sensitive to piperacillin/tazobactam, imipenem, ertapenem, and amikacin, but showed high resistance rates to cefazolin, ceftriaxone, ciprofloxacin, levofloxacin, gentamicin, and trimethoprim/ sulfamethoxazole (all over 50%).The positivity rate of ESBLs in the ST131 strains was 77.8%, and the multidrug resistance rate reached 88.9%, which was higher than that of non-ST131 isolates, but the difference was not statistically significant. CONCLUSIONS The most common phylogenetic groups of isolates from patients with bloodstream infections are group B2 and F, and the positivity rate of ST131 is low.We for the first time detected O16-ST131 in patients with blood-borne infections in China.The clinical features of ST131-infected patients are similar to those of non-ST131-infected patients.The positivity rate of ESBLs and the multidrug resistance rate are high in ST131 strains, which may raise concerns in the future.
Anti-Bacterial Agents ; therapeutic use ; Bacteremia ; drug therapy ; epidemiology ; microbiology ; China ; Drug Resistance, Bacterial ; Escherichia coli ; classification ; drug effects ; genetics ; Escherichia coli Infections ; drug therapy ; epidemiology ; microbiology ; Genotype ; Humans ; Microbial Sensitivity Tests ; Molecular Epidemiology ; Phylogeny ; Species Specificity

OBJECTIVETo study the changes in gut microbiota and serum D-lactate level and their significance in children with recurrent pneumonia.

METHODSThe stool and blood samples were collected from 30 children with recurrent pneumonia (recurrent group), 30 children with acute pneumonia (acute group), and 15 children receiving surgical operation (surgery group). The 16S rRNA fluorescent quantitative polymerase chain reaction (FQ-PCR) was applied to determine the numbers of Bifidobacterium and Escherichia coli in stool samples, and the ratio between the logarithmic values of the numbers of Bifidobacterium and Escherichia coli (B/E value) was calculated. The serum D-lactate level was measured, and correlation analysis was performed.

RESULTSThe recurrent group had a significantly lower number of Bifidobacterium and a significantly lower B/E value than the acute group and the surgery group (P<0.05), as well as a significantly higher number of Escherichia coli than the surgery group (P<0.05). There was no significant difference in the number of Escherichia coli between the recurrent group and the acute group. The recurrent group had a significantly higher serum D-lactate level than the acute group and the surgery group (P<0.05). In the recurrent group, B/E value was negatively correlated with serum D-lactate level (r=-0.539, P<0.05).

CONCLUSIONSChildren with recurrent pneumonia may have biological and mechanical barrier damage in the intestinal mucosa.

Bacteria ; classification ; genetics ; isolation & purification ; Bifidobacterium ; genetics ; isolation & purification ; Child ; Child, Preschool ; Escherichia coli ; genetics ; isolation & purification ; Feces ; microbiology ; Female ; Gastrointestinal Microbiome ; Humans ; Lactates ; blood ; Pneumonia ; blood ; microbiology ; pathology ; Recurrence

OBJECTIVETo understand the epidemiologic and etiologic characteristics of diarrheagenic Escherichia (E.) coli infections in Shenzhen.

METHODSStool samples were collected from acute diarrheal patients in four sentinel hospitals in Shenzhen and diarrheagenic E. coli strains were isolated and identified with multiplex real-time PCR. Serotyping and pulsed-field gel electrophoresis (PFGE) typing were conducted for the diarrheagenic E. coli isolates.

RESULTSA total of 74 diarrheagenic E. coli strains were isolated from 1 823 stool samples (4.06%). The patients were mainly young children aged <3 years and adults aged 20-39 years, and the infections mainly occurred during May-September of a year. Enterotoxigenic E. coli (ETEC) and enteropathognic E. coli (EPEC) were predominant (45.9% and 31.1%). Serogroups and PFGE patterns varied among the diarrheagenic E. coli isolates. However, serogroup O159 were predominant in ETEC and there were 5 clusters with ≥2 strains sharing same PFGE patterns.

CONCLUSIONSETEC and EPEC were predominant in diarrheagenic E. coli strains isolated from diarrheal patients in Shenzhen. Age and season specific characteristics of diarrheagenic E. coli infections were observed. The serotypes and PFGE patterns of diarrheagenic E. coli strains varied. Close attention should be paid to the possible ETEC outbreak.

Adult ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; microbiology ; Disease Outbreaks ; Electrophoresis, Gel, Pulsed-Field ; Enterotoxigenic Escherichia coli ; classification ; isolation & purification ; Escherichia coli Infections ; epidemiology ; Humans ; Real-Time Polymerase Chain Reaction ; Serotyping ; Young Adult

MCL-1 is encoded by myeloid cell leukemia-1 gene (mcl-1), which is one of the anti-apoptotic members of bcl-2 cell apoptotic gene superfamily. ChanSu is made of dorsal secretions of several Bufo species, commonly used in the prescriptions of traditional Chinese medicine for treating many diseases including cancer. To clarify if mcl-1 is expressed in the dorsal skin of B. gargarizans, the PCR (polymerase chain reaction) was performed with its dorsal skin first strand cDNA as the template and a pair of specific primers of mcl-1, and PCR products were cloned into the pGM-T vector. DNA sequencing indicated that the ORF length was 639 bp encoding 212 amino acid residues, and the homology of 44%-95% with the MCL-1 of several other animals. For the further studies on MCL-1 biological functions during the oncogenesis and preparation of its antibody, the prokaryotic expression construct of pET-28b-mcl-1 was prepared which was confirmed by DNA sequencing, and its recombinant protein expression (0.02% wet weight) in E. coli BL21 (DE3) strain was confirmed by SDS-PAGE and Western blotting.
Amino Acid Sequence ; Animals ; Bufonidae ; classification ; genetics ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; metabolism ; Myeloid Cell Leukemia Sequence 1 Protein ; genetics ; metabolism ; Open Reading Frames ; genetics ; Phylogeny ; Recombinant Proteins ; genetics ; metabolism ; Sequence Alignment ; Sequence Homology ; Skin ; metabolism

Hespintor is an unknown function protein that was got from hepatoblastoma cell lines HepG2 by suppression subtractive hybridization technique (SSH), sequence analysis showed that the protein is a new member of secretory type of Kazal type serine protease inhibitor (Serpin) family, and has high homology with esophageal cancer related gene 2 (ECRG2). The coding sequence of Hespintor's Kazal domain was subcloned into prokaryotic expression vector pET-40b(+), then transformed into Rosetta (DE3). A recombinant protein about 42 kDa in the form of inclusion body was optimization expressed by inducing with 0.25 mmol/L IPTG, 30 degrees C for 5 h. and its specificity was confirmed via Western blotting. The recombinant protein was purified by metal chelate affinity chromatography (MCAC) and anion-exchange chromatography. The preliminary experimental result showed that the recombinant protein can inhibit trysin hydrolysis activity specifically. The result clearly demonstrated that Hespintor, as a novel member of Serpin, would be valuable in developing anti-tumor agents.
Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Hep G2 Cells ; Humans ; Proteinase Inhibitory Proteins, Secretory ; Recombinant Proteins ; biosynthesis ; genetics ; Serine Peptidase Inhibitors, Kazal Type ; Serine Proteinase Inhibitors ; biosynthesis ; classification ; genetics

A repetitive DNA fragment, named P1, was amplified by PCR with the full-length Minor Ampullate Spidroin gene sequence of Araneus ventricosus as template. P1 was ligated with pPic3.5 and PKT expression vectors and transferred into GS115 and BL21(DE3) competence cells, respectively. SDS-PAGE and Western blot were used to analyze the recombinant his-tag fusion protein. With expressed in different expression systems, soluble P1 induced proteins could be obtained as the same size. Furthermore, the expression level and purification recovery efficiency were also higher in GS115 than that of BL21(DE3). Additionally, the expression level could be improved after optimizing the incubation and induction conditions of GS115. In this research, Pichia pastoris expression system is more suitable for the native repetitive Gly/Ala-rich spider spidroin gene sequence expression than Escherichia coli system. The data can help the native full-length MiSp gene expression and large-scale exploitation of recombinant of spider silk proteins.
Animals ; Escherichia coli ; genetics ; metabolism ; Fibroins ; biosynthesis ; classification ; genetics ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; Spiders ; classification ; genetics ; metabolism

Muscovy ducks reovirus (DRV) is an important pathogen with a high mortality rate in Muscovy ducks, the researches in the test and the immunity were useful for the prevention and control of DRV infection. In this study, the S3 genes of the three Fujian DRVs were cloned by RT-PCR and sequencing technology. It was found that DRV-YH and YJL were close to avian reovirus (ARV) in the genetic distance, with high identities ranged from 94. 6% to 98. 9%, however, the identities of DRV-YB strain and reference ARV strains in the S3 gene were only 60.6% - 61.7%. The expression vector pET-30a-S3 harboring DRV YB strain S3 gene was constructed and transformed into E. coli BL21, and then the fusion sigmaB protein expression was induced with IPTG. The SDS-PAGE of the expressed products indicated that the fusion protein of approximately 42ku in molecular weight was expressed highly in inclusion body, and made up 67. 7% of the total proteins. The most efficient concentration of IPTG and inducing time were 0. 1 mM and 5h respectively, while the best temperature for expression was 37 degrees C. After purification with the Ni2+ affinity chromatography, the fusion sigmaB protein was 93% of the total proteins, and the purified protein amounted to 0. 86g/L. The Western blot analysis showed that the fusion aB protein was recognized specifically by the antiserum against DRV, confirming that the recombinant fusion protein had good immunoreactivity.
Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Molecular Sequence Data ; Orthoreovirus, Avian ; chemistry ; classification ; genetics ; isolation & purification ; Phylogeny ; Poultry Diseases ; virology ; RNA-Binding Proteins ; chemistry ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Reoviridae Infections ; veterinary ; virology ; Sequence Analysis ; Sequence Homology, Amino Acid

OBJECTIVESecreted proteins of Helicobacter pylori (H. pylori) interact with gastric epithelium cells and may contribute to cell damage. Considering the fact that HP0175 and hypothetical conserved protein HP1286 are included in the group and that HP0175 is a well-known apoptosis-induced factor, the present study aims to clarify whether HP1286 plays a role in bacterial pathogenicity or even functions as an apoptosis-induced factor in human stomach.

METHODSTwo genes encoding HP1286 and HP0175 were cloned into pET32a vector and expressed as recombinant proteins in Escherichia coli (E. coli) BL21. Signal peptide sequences were not included. The recombinant proteins were purified with His SpinTrap and desalted by using HiTrap Desalting. Immunoreactivity of the proteins was determined by Western blot. Human gastric epithelial cell AGS was challenged with these endotoxin-free proteins; and apoptosis of cells was assayed with the Cell Death ELISA kit.

RESULTSRecombinant proteins and their respective products whose N-terminal his-tag were removed with thrombin were recognized by serum from the patient infected with H. pylori. Apoptotic AGS cells challenged by HP1286 of H. pylori 26695 were four times more than untreated cells. In addition, apoptosis-induced ability of HP1286 of SS1 was not as strong as that of H. pylori 26695 strain.

CONCLUSIONHP1286 of H. pylori 26695 induces apoptosis of AGS cells in a time-dependent manner, however the apoptosis-induced ability of HP1286 may differ due to variations between different strains.

Amino Acid Sequence ; Apoptosis ; drug effects ; Bacterial Proteins ; metabolism ; pharmacology ; Cell Line, Tumor ; Cloning, Molecular ; Escherichia coli ; metabolism ; Gene Expression Regulation, Bacterial ; Genetic Variation ; Helicobacter pylori ; classification ; genetics ; metabolism ; Humans ; Molecular Sequence Data ; Recombinant Proteins ; Stomach Neoplasms