DNA Viral Size Fraction Metagenomics for Human Stool Samples.

INTRODUCTION. Marburg and Ebola viruses cause severe haemorrhagic fever in humans and primates. Currently, there are no licensed prophylactic vaccines that can simultaneously prevent the spread or reduce the severity of both diseases caused by these filoviruses. The development of effective prophylactic vaccines requires studies aimed at selecting the most immunogenic forms of protective antigens.AIM. This study aimed to evaluate humoral immune induction in animals after administration of recombinant adenoviral vectors expressing various forms of Ebola and Marburg virus glycoproteins (GPs).MATERIALS AND METHODS. Samples of recombinant human adenovirus type 5 (rAd5) were obtained using homologous recombination in Escherichia coli, growth in HEK293 cells, and purification by CsCl gradient ultracentrifugation. The resulting rAd5 samples were characterised in terms of their identity (PCR and whole-genome sequencing), the concentration of viral particles (fluorescence spectroscopy), and the concentration of infectious viral particles (TCID50 assay). Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the GP-specific IgG titres in the sera of immunised mice.RESULTS. The authors constructed rAd5 samples, and each construct contained an expression cassette with a GP gene form encoding a full-length GP, a GP without the mucin-like domain, or a GP without both the glycan cap and the mucin-like domain. Each of these forms was studied using the GPs of four filoviruses, including Zaire Ebola virus, Sudan Ebola virus, Bundibugyo Ebola virus, and Marburg virus. Neither of the forms had a critical effect on the rAd5 replicative capacity. Three weeks after immunisation, the highest GP-specific IgG production was induced by the rAd5 samples encoding either the full-length GP or the GP without the mucin-like domain. The GP without both the glycan cap and the mucin-like domain was the least immunogenic antigen regardless of the filovirus species.CONCLUSIONS. The most promising constructs for the development of filovirus vaccines based on recombinant adenoviral vectors are the constructs that include the genes encoding the fulllength GP or the GP without the mucin-like domain.

Adenoviral, adeno-associated viral, and retroviral particles are chosen as gene delivery shuttles in more than 50% of all gene therapy clinical trials. Bulk availability of clinical-grade viral particles and their efficiency to transduce the therapeutic cargo into specific target cells remain the most critical bottlenecks in gene therapy applications to date. Capitalizing on the flame-spray technology for the reproducible economic large-scale production of amorphous tricalcium phosphate nanoparticulate powders (ATCP), we designed a scalable ready-to-use gravity-flow column set-up for the straightforward concentration and purification of transgenic adenoviral, adeno-associated viral, and lentiviral particles. Specific elution buffers enabled rapid release of viral particles from the ATCP matrix of the column and provided high-titer virus preparations in an unsurpassed period of time. The interaction of ATCP with adenoviral, adeno-associated viral, and lentiviral particles in solution increased the transduction kinetics of several mammalian cell lines in culture. The nanoparticles were also able to modify the tropism of murine leukemia virus (MLV) towards transduction of human cells. Based on these findings, we believe that the use of flame-spray tricalcium phosphate nanoparticles will lead to important progress in the development of future gene therapy initiatives.

Exploring viral particle, soil, and extraction buffer physicochemical characteristics and their impacts on extractable viral communities

Our study aimed to determine the prevalence of Campylobacter jejuni isolated from raw milk, cheese, and human stool samples in Beni-Suef Governorate, Egypt, and to characterize the antibiotic resistance profile and virulence genes of the isolates. An additional objective was to evaluate the effectiveness of cinnamon oil and Lactobacillus acidophilus La5 for controlling C. jejuni in cheese. A total of 200 samples of raw milk and dairy products, including 50 samples of raw milk and 150 samples of three different types of cheese were used. Fifty-three human stool samples were also collected. The samples were tested for the presence of C. jejuni using culture and molecular methods. Campylobacter spp. were isolated from 9.5% (19/200) of the raw milk and cheese samples. The highest prevalence was observed in milk samples (18%), followed by Kareish cheese (14%) and Talaga cheese (6%). In contrast, C. jejuni was not found in any of the Feta cheese samples. Of the human stool samples, 21 (39.6%) were positive for C. jejuni. Of the isolates, 60-90% were highly resistant to the antimicrobial agents tested, that is, nalidixic acid, ciprofloxacin, and tetracycline. Virulent cadF and cdtA genes were detected in all isolates. As milk and dairy products are important sources of contamination, reducing the level of C. jejuni in them will lower the risk to consumers. We showed that L. acidophilus La5 was able to control C. jejuni in Kareish cheese, but cinnamon oil was less effective.

Can viruses form biofilms?

To delineate the interaction between in vivo HIV replication and host antiviral immunity during disease progression in order to elucidate the pathogenesis of AIDS. In a cohort of HIV-seropositive patients, the serum concentration of viral particles, the blood concentration of mononuclear cells harbouring infectious virus and the serum titre of isolate-specific neutralizing antibodies were correlated with the rates of CD4+ T-cell depletion and disease progression. Using a quantitative reverse-transcriptase linked polymerase chain reaction assay, the concentration of viral particles was measured in blood samples from 103 initially symptom-free subjects who were followed up for > or = 24 months. The concentration of infectious virus and the neutralizing antibodies to autologous HIV isolates were assessed in 37 out of the 103 subjects. The rate of decrease in CD4 cells over the 24 months was calculated for each subject. Rapidly progressing patients (rate of decrease in CD4 cells > or = 60%) had a high concentration of viral particles and a high concentration of infectious virus associated with an undetectable serum titre of isolate-specific neutralizing antibodies. Stable patients (rate of decrease in CD4 cells < 30%) had a low concentration of infectious virus and either a low concentration of viral particles with the absence of isolate-specific neutralizing antibodies or a high concentration of viral particles with the presence of isolate-specific neutralizing antibodies. Slowly progressing patients (rate of decrease in CD4 cells > or = 30 and < 60%) showed an intermediate profile. Progression to AIDS is associated with a shift in the balance between viral replication and host immunity that increases the concentration of infected cells and destroys the CD4+ T-lymphocyte population.

A total of 350 samples including 175 faecal swabs from livestock, 125 samples from animal-derived foods, and 50 human stool samples, were examined. Both bacterial culture methods and PCR techniques were employed to detect Clostridium difficile species. The analysis indicated that 2.8% of the tested samples were positive for C. difficile through species-specific PCR. Among the faecal samples from livestock, pigs and calves showed a higher prevalence (4%). In the category of animal-derived foods, chicken samples had a notable prevalence (8%), while mutton samples had a lower prevalence (2%). Additionally, 8% of the human stool samples tested positive for difficile. Using bacterial culture methods, 52 isolates (14.8%) of C. difficile were identified. The application of species-specific PCR, targeting gene (tpi) further validated the presence of C. difficile in faecal samples from animals, animal-derived foods, and humans in Andhra Pradesh, India.

Concentration and purification of rubella virus using monolithic chromatographic support

We have used dielectrophoretic filters within microfluidic biochips to capture from a flow and image virus particles in real-time. The verification of capture was performed by fluorescently labeling the particles using dual or triple labeling. These nonmechanical filters can be very valuable in the sample preparation, purification, and concentration of viral particles from a mixed sample. The described imaging methodology can be used for real-time imaging of nanometer scale virus particles for analysis of capture, detection, and characterization of these particles within micro and nanoscale sensors.

Specific cytoplasmic fluorescence resulted when RLV- or MLV-infected cells were treated with their respective antiviral sera in the indirect immunofluorescence test. The fluorescence was especially intense in the cytoplasm of megakaryocytes, where abundant virus particles were seen by electron microscopy. No antigenic cross reactivity of RLV and MLV could be demonstrated by this test. Inoculation of mouse fetal cell monolayers with RLV or MLV led to the appearance of immunofluorescent cells, which increased in percentage with increasing length of incubation time. Electron microscopy of the infected cells demonstrated that the initial appearance of virus at cell membranes was related to the appearance of fluorescence and that increasing numbers of virus-forming cells and concentrations of virus particles between and within cells were associated with the increasing percentage of fluorescent cells. The indirect immunofluorescence method is useful for detection of MLV and RLV and may be applicable to the quantitative assay of these viruses in cell culture.

SUMMARY A field isolate, 3b1a, of the wheat take-all fungus Gaeumannomyces graminis var. tritici, was previously shown to be infected with three serologically unrelated viruses, A, B, and C. It is now shown that virus B can be separated into two distinct strains, designated B1 and B2. All four viruses, A, B1, B2 and C, were faithfully transmitted into conidia. However, six out of eight single ascospore cultures derived from 3b1a single conidial cultures were shown to be virus-free. The remaining two single ascospore cultures each contained only one virus, which appeared to be the same in each culture. This virus was serologically indistinguishable from virus B1, but had dsRNA components of mol. wt. lower than those of virus B1 and had only the smaller of the two capsid polypeptide species of virus B1. No DNA provirus molecules homologous to viruses B1, B2 or C could be detected in two of the virus-free ascospore cultures or in one of the virus-infected ascospore cultures. Very low concentrations of virus particles were detected in hyphal tip isolates of G. graminis. After prolonged storage and subculturing of these isolates, the concentration of virus particles had increased to the level of the parent culture from which the hyphal tip isolates were derived.

Quantification of short-chain fatty acids in human stool samples by LC-MS/MS following derivatization with aniline analogues

Statins exert anti-inflammatory effects independently of cholesterol-lowering properties. Cytomegalovirus (CMV) infection appears to be implicated in the pathophysiology of atherosclerosis by inducing inflammatory modifications in endothelial cells, especially in immunosuppressed patients. We investigated whether the activity of statins can inhibit replication of CMV in human endothelial cells. Human umbilical vein endothelial cells (HUVECs) were infected with CMV and coincubated with fluvastatin at 0.1 and 0.2 micromol/L. Fluvastatin inhibited (P<0.001) CMV antigen expression, and this effect was dose related (P<0.001). Quantitative polymerase chain reaction showed that CMV DNA concentration was consistently lower in supernatants from fluvastatin-treated cells than in infected controls, and viral particle concentration was up to 30 times lower in 0.2 micromol/L fluvastatin-treated cells than in infected controls (10.5+/-0.9 versus 0.34+/-0.03 per 10(3) pfu/mL, P<0.001). Addition of mevalonate to treated cultures almost completely abolished fluvastatin inhibition of viral growth. Electrophoretic mobility shift assay showed that fluvastatin reduced nuclear factor-kappaB binding activity in CMV-infected cells. HMG-CoA inhibition by fluvastatin restrains CMV replication in HUVECs by inhibiting viral antigen expression, DNA synthesis, and viral particle production, conceivably by involving a reduction of nuclear factor-kappaB binding activity.

Cancer is a major cause of morbidity and mortality worldwide. The proliferation of cancer cells depends largely on glutamine for survival and proliferation. Glutamine serves as a carbon source for the synthesis of metabolites and lipids via the TCA cycle, as well as a source of nitrogen for the synthesis of amino acids and nucleotides. Recently, the role of glutamine metabolism in cancer has been explored in many studies. Therefore, it provides a scientific relationship for targeting glutamine metabolism for cancer treatment. L-glutaminase which is a powerful anticancer medication that is widely used around the world, works by removing L-glutamine from cancerous cells. L-glutaminase has been cited as the most potent molecule that inhibits the proliferation of cancer cells, which significantly raises the possible applicability of cancer therapy and the possibility of its application as an alternative drug to chemotherapy. The first investigation into the antitumor property of L-glutaminase revealed its inhibitory effect on the growth of Gardner lymphosarcoma (6C3HED) and L-1210 leukemia cells. In the same study, glutaminase from Pseudomonas spp., in combination with azaserine enhanced the degree of tumor growth inhibition. Subsequently, L-glutaminase was administered intravenously in patients with acute lymphoblastic leukemia and acute myeloid leukemia. Recently, a purified L-glutaminase from Streptomyces sp. D214 was shown to be the most effective, with an IC50 value of 10mg/ml against the MCF-7 tumor cell line. Also, various in vitro studies have revealed that the activity of glutaminase against the proliferation of tumor cell lines using the MTT (3-(4,5- dimethylthiazol-2-yl)- 2, 5-diphenyltetrazolium bromide) cell proliferation assay. Alcaligenes faecalis KLU102 glutaminase was able to reduce the viability of HeLa cells in a dose-dependent manner, with an IC50 value of 12.5mg/ml within 24h. In this study, a bacterium extracellular from human stool samples was extracted and identified using morphological, biochemical, and molecular methods. The 16S rRNA gene was 100% identical to the sequence from Klebsiella pneumoniae and was submitted to GenBank under accession number OQ703039. Thus, this strain was named Klebsiella pneumoniae AS KP 23. Further kinetic studies on the purified enzyme were performed. In addition, the pH stability of the L-glutaminase enzyme was slightly affected over the pH range of 7.0-9.0 after 2h of pre-incubation, and the rate of thermal inactivation of the L-glutaminase enzyme increased with higher temperatures and longer preheating periods. In addition, the stability of the tested enzyme decreased with an increasing storage period at -20°C. The SDS-PAGE revealed that the L-glutaminase subunits had a molecular weight of around 97kDa. L-glutaminase was purified 1.33-fold with a final specific activity of 799.9 U/mg protein using gel filtration chromatography. The enzyme´s cytotoxic activity showed severe toxicity against the HepG-2 human hepatocellular and breast cancer cell lines. Klebsiella pneumoniae glutaminase was able to reduce the viability of HeLa cells in a dose-dependent manner, with an IC50 value of 305.78µg/ml in human hepatocellular carcinoma and an IC50 value of 400.51µg/ml in breast cancer cell lines. Klebsiella pneumoniae AS KP 23 was a genetically determined microbial species isolated from human stool samples. The production of extracellular enzymes was examined. Additionally, purified L-glutaminase inhibited the growth of normal cells and showed potent anticancer activity against numerous cancer cell lines in the study. Its broad pH and temperature range, combined with its unique and highly stable catalytic activity, make it an excellent choice for use as an effective cancer inhibitor.

Roseburia intestinalis has received attention as a potential probiotic bacterium. Recent studies have demonstrated that changes in its intestinal abundance can cause various diseases, such as obesity, enteritis and atherosclerosis. Probiotic administration or fecal transplantation alter the structure of the intestinal flora, offering possibilities for the prevention and treatment of these diseases. However, current monitoring methods, such as 16S rRNA sequencing, are complex and costly and require specialized personnel to perform the tests, making it difficult to continuously monitor patients during treatment. Hence, the rapid and cost-effective quantification of intestinal bacteria has become an urgent problem to be solved. Aptamers are of emerging interest because their stability, low immunogenicity and ease of modification are attractive properties for a variety of applications. We report a FluCell-SELEX polyclonal aptamer library specific for R. intestinalis isolated after seven evolution rounds, that can bind and label this organism for fluorescence microscopy and binding assays. Moreover, R. intestinalis can be distinguished from other major intestinal bacteria in complex defined mixtures and in human stool samples. We believe that this preliminary evidence opens new avenues towards aptamer-based electronic biosensors as new powerful and inexpensive diagnostic tools for the relative quantitative monitoring of R. intestinalis in gut microbiomes.