The secretomes of a virulent Bacillus anthracis strain and of avirulent strains (cured of the virulence plasmids pXO1 and pXO2), cultured in rich and minimal media, were studied by a comparative proteomic approach. More than 400 protein spots, representing the products of 64 genes, were identified, and a unique pattern of protein relative abundance with respect to the presence of the virulence plasmids was revealed. In minimal medium under high CO 2 tension, conditions considered to simulate those encountered in the host, the presence of the plasmids leads to enhanced expression of 12 chromosome-carried genes (10 of which could not be detected in the absence of the plasmids) in addition to expression of 5 pXO1-encoded proteins. Furthermore, under these conditions, the presence of the pXO1 and pXO2 plasmids leads to the repression of 14 chromosomal genes. On the other hand, in minimal aerobic medium not supplemented with CO 2 , the virulent and avirulent B. anthracis strains manifest very similar protein signatures, and most strikingly, two proteins (the metalloproteases InhA1 and NprB, orthologs of gene products attributed to the Bacillus cereus group PlcR regulon) represent over 90% of the total secretome. Interestingly, of the 64 identified gene products, at least 31 harbor features characteristic of virulence determinants (such as toxins, proteases, nucleotidases, sulfatases, transporters, and detoxification factors), 22 of which are differentially regulated in a plasmid-dependent manner. The nature and the expression patterns of proteins in the various secretomes suggest that distinct CO 2 -responsive chromosome-and plasmid-encoded regulatory factors modulate the secretion of potential novel virulence factors, most of which are associated with extracellular proteolytic activities.Bacillus anthracis is a gram-positive spore-forming bacterium that is the etiological agent of anthrax, a lethal disease sporadically affecting humans and animals, in particular herbivores. In its most severe manifestation, B. anthracis infection is initiated by inhalation of spores, which are taken up by alveolar macrophages and germinate into fast-dividing vegetative cells which secrete toxins and virulence factors during growth (81,99). If untreated by prompt antibiotic administration, the bacteria invade the bloodstream, resulting in massive bacteremia and consequently generalized systemic failure and death. B. anthracis is considered to represent a potential biothreat agent, owing to the severity of the anthrax disease, the ease of respiratory contamination, and the perpetual environmental stability of the infective spores. The recent deliberate dissemination of B. anthracis (15) accelerated the efforts to identify new B. anthracis virulence-related determinants for the design of novel diagnostic, preventive, and/or therapeutic strategies.Fully virulent B. anthracis strains harbor two native plasmids, pXO1 and pXO2, which encode critical pathogenicity factors. The absence of either one of the two plasmids results in a pronounce...
The COVID-19 pandemic and the fast global spread of the disease resulted in unprecedented decline in world trade and travel. A critical priority is, therefore, to quickly develop serological diagnostic capacity and identify individuals with past exposure to SARS-CoV-2. In this study serum samples obtained from 309 persons infected by SARS-CoV-2 and 324 of healthy, uninfected individuals as well as serum from 7 COVID-19 patients with 4–7 samples each ranging between 1–92 days post first positive PCR were tested by an “in house” ELISA which detects IgM, IgA and IgG antibodies against the receptor binding domain (RBD) of SARS-CoV-2. Sensitivity of 47%, 80% and 88% and specificity of 100%, 98% and 98% in detection of IgM, IgA and IgG antibodies, respectively, were observed. IgG antibody levels against the RBD were demonstrated to be up regulated between 1–7 days after COVID-19 detection, earlier than both IgM and IgA antibodies. Study of the antibody kinetics of seven COVID 19 patients revealed that while IgG levels are high and maintained for at least 3 months, IgM and IgA levels decline after a 35–50 days following infection. Altogether, these results highlight the usefulness of the RBD based ELISA, which is both easy and cheap to prepare, to identify COVID-19 patients even at the acute phase. Most importantly our results demonstrate that measuring IgG levels alone is both sufficient and necessary to diagnose past exposure to SARS-CoV-2.
, and good agreement were observed among all assays, although the Xpert and Aptima assays, which provided the most similar outputs (estimated mean viral loads of 2.67 log copies/ml [95% confidence interval [CI], 2.50 to 2.84 log copies/ml] and 2.68 log copies/ml [95% CI, 2.49 to 2.86 log copies/ml], respectively), correlated best with the RealTime assay (89.8% concordance, with Pearson r values of 0.97 to 0.98). These three assays exhibited greater precision than the NucliSens v2.0 assay. All assays were equally sensitive for subtype B and AG/G samples and for samples with viral loads of 1.60 to 3.00 log copies/ml. The NucliSens v2.0 assay underestimated A1 samples and those with viral loads of >3.00 log copies/ml. The RealTime assay tended to underquantify subtype C (compared to the Xpert and Aptima assays) and subtype A1 samples. The Xpert and Aptima assays were equally efficient for detection of all subtypes and viral loads, which renders these new assays most suitable for clinical HIV laboratories.
Between December 2016 and June 2017, 19 Hepatitis A virus (HAV)-positive cases, 17 of which were among men who have sex with men (MSM) were identified in the Tel Aviv area. Seven of the 15 sewage samples collected between January and June 2017 were also HAV-positive. All sequences clustered with two of the three strains identified in the current European HAV outbreak. We demonstrate that despite an efficient vaccination programme, HAV can still be transmitted to an unvaccinated high-risk population.
The overall significant prevalence of HDV (6.5%) mandates HDV RNA testing for all coinfected patients. Patients positive for HDV RNA (characterized by low HBV DNA blood levels) carried HDV genotype 1. Taken together, the significant HDV seroprevalence and the lack of effective anti-HDV therapy, necessitates strict clinical surveillance especially in patients with higher HDV viral loads and increased viral evolution.
Recently, considerable interest has been directed to red-fluorescence photodiagnosis of brain and other tumours during surgery using the protoporphyrin IX natural precursor, 5-aminolaevulinic acid. In the present study we focused on the role of the rate-limiting enzyme porphobilinogen deaminase in glioma C6 cell activity, differentiation and sub-cellular distribution. Over-expression of the human housekeeping porphobilinogen deaminase in the glioma cells, using the housekeepingporphobilinogen deaminase plasmid, induced a G1 cell cycle attenuation accompanied by increases in enzyme activity and c6 differentiation toward astrocytes. Visualisation of subcellular localisation of the porphobilinogen deaminase using the independent techniques of fluorescence immuno-staining with specific anti-human porphobilinogen deaminase antibodies and cellular expression of porphobilinogen deaminase fused to green fluorescent protein, revealed (unexpectedly) a major fraction of porphobilinogen deaminase in the nucleus and only a minor fraction in the cytoplasm. Both C and N terminals of porphobilinogen deaminase fused to green fluorescent protein revealed a major fraction of the newly synthesized fused porphobilinogen deaminase in the nucleus. Furthermore, newborn rat brain cells grown in a primary culture showed the same localisation pattern of porphobilinogen deaminase in the nuclei. Stimulation of C6 glioma cell differentiation by butyrate induced a marked decrease in porphobilinogen deaminase both in the nucleus and in the cytoplasm as determined by Western blotting and fluorescence immuno-localisation. These findings suggest a possible dual role for housekeeping porphobilinogen deaminase in fast dividing glioma cells, one related to the porphyrin synthesis pathway and another coupled to nuclear function, which might be linked to tumorigenesis.
Porphobilinogen deaminase (PBGD) is a rate-limiting enzyme of the heme biosynthesis pathway, whose level is elevated in various human tumors. PBGD was observed in both nuclear and cytoplasmic fractions of C6 glioma cells by immunostaining. During mitosis, chromatids were intensely stained for PBGD in comparison to the interphase chromatin. Using the yeast two-hybrid system, we identified RanBPM, the nuclear Ran-binding protein, as an interacting partner of PBGD. During butyrateinduced differentiation of C6, both nuclear and cytoplasmic PBGD levels declined as did Ran protein and its nucleotide exchange factor RCC1. N,N 0 -hexamethylene bis-acetamide-dependent differentiation resulted in an increase of the cytoplasmic PBGD, whereas nuclear PBGD, Ran protein and RCC1 remained unchanged. mRNA levels of PBGD remained unchanged during stimulation with both butyrate and N,N 0 -hexamethylene bis-acetamide. The enzymatic activity of PBGD and protoporphyrin IX synthesis in C6 cells were dependent on the differentiation induction agent. We conclude that PBGD possibly has a nuclear role in addition to its cytosolic enzymatic activity required for heme synthesis, which is related to cell transformation and differentiation.
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