Visualization of viral DNA assembly using nonfluorescent staining

ABSTRACT

We present an easy test for rapid visualization of viral DNA assemblies in infected cell cytoplasm. We selected the best stains for nuclear staining: Nile blue A, Bismarck brown, gallocyanin chrome alum, methyl green pyronin and azure II. None of the staining techniques is fluorescent, which facilitates their use in everyday experiments. Methyl green is most promising for routine detection of viral DNA assemblies in the cytoplasm; the procedure enables ready detection of viral DNA accumulation in the cytoplasm.     

Antiviral Activity of Nanocomplexes of Antisense Oligonucleotides Targeting VP72 Protein in Vero Cells Infected by African Swine Fever Virus

Russian Journal of Bioorganic Chemistry - Antiviral activity of antisense oligodeoxyribonucleotides with phosphorothioate or mesyl phosphoramidate internucleotidic groups targeting the main capsid...     

Unbiased Approach for Virus Detection in Skin Lesions

To assess presence of virus DNA in skin lesions, swab samples from 82 squamous cell carcinomas of the skin (SCCs), 60 actinic keratoses (AKs), paraffin-embedded biopsies from 28 SCCs and 72 kerathoacanthomas (KAs) and fresh-frozen biopsies from 92 KAs, 85 SCCs and 92 AKs were analyzed by high throughput sequencing (HTS) using 454 or Ion Torrent technology. We found total of 4,284 viral reads, out of which 4,168 were Human Papillomavirus (HPV)-related, belonging to 15 known (HPV8, HPV12, HPV20, HPV36, HPV38, HPV45, HPV57, HPV59, HPV104, HPV105, HPV107, HPV109, HPV124, HPV138, HPV147), four previously described putative (HPV 915 F 06 007 FD1, FA73, FA101, SE42) and two putatively new HPV types (SE46, SE47). SE42 was cloned, sequenced, designated as HPV155 and found to have 76% similarity to the most closely related known HPV type. In conclusion, an unbiased approach for viral DNA detection in skin tumors has found that, although some new putative HPVs were found, known HPV types constituted most of the viral DNA.     

Exposure assessment of potentially toxic trace elements via consumption of fruits and vegetables grown under the impact of Alaverdi's mining complex

This study is aimed to investigate the transfer of potentially toxic trace elements from soils to plants grown under the impact of Alaverdi's mining complex and assess the related dietary exposure to local residents. Contamination levels of potentially toxic trace elements (Cu, Ni, Pb, Zn, Hg, As, Cd) in soils and plants were determined and afterwards, transfer factors, estimated daily intakes, target hazard quotients, and hazard indexes were calculated.

Some trace elements (Pb, Zn, Cd) exceeded the maximum allowable levels. EDIs of Cu, Ni, Hg for the majority of studied fruits and vegetables exceeded the health-based guideline values. Meanwhile, in case of combined consumption of the studied food items, the estimated cumulative daily intakes exceeded health-based guideline values not only for the aforementioned trace elements but also for Zn in the following sequence: Zn > Hg > Ni > Cu. HI > 1 values highlighted the potential adverse health effects for local population through more than one trace element.

Detailed investigations need to be done for the overall assessment of health risks, taking into consideration not only adverse health effects posed by more than one toxic trace element but also through other exposure pathways.     

Morphometry of nuclear and nucleolar structures in a CaCo-2 cell line

The number of the nucleoli in a CaCo-2 cell nucleus does not generally depend on the quantity of DNA in the nucleus, but nucleolar DNA content is directly proportional to total nuclear DNA. However, in multinucleolar cells (three or more nucleoli), the nucleolar DNA content increases after 96 h incubation in culture without concomitant quantitative changes in nuclear DNA. The percentage of multinucleolar cells and the average number of nucleoli per nucleus increase with increasing incubation time. After 72 and 96 h in culture, multinucleolar cells show distinctive morphologies. The ratio of the sum of nucleolar perimeters to the nuclear perimeter increases linearly when the number of nucleoli in a nucleus increases, but there is no concomitant increase in total nucleolar area or DNA content, except in the 72 and 96 h populations. When the number of nucleoli in CaCo-2 cells increases after 48 and 60 h in culture, the amount of DNA per nucleolus decreases.     

Evaluating the Impact of Zimbabwe’s Prevention of Mother-to-Child HIV Transmission Program: Population-Level Estimates of HIV-Free Infant Survival Pre-Option A

Objective

We estimated HIV-free infant survival and mother-to-child HIV transmission (MTCT) rates in Zimbabwe, some of the first community-based estimates from a UNAIDS priority country.

Methods

In 2012 we surveyed mother-infant pairs residing in the catchment areas of 157 health facilities randomly selected from 5 of 10 provinces in Zimbabwe. Enrolled infants were born 9–18 months before the survey. We collected questionnaires, blood samples for HIV testing, and verbal autopsies for deceased mothers/infants. Estimates were assessed among i) all HIV-exposed infants, as part of an impact evaluation of Option A of the 2010 WHO guidelines (rolled out in Zimbabwe in 2011), and ii) the subgroup of infants unexposed to Option A. We compared province-level MTCT rates measured among women in the community with MTCT rates measured using program monitoring data from facilities serving those communities.

Findings

Among 8568 women with known HIV serostatus, 1107 (12.9%) were HIV-infected. Among all HIV-exposed infants, HIV-free infant survival was 90.9% (95% confidence interval (CI): 88.7–92.7) and MTCT was 8.8% (95% CI: 6.9–11.1). Sixty-six percent of HIV-exposed infants were still breastfeeding. Among the 762 infants born before Option A was implemented, 90.5% (95% CI: 88.1–92.5) were alive and HIV-uninfected at 9–18 months of age, and 9.1% (95%CI: 7.1–11.7) were HIV-infected. In four provinces, the community-based MTCT rate was higher than the facility-based MTCT rate. In Harare, the community and facility-based rates were 6.0% and 9.1%, respectively.

Conclusion

By 2012 Zimbabwe had made substantial progress towards the elimination of MTCT. Our HIV-free infant survival and MTCT estimates capture HIV transmissions during pregnancy, delivery and breastfeeding regardless of whether or not mothers accessed health services. These estimates also provide a baseline against which to measure the impact of Option A guidelines (and subsequently Option B+).     

Association of the FADS2 gene with omega-6 and omega-3 PUFA Concentration in the egg yolk of Japanese quail

This study focused on the association of polymorphisms of the FADS2 gene with fatty acid profiles in egg yolk of eight Japanese quail lines selected for high and low omega-6:omega-3 PUFA ratio (h2 = 0.36-0.38). For the identification of polymorphisms within the FADS2 gene 1350 bp of cDNA sequence were obtained encoding 404 amino acids. Five synonymous SNPs were found by comparative sequencing of animals of the high and low lines. These SNPs were genotyped by single base extension on 160 Japanese quail. The association analysis, comprising analysis of variance and family based association test (FBAT), revealed significant effects of SNP3 and SNP4 genotypes on the egg yolk fatty acid profiles, especially the omega-6 and omega-3 PUFAs (P < 0.05). No effects of the other SNPs were found - indicating that these are not in linkage disequilibrium with the causal polymorphism. The results of this study promote FADS2 as a functional candidate gene for traits related to omega-6 and omega-3 PUFA concentration in the egg yolk.     

Proton translocation coupled to formate oxidation in anaerobically grown fermenting Escherichia coli

Proton translocation, coupled to formate oxidation and hydrogen evolution, was studied in anaerobically grown fermenting Escherichia coli JW136 carrying hydrogenase 1 (hya) and hydrogenase 2 (hyb) double deletions. Rapid acidification of the medium by EDTA-treated anaerobic suspension of the whole cells or its alkalization by inverted membranes was observed in response to application of formate. The formate-dependent proton translocation and 2H(+)-K(+) exchange coupled to H(2) evolution were sensitive to the uncoupler, carbonylcyanide-m-chlorophenylhydrazone, and to copper ions, inhibitors of hydrogenases. No pH changes were observed in a suspension of formate-pulsed aerobically grown ("respiring") cells. The apparent H(+)/formate ratio of 1.3 was obtained in cells oxidizing formate. The 2H(+)-K(+) exchange of the ATP synthase inhibitor N,N'-dicyclohexylcarbodiimide-sensitive ion fluxes does take place in JW136 cell suspension. Hydrogen formation from formate by cell suspensions of E. coli JW136 resulted in the formation of a membrane potential (Deltapsi) across the cytoplasmic membrane of -130 mV (inside negative). This was abolished in the presence of copper ions, although they had little effect on the value of Deltapsi generated by E. coli under respiration. We conclude that the hydrogen production by hydrogenase 3 is coupled to formate-dependent proton pumping that regulates 2H(+)-K(+) exchange in fermenting bacteria.     

Comparison of impact of EMCV replication on the nuclear apparatus of NIH 3T3 and HEp-2 cells

We have investigated differences between the actions of encephalomyocarditis virus (EMCV) on cytometric indices in cultured NIH 3T3 and HEp-2 cells, which are characterized by different levels of transformation. HEp-2 cells surviving 48 h after EMCV infection showed lower nuclear ploidy, reduced nuclear area, fewer nucleoli and a higher percentage of euploid cells. There was a significant increase of nucleolar/nuclear DNA 6-24 h after EMCV infection. However, EMCV had markedly different effects on NIH 3T3 cells: there was a consistent increase in population ploidy, but the average number of nucleoli and the number of euploid cells in the population remained constant. The nucleolar/nuclear DNA ratio was almost unchanged. These different viral effects might be explained by the contrasting levels of differentiation of the cultured cell lines. The number of nucleoli does not depend on the amount of nuclear DNA in either viral-infected or intact cells but on the euploidy-to-aneuploidy ratio. The ratio of the sums of the nucleolar perimeters to the nuclear perimeter increases linearly with the number of nucleoli per nucleus in both intact and virus-infected cells. In both cell lines, the amount of DNA per nucleolus decreases as the number of nucleoli increases.     

Infant mortality in Armenia, 1992–2003

Health system reforms have been taken in Armenia during a time of dramatic economic and fiscal distress. It is important to assess trends in health indicators and ascertain if the changes in socio-economic systems affected the health status of infants as the most vulnerable part of the population. We find that infant mortality has fallen during the period c. 1992-2003 in spite of the difficult economic circumstances because of health-care procedures that were introduced. Particular attention is paid to the underreporting of infants' death cases in the state registration system as well as to estimating the role of different factors influencing infant mortality in Armenia.