Importance of the confirmatory assay for the detection of the HBsAg in the epidemiological studies and in the diagnosis of the viral Hepatitis B

Several epidemiological studies have reported high prevalence of HBsAg among pregnant women in Burkina Faso. They used various algorithms, as it is also done for the routine diagnostic. Knowing this antigen carriage rate in such a population or in other clinic attendees is important for the implementation of a national immunisation programme and the monitoring of patients with hepatitis B. Often, the screening tests were not confirmed in spite of the existence of known false positive and false negative results. The aim of this study was to determine a more accurate prevalence of HBsAg, among the pregnant women in Burkina Faso. From October 2006 to January 2007, blood samples were collected from 1139 pregnant women. Each sample was analyzed for HBsAg, using two assays and according to manufacturers’ instructions vis, Hepanostika®HBsAg Uniform II B9 (Bio-Mérieux; France) and HBsAg (V2) Abbott AxSYM® system (Abbott Diagnostics). All the positive samples were tested with a confirmatory neutralization assay- Hepanostika®HBsAg Uniform II B9 Confirmatory (Bio-Merieux). The mean age of the pregnant women was 24.85years [range: 15-45years] and the age range of 20-24 (37%) and 25-29 (25.4%) years were the most represented. The overall rate of HBsAg-positive pregnant women with the two screening assays was 20.9%. The HBsAg detection rate was significantly higher with Hepanostika® UniformII B9 (16.9%) than with HBsAg (V2) AxSYM system assay (12.1%), with

Macrophage tropism of HIV-1 depends on efficient cellular dNTP utilization by reverse transcriptase

Retroviruses utilize cellular dNTPs to perform proviral DNA synthesis in infected host cells. Unlike oncoretroviruses, which replicate in dividing cells, lentiviruses, such as human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus, are capable of efficiently replicating in non-dividing cells (terminally differentiated macrophages) as well as dividing cells (i.e. activated CD4+ T cells). In general, non-dividing cells are likely to have low cellular dNTP content compared with dividing cells. Here, by employing a novel assay for cellular dNTP content, we determined the dNTP concentrations in two HIV-1 target cells, macrophages and activated CD4+ T cells. We found that human macrophages contained 130-250-fold lower dNTP concentrations than activated human CD4+ T cells. Biochemical analysis revealed that, unlike oncoretroviral reverse transcriptases (RTs), lentiviral RTs efficiently synthesize DNA even in the presence of the low dNTP concentrations equivalent to those found in macrophages. In keeping with this observation, HIV-1 vectors containing mutant HIV-1 RTs, which kinetically mimic oncoretroviral RTs, failed to transduce human macrophages despite retaining normal infectivity for activated CD4+ T cells and other dividing cells. These results suggest that the ability of HIV-1 to infect macrophages, which is essential to establishing the early pathogenesis of HIV-1 infection, depends, at least in part, on enzymatic adaptation of HIV-1 RT to efficiently catalyze DNA synthesis in limited cellular dNTP substrate environments

Characterizing cassava farmer typologies and their seed sourcing practices to explore opportunities for economically sustainable seed business models in Rwanda

Open Access Article; Published online: 30 Nov 2021The overdependency on local cassava varieties and informal seed sources by farmers in Rwanda has contributed to the spread of cassava viral diseases. The use of improved planting materials made available through formal seed sources, that assure seed quality, is one way to prevent future disease outbreaks. In order to increase the availability of, and farmers access to, such materials there is increasing interest to develop seed business models. This study aims to understand seed sourcing practices of different farm typologies to inform the development of tailored seed business models. A total of 390 farmers were interviewed and the collected data was analyzed into clusters, resulting in seven farm typologies. Seed sourcing strategies, seed replacement dynamics and purchasing behavior of these typologies were explored via a seed tracing study. We find that more commercial oriented farmers have better access to formal seed sources. Nevertheless, the majority of farmers in all typologies accessed new varieties and quality cassava seed via informal channels. At both formal and informal sources, cash investments in seed were mainly made by the categories of better-off farmers, and were one-time investments to acquire a new variety. Based on farmers current seed sourcing practices, clarifications on the differences between farmers and their willingness-to-pay, the roles of seed degeneration, cost-benefit analysis, value propositions and profit formulas seem important requirements for the further development of viable cassava seed business models. We conclude that tailoring seed business models can have a high potential as it acknowledges differences among farmers, but that careful coordination is needed to ensure that one approach or intervention does not contrast with and/or undermine the others

Thermostable DNA Polymerase from a Viral Metagenome Is a Potent RT-PCR Enzyme

Viral metagenomic libraries are a promising but previously untapped source of new reagent enzymes. Deep sequencing and functional screening of viral metagenomic DNA from a near-boiling thermal pool identified clones expressing thermostable DNA polymerase (Pol) activity. Among these, 3173 Pol demonstrated both high thermostability and innate reverse transcriptase (RT) activity. We describe the biochemistry of 3173 Pol and report its use in single-enzyme reverse transcription PCR (RT-PCR). Wild-type 3173 Pol contains a proofreading 3′-5′ exonuclease domain that confers high fidelity in PCR. An easier-to-use exonuclease-deficient derivative was incorporated into a PyroScript RT-PCR master mix and compared to one-enzyme (Tth) and two-enzyme (MMLV RT/Taq) RT-PCR systems for quantitative detection of MS2 RNA, influenza A RNA, and mRNA targets. Specificity and sensitivity of 3173 Pol-based RT-PCR were higher than Tth Pol and comparable to three common two-enzyme systems. The performance and simplified set-up make this enzyme a potential alternative for research and molecular diagnostics

Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

BACKGROUND: Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. METHODOLOGY: Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. PRINCIPAL FINDINGS: Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. CONCLUSIONS: The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage

Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein– substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed

Preliminary Evidence for Cell Membrane Amelioration in Children with Cystic Fibrosis by 5-MTHF and Vitamin B12 Supplementation: A Single Arm Trial

Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis.A single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K(+) content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association.5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF.ClinicalTrials.gov NCT00730509

The Yeast Pif1 Helicase Prevents Genomic Instability Caused by G-Quadruplex-Forming CEB1 Sequences In Vivo

In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences

Becoming a black researcher: reflections on racialised identity and knowledge production

Critical race theory (CRT) emerged from the U.S. context, and many question the validity of its application to spaces beyond the United States; however, for many black academics in the UK, it has a powerful resonance. Where many in the academy have dismissed the viability of the concept of race in favour of the term ethnicity – or they privilege class – in any discussion of inequalities, CRT recognises the salience of race, centralising it and analysing the ways in which race and racism continue to shape life experiences. CRT has provided an intellectual space for a growing community of academics in England to explore not only our own racial positioning within the academy and wider society but also that of the communities we work with in our research to achieve greater social justice. This paper explores the significance of CRT to the author’s biography and intellectual journey