Pif1 Helicase Lengthens Some Okazaki Fragment Flaps Necessitating Dna2 Nuclease/Helicase Action in the Two-nuclease Processing Pathway

We have developed a system to reconstitute all of the proposed steps of Okazaki fragment processing using purified yeast proteins and model substrates. DNA polymerase δ was shown to extend an upstream fragment to displace a downstream fragment into a flap. In most cases, the flap was removed by flap endonuclease 1 (FEN1), in a reaction required to remove initiator RNA in vivo. The nick left after flap removal could be sealed by DNA ligase I to complete fragment joining. An alternative pathway involving FEN1 and the nuclease/helicase Dna2 has been proposed for flaps that become long enough to bind replication protein A (RPA). RPA binding can inhibit FEN1, but Dna2 can shorten RPA-bound flaps so that RPA dissociates. Recent reconstitution results indicated that Pif1 helicase, a known component of fragment processing, accelerated flap displacement, allowing the inhibitory action of RPA. In results presented here, Pif1 promoted DNA polymerase δ to displace strands that achieve a length to bind RPA, but also to be Dna2 substrates. Significantly, RPA binding to long flaps inhibited the formation of the final ligation products in the reconstituted system without Dna2. However, Dna2 reversed that inhibition to restore efficient ligation. These results suggest that the two-nuclease pathway is employed in cells to process long flap intermediates promoted by Pif1

An Alternative Pathway for Okazaki Fragment Processing - Resolution of Fold-Back Flaps by Pif1 Helicase

Two pathways have been proposed for eukaryotic Okazaki fragment RNA primer removal. Results presented here provide evidence for an alternative pathway. Primer extension by DNA polymerase δ (pol δ) displaces the downstream fragment into an RNA-initiated flap. Most flaps are cleaved by flap endonuclease 1 (FEN1) while short, and the remaining nicks joined in the first pathway. A small fraction escapes immediate FEN1 cleavage and is further lengthened by Pif1 helicase. Long flaps are bound by replication protein A (RPA), which inhibits FEN1. In the second pathway, Dna2 nuclease cleaves an RPA-bound flap and displaces RPA, leaving a short flap for FEN1. Pif1 flap lengthening creates a requirement for Dna2. This relationship should not have evolved unless Pif1 had an important role in fragment processing. In this study, biochemical reconstitution experiments were used to gain insight into this role. Pif1 did not promote synthesis through GC-rich sequences, which impede strand displacement. Pif1 was also unable to open fold-back flaps that are immune to cleavage by either FEN1 or Dna2 and cannot be bound by RPA. However, Pif1 working with pol δ readily unwound a full-length Okazaki fragment initiated by a fold-back flap. Additionally, a fold-back in the template slowed pol δ synthesis, so that the fragment could be removed before ligation to the lagging strand. These results suggest an alternative pathway in which Pif1 removes Okazaki fragments initiated by fold-back flaps in vivo

Otomastoïdites aiguës dans le Service d’ORL du CHU Yalgadode Ouagadougou: à propos de 44 cas

Analyser les aspects diagnostiques et thérapeutiques des otomastoïdites dans le Service d’ORL du CHU-YO de Ouagadougou. Méthode : Il s’est agit d’une étude rétrospective de 10 ans, qui a porté sur 44 cas. Résultats: l’incidence annuelle était de 4,4 cas et l’âge moyen de 13,41 ans. Les mastoïdites étaient majoritairement fistulisées (59,09%) et les principaux germes incriminés étaient Staphylococcus aureus (35,29%), Proteus mirabilis (23,53%) et Pseudomonas aeruginosa (23,53%). Ils étaient surtout sensibles à la ciprofloxacine (82,35%) et la ceftriaxone (58,82%). Le traitement a été médico-chirurgical dans 86,36% des cas et l’évolution favorable dans 72,73%. Douze cas (27,27%) de décès par complication systémique et endocrânienne ont été enregistrés. Conclusion : Il convient de prioriser les mesures préventives et la prise en charge précoce des otites infectieuses.Mots clés: Otomastoïdite, épidémiologie, diagnostic, thérapeutique

Risk factors associated with HIV prevalence in pregnant women in Burkina Faso, from 2006 to 2014

Purpose of the study: To determine the socio-demographic factors influencing the dynamics of HIV prevalence among pregnant women in Burkina Faso.Material and methods: A total of 66,597 pregnant women from the 13 health regions of Burkina Faso were included in this study conducted between 2006 and 2014. Venous blood samples were collected and analyzed for the detection of HIV antibodies according to WHO / UNAIDS strategy II, using the mixed test Vironostika HIV Uniform II Plus O (Bio-Mérieux) and the test discriminating ImmunoCombII HIV-1 & 2 BiSpot (Orgenics). Samples with discordant results between the two tests, as well as those positive to HIV-2 or HIV-1 + 2, were retested with HIV BLOT 2.2 (MP Diagnostics). Sociodemographic data collected from the participants were correlated with their HIV status to determine key risk factors influencing HIV infection prevalence in Burkina Faso.Results: Sociodemographic data showed that the study population consisted mainly of married women (91.2%) at their first pregnancy (27.1%) with a large majority of them being housewives (86.2%) who did not attend any form of schooling (69.4%). About 88.4% had stayed longer than a year in the health region where they initially participated in the study and 55.8% were between 20 and 29 years of age. Overall HIV prevalence significantly dropped from 2.7 % in 2006 to 1.3% in 2014. However HIV seroprevalence in this study has varied significantly according to socio-demographic characteristics including marital status, parity, occupation, education, age group and the length of stay in the women's health community (p <0.0001). Factors sustaining HIV transmission included the status of being unmarried (OR=1.67 [1.42-1.97]), primigest (OR=1.64 [1.41-1.89]), having other occupations except being student (OR = 1.68 [1.20-2.33]), aged between 20-49 years (OR=3.14 [2.51-3.93]) and the duration of stay less than a year in their locality (OR=5.33 [4.61-10.16]) and these factors were identified as main risk factors associated with HIV prevalence.Conclusion: Burkina Faso remains among the countries with concentrated epidemics despite a significant reduction in the prevalence observed in this study. The inclusion of identified risk factors in the national HIV program could improve the quality of the response to the epidemic.Keywords: HIV-Pregnant Women-Risk Factors-Burkina Fas

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

Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype

Prioritising public health: a qualitative study of decision making to reduce health inequalities

<p>Abstract</p> <p>Background</p> <p>The public health system in England is currently facing dramatic change. Renewed attention has recently been paid to the best approaches for tackling the health inequalities which remain entrenched within British society and across the globe. In order to consider the opportunities and challenges facing the new public health system in England, we explored the current experiences of those involved in decision making to reduce health inequalities, taking cardiovascular disease (CVD) as a case study.</p> <p>Methods</p> <p>We conducted an in-depth qualitative study employing 40 semi-structured interviews and three focus group discussions. Participants were public health policy makers and planners in CVD in the UK, including: Primary Care Trust and Local Authority staff (in various roles); General Practice commissioners; public health academics; consultant cardiologists; national guideline managers; members of guideline development groups, civil servants; and CVD third sector staff.</p> <p>Results</p> <p>The short term target- and outcome-led culture of the NHS and the drive to achieve "more for less", combined with the need to address public demand for acute services often lead to investment in "downstream" public health intervention, rather than the "upstream" approaches that are most effective at reducing inequalities. Despite most public health decision makers wishing to redress this imbalance, they felt constrained due to difficulties in partnership working and the over-riding influence of other stakeholders in decision making processes. The proposed public health reforms in England present an opportunity for public health to move away from the medical paradigm of the NHS. However, they also reveal a reluctance of central government to contribute to shifting social norms.</p> <p>Conclusions</p> <p>It is vital that the effectiveness and cost effectiveness of all new and existing policies and services affecting public health are measured in terms of their impact on the social determinants of health and health inequalities. Researchers have a vital role to play in providing the complex evidence required to compare different models of prevention and service delivery. Those working in public health must develop leadership to raise the profile of health inequalities as an issue that merits attention, resources and workforce capacity; and advocate for central government to play a key role in shifting social norms.</p

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

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

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