Use of streptavidin bound to biotinylated DNA structures as model substrates for analysis of nucleoprotein complex disruption by helicases

Helicases are a subfamily of translocases that couple the directional translocation along a nucleic acid lattice to the separation of nucleic acid duplexes using the energy derived from nucleoside triphosphate hydrolysis. These enzymes perform essential functions in all aspects of nucleic acid metabolism by unwinding and remodelling DNA or RNA in DNA replication, repair, recombination and transcription. Most classical biochemical studies assay the ability of these enzymes to separate naked nucleic acids. However, many different types of proteins form non-covalent interactions with nucleic acids in vivo and so the true substrates of helicases are protein-nucleic acid complexes rather than naked DNA and RNA. Studies over the last decade have revealed that bound proteins can have substantial inhibitory effects on the ability of helicases to unwind nucleic acids. Any analysis of helicase mechanisms in vitro must therefore consider helicase function within the context of nucleoprotein substrates rather than just DNA or RNA. Here we discuss how to analyse the impact of bound proteins on the ability of helicases to unwind DNA substrates in vitro

Top predators, habitat complexity and the biodiversity of litter-dwelling ants

Trabalho final de mestrado integrado em Medicina (Geriatria), apresentado á Faculdade de Medicina da Universidade de CoimbraA hipertensão arterial é o principal fator de risco modificável para a morbimortalidade por doenças cardiovasculares, a principal causa de morte a nível mundial. Dado o aumento da sua prevalência com a idade, a hipertensão arterial no idoso é uma patologia cada vez mais frequente. A rigidez arterial e a disfunção endotelial são a base fisiopatológica da hipertensão arterial no idoso, não devendo contudo ser descurada a maior incidência de causas secundárias. O tratamento farmacológico da hipertensão arterial no idoso é recomendado tendo em consideração o seu efeito na redução da mortalidade e morbilidade cardiovascular. O valor-alvo de tensão arterial recomendado para estes doentes é 150/90 mmHg, pela ausência de benefícios com um controlo tensional mais restrito. Não há evidência que suporte a utilização preferencial de uma classe ou combinação farmacológica, devendo o grande enfoque terapêutico ser a redução tensional e não os agentes utilizados. Doentes com comorbilidades ou pertencentes a populações especiais podem apresentar indicações farmacológicas específicas e valores-alvo diferentes. As reações adversas à terapêutica são mais frequentes no idoso. Assim, os idosos hipertensos devem manter vigilância para identificação precoce de reações adversas e aumentar a adesão terapêutica.The arterial hypertension is the main modifiable risk factor for morbimortality of cardiovascular diseases, the main cause of death worldwide. Given the increase of its prevalence with age, the arterial hypertension in the elderly is becoming increasingly frequent. The arterial stiffness and endothelial dysfunction are the pathophysiological basis of the arterial hypertension in the elderly, although it cannot be neglect the higher incidence of secondary causes. The pharmacological treatment of arterial hypertension in the elderly is recommended considering its effects on the reduction of cardiovascular mortality and morbidity. The blood pressure target recommended for these patients is 150/90 mmHg, due to the lack of benefits in a stricter blood pressure control. There is no evidence supporting the preferential utilization of a pharmacological class or combination. The major focus should be on blood pressure reduction and not on the agent used. Patients with comorbidities or from special populations may have specific pharmacologial indications and different target values. The therapy’s adverse reactions are more frequent in the elderly. Thus, the hypertensive elderly must maintain vigilance to identify early the adverse reactions and increase the therapeutic adherenc

Dynamical signatures of freezing: stable fluids, metastable fluids, and crystals

Mean squared displacements and velocity auto correlation functions are calculated using molecular dynamics for hard spheres under a range of conditions (i) for the equilibrium fluid below freezing; (ii) for the metastable fluid above freezing; and (iii) for the hard sphere crystal, both in the metastable region between freezing and melting, and in the stable region above melting. In addition, simulations are carried out for a metastable Lennard-Jones system. The results confirm recent studies that indicated the disappearance of the classical Alder long-time tail, and show that they apply to systems other than the metastable hard sphere fluid. The implications of these results for our understanding of crystallization and the glass transition are discussed

RecG interacts directly with SSB: implications for stalled replication fork regression

RecG and RuvAB are proposed to act at stalled DNA replication forks to facilitate replication restart. To define the roles of these proteins in fork regression, we used a combination of assays to determine whether RecG, RuvAB or both are capable of acting at a stalled fork. The results show that RecG binds to the C-terminus of single-stranded DNA binding protein (SSB) forming a stoichiometric complex of 2 RecG monomers per SSB tetramer. This binding occurs in solution and to SSB protein bound to single stranded DNA (ssDNA). The result of this binding is stabilization of the interaction of RecG with ssDNA. In contrast, RuvAB does not bind to SSB. Side-by-side analysis of the catalytic efficiency of the ATPase activity of each enzyme revealed that (−)scDNA and ssDNA are potent stimulators of the ATPase activity of RecG but not for RuvAB, whereas relaxed circular DNA is a poor cofactor for RecG but an excellent one for RuvAB. Collectively, these data suggest that the timing of repair protein access to the DNA at stalled forks is determined by the nature of the DNA available at the fork. We propose that RecG acts first, with RuvAB acting either after RecG or in a separate pathway following protein-independent fork regression

DNA replication roadblocks caused by Cascade Interference complexes are alleviated by RecG DNA repair helicase

Cascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating "adaptation" reactions that update immunity and by initiating "interference" reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2. DNA capture processes are unclear but may involve RecG helicase, which stimulates adaptation during its role responding to genome instability. We show that Cascade is a potential source of genome instability because it blocks DNA replication and that RecG helicase alleviates this by dissociating Cascade. This highlights how integrating in vitro CRISPR-Cas interference and adaptation reactions with DNA replication and repair reactions will help to determine precise mechanisms underpinning prokaryotic adaptive immunity

Acceptability and feasibility of point-of-care CD4 testing on HIV continuum of care in low and middle income countries: a systematic review

Background: CD4 testing is, and will remain an important part of HIV treatment and care in low and middle income countries (LMICs). We report the findings of a systematic review assessing acceptability and feasibility of POC CD4 testing in field settings. Methods: Electronic databases were searched for studies published in English between 2005 and 2015 that describe POC CD4 platforms. Studies conducted in LMICs and under field conditions outside a laboratory environment were eligible. Qualitative and descriptive data analysis was used to present the findings. Results: Twelve studies were included, 11 of which were conducted in sub-Saharan countries and used one POC CD4 test (The Alere Pima CD4). Patients reported positively regarding the implementation of POC CD4 testing at primary health care and community level with ≥90 % of patients accepting the test across various study settings. Health service providers expressed preference toward POC CD4 testing as it is easy-to-use, efficient and satisfied patients\u27 needs to a greater extent as compared to conventional methods. However, operational challenges including preference toward venous blood rather than finger-prick sampling, frequent device failures and operator errors, quality of training for test operators and supervisors, and increased staff workload were also identified. Conclusions: POC CD4 testing seems acceptable and feasible in LIMCs under field conditions. Further studies using different POC CD4 tests available on the market are required to provide critical data to support countries in selection and implementation of appropriate POC CD4 technologies