Carcinogenic adducts induce distinct DNA polymerase binding orientations

DNA polymerases must accurately replicate DNA to maintain genome integrity. Carcinogenic adducts, such as 2-aminofluorene (AF) and N-acetyl-2-aminofluorene (AAF), covalently bind DNA bases and promote mutagenesis near the adduct site. The mechanism by which carcinogenic adducts inhibit DNA synthesis and cause mutagenesis remains unclear. Here, we measure interactions between a DNA polymerase and carcinogenic DNA adducts in real-time by single-molecule fluorescence. We find the degree to which an adduct affects polymerase binding to the DNA depends on the adduct location with respect to the primer terminus, the adduct structure and the nucleotides present in the solution. Not only do the adducts influence the polymerase dwell time on the DNA but also its binding position and orientation. Finally, we have directly observed an adduct- and mismatch-induced intermediate state, which may be an obligatory step in the DNA polymerase proofreading mechanism.     

Atomistic details of effect of disulfide bond reduction on active site of Phytase B from Aspergillus niger: A MD Study

The molecular integrity of the active site of phytases from fungi is critical for maintaining phytase function as efficient catalytic machines. In this study, the molecular dynamics (MD) of two monomers of phytase B from Aspergillus niger, the disulfide intact monomer (NAP) and a monomer with broken disulfide bonds (RAP), were simulated to explore the conformational basis of the loss of catalytic activity when disulfide bonds are broken. The simulations indicated that the overall secondary and tertiary structures of the two monomers were nearly identical but differed in some crucial secondary–structural elements in the vicinity of the disulfide bonds and catalytic site. Disulfide bonds stabilize the β-sheet that contains residue Arg66 of the active site and destabilize the α-helix that contains the catalytic residue Asp319. This stabilization and destabilization lead to changes in the shape of the active–site pocket. Functionally important hydrogen bonds and atomic fluctuations in the catalytic pocket change during the RAP simulation. None of the disulfide bonds are in or near the catalytic pocket but are most likely essential for maintaining the native conformation of the catalytic site.

Abbreviations

PhyB - 2.5 pH acid phophatese from Aspergillus niger, NAP - disulphide intact monomer of Phytase B, RAP - disulphide reduced monomer of Phytase B, Rg - radius of gyration, RMSD - root mean square deviation, MD - molecular dynamics.     

Effect of weight loss and exercise on angiogenic factors in the circulation and in adipose tissue in obese subjects

Background:

Vascular growth is a prerequisite for adipose tissue (AT) development and expansion. Some AT cytokines and hormones have effects on vascular development, like vascular endothelial growth factor (VEGF‐A), angiopoietin (ANG‐1), ANG‐2 and angiopoietin‐like protein‐4 (ANGPTL‐4).

Methods:

In this study, the independent and combined effects of diet‐induced weight loss and exercise on AT gene expression and proteins levels of those angiogenic factors were investigated. Seventy‐nine obese males and females were randomized to: 1. Exercise‐only (EXO; 12‐weeks exercise without diet‐restriction), 2. Hypocaloric diet (DIO; 8‐weeks very low energy diet (VLED) + 4‐weeks weight maintenance diet) and 3. Hypocaloric diet and exercise (DEX; 8‐weeks VLED + 4‐weeks weight maintenance diet combined with exercise throughout the 12 weeks). Blood samples and fat biopsies were taken before and after the intervention.

Results:

Weight loss was 3.5 kg in the EXO group and 12.3 kg in the DIO and DEX groups. VEGF‐A protein was non‐significantly reduced in the weight loss groups. ANG‐1 protein levels were significantly reduced 22‐25% after all three interventions (P < 0.01). The ANG‐1/ANG‐2 ratio was also decreased in all three groups (P < 0.05) by 27‐38%. ANGPTL‐4 was increased in the EXO group (15%, P < 0.05) and 9% (P < 0.05) in the DIO group. VEGF‐A, ANG‐1, and ANGPTL‐4 were all expressed in human AT, but only ANGPTL‐4 was influenced by the interventions.

Conclusions:

Our data show that serum VEGF‐A, ANG‐1, ANG‐2, and ANGPTL‐4 levels are influenced by weight changes, indicating the involvement of these factors in the obese state. Moreover, it was found that weight loss generally was associated with a reduced angiogenic activity in the circulation.     

The status and conservation of the Cape Gannet Morus capensis

The Cape Gannet Morus capensis is one of several seabird species endemic to the Benguela upwelling ecosystem (BUS) but whose population has recently decreased, leading to an unfavourable IUCN Red List assessment. Application of ‘JARA’ (‘Just Another Red-List Assessment,’ a Bayesian state-space tool used for IUCN Red List assessments) to updated information on the areas occupied by Cape Gannets and the nest densities of breeding birds at their six colonies, suggested that the species should be classified as Vulnerable. However, the rate of decrease of Cape Gannets in their most-recent generation exceeded that of the previous generation, primarily as a result of large decreases at Bird Island, Lambert’s Bay, and Malgas Island, off South Africa’s west coast (the western part of their range). Since the 1960s, there has been an ongoing redistribution of the species from northwest to southeast around southern Africa, and ～70% of the population now occurs on the south coast of South Africa, at Bird Island in Algoa Bay, on the eastern border of the BUS. Recruitment rather than adult survival may be limiting the present population; however, information on the seabird’s demographic parameters and mortality in fisheries is lacking for colonies in the northern part of the BUS. Presently, major threats to Cape Gannet include: substantially decreased availability of their preferred prey in the west; heavy mortalities of eggs, chicks and fledglings at and around colonies, inflicted by Cape Fur Seals Arctocephalus pusillus and other seabirds; substantial disturbance at colonies caused by Cape Fur Seals attacking adult gannets ashore; oiling; and disease.     

Engagement of alpha4beta7 integrins by monoclonal antibodies or ligands enhances survival of human eosinophils in vitro

Asthma is characterized by an airway inflammatory infiltrate that is rich in eosinophilic leukocytes. Cellular fibronectin and VCAM-1, ligands for alpha4 integrins, are enriched in the fluid of airways of allergic patients subjected to Ag challenge. We therefore hypothesized that ligands of alpha4 integrins can promote eosinophil survival independent of cell adhesion. Cellular fibronectin and VCAM-1 increased viability of human peripheral blood eosinophil in a dose- and time-dependant manner whether the ligand was coated on the culture well or added to the medium at the beginning of the assay. Eosinophils cultured with cellular fibronectin were not adherent to the bottom of culture wells after 3 days. Treatment with mAb Fib 30 to beta7, but not mAb P4C10 or TS2/16 to beta1, increased eosinophil survival. The increased survival of eosinophils incubated with Fib 30 was blocked by Fab fragments of another anti-beta7 mAb, Fib 504. Eosinophils incubated with soluble cellular fibronectin or mAb Fib 30 for 6 h demonstrated a higher level of GM-CSF mRNA than eosinophils incubated with medium alone. Addition of neutralizing mAb to GM-CSF during incubation, but not mAbs to IL-3 or IL-5, reduced the enhancement of eosinophil survival by soluble cellular fibronectin or mAb Fib 30 to control levels. Thus, viability of eosinophils incubated with cellular fibronectin or VCAM-1 is due to engagement, probably followed by cross-linking, of alpha4beta7 by soluble ligand (or mAb) that stimulates autocrine production of GM-CSF and promotes eosinophil survival.     

Decreased expression of membrane IL-5 receptor alpha on human eosinophils: I. Loss of membrane IL-5 receptor alpha on airway eosinophils and increased soluble IL-5 receptor alpha in the airway after allergen challenge

IL-5 is a key cytokine for eosinophil maturation, recruitment, activation, and possibly the development of inflammation in asthma. High concentrations of IL-5 are present in the airway after Ag challenge, but the responsiveness of airway eosinophils to IL-5 is not well characterized. The objectives of this study were to establish, following airway Ag challenge: 1) the expression of membrane (m)IL-5Ralpha on bronchoalveolar lavage (BAL) eosinophils; 2) the responsiveness of these cells to exogenous IL-5; and 3) the presence of soluble (s)IL-5Ralpha in BAL fluid. To accomplish these goals, blood and BAL eosinophils were obtained from atopic subjects 48 h after segmental bronchoprovocation with Ag. There was a striking reduction in mIL-5Ralpha on airway eosinophils compared with circulating cells. Furthermore, sIL-5Ralpha concentrations were elevated in BAL fluid, but steady state levels of sIL-5Ralpha mRNA were not increased in BAL compared with blood eosinophils. Finally, BAL eosinophils were refractory to IL-5 for ex vivo degranulation, suggesting that the reduction in mIL-5Ralpha on BAL eosinophils may regulate IL-5-mediated eosinophil functions. Together, the loss of mIL-5Ralpha, the presence of sIL-5Ralpha, and the blunted functional response (degranulation) of eosinophils to IL-5 suggest that when eosinophils are recruited to the airway, regulation of their functions becomes IL-5 independent. These observations provide a potential explanation for the inability of anti-IL-5 therapy to suppress airway hyperresponsiveness to inhaled Ag, despite a reduction in eosinophil recruitment.     

How can malaria rapid diagnostic tests achieve their potential? A qualitative study of a trial at health facilities in Ghana

Malaria is still a major public health problem in Brazil, with approximately 306 000 registered cases in 2009, but it is estimated that in the early 1940s, around six million cases of malaria occurred each year. As a result of the fight against the disease, the number of malaria cases decreased over the years and the smallest numbers of cases to-date were recorded in the 1960s. From the mid-1960s onwards, Brazil underwent a rapid and disorganized settlement process in the Amazon and this migratory movement led to a progressive increase in the number of reported cases. Although the main mosquito vector (Anopheles darlingi) is present in about 80% of the country, currently the incidence of malaria in Brazil is almost exclusively (99,8% of the cases) restricted to the region of the Amazon Basin, where a number of combined factors favors disease transmission and impair the use of standard control procedures. Plasmodium vivax accounts for 83,7% of registered cases, while Plasmodium falciparum is responsible for 16,3% and Plasmodium malariae is seldom observed. Although vivax malaria is thought to cause little mortality, compared to falciparum malaria, it accounts for much of the morbidity and for huge burdens on the prosperity of endemic communities. However, in the last few years a pattern of unusual clinical complications with fatal cases associated with P. vivax have been reported in Brazil and this is a matter of concern for Brazilian malariologists. In addition, the emergence of P. vivax strains resistant to chloroquine in some reports needs to be further investigated. In contrast, asymptomatic infection by P. falciparum and P. vivax has been detected in epidemiological studies in the states of Rondonia and Amazonas, indicating probably a pattern of clinical immunity in both autochthonous and migrant populations. Seropidemiological studies investigating the type of immune responses elicited in naturally-exposed populations to several malaria vaccine candidates in Brazilian populations have also been providing important information on whether immune responses specific to these antigens are generated in natural infections and their immunogenic potential as vaccine candidates. The present difficulties in reducing economic and social risk factors that determine the incidence of malaria in the Amazon Region render impracticable its elimination in the region. As a result, a malaria-integrated control effort - as a joint action on the part of the government and the population - directed towards the elimination or reduction of the risks of death or illness, is the direction adopted by the Brazilian government in the fight against the disease.     

Mapping of citrullinated fibrinogen B-cell epitopes in rheumatoid arthritis by imaging surface plasmon resonance

Introduction  

Rheumatoid arthritis (RA) frequently involves the loss of tolerance to citrullinated antigens, which may play a role in pathogenicity. Citrullinated fibrinogen is commonly found in inflamed synovial tissue and is a frequent target of autoantibodies in RA patients. To obtain insight into the B-cell response to citrullinated fibrinogen in RA, its autoepitopes were systematically mapped using a new methodology.