The Effect of Freeze-Thaw Cycles on Gene Expression Levels in Lymphoblastoid Cell Lines

Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene expression. We report a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene expression levels in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. We found that previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that while insight drawn from gene regulatory studies in mature LCLs may generally not be affected by the artificial nature of the LCL model system, many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures.     

Impact of NGO Training and Support Intervention on Diarrhoea Management Practices in a Rural Community of Bangladesh: An Uncontrolled,Single-Arm Trial

Purpose/Objective

The evolving Non-Governmental Organization (NGO) sector in Bangladesh provides health services directly, however some NGOs indirectly provide services by working with unlicensed providers. The primary objective of this study was to examine the impact of NGO training of unlicensed providers on diarrhoea management and the scale up of zinc treatment in rural populations.

Methods

An uncontrolled, single-arm trial for a training and support intervention on diarrhoea outcomes was employed in a rural sub-district of Bangladesh during 2008. Two local NGOs and their catchment populations were chosen for the study. The intervention included training of unlicensed health care providers in the management of acute childhood diarrhoea, particularly emphasizing zinc treatment. In addition, community-based promotion of zinc treatment was carried out. Baseline and endline ecologic surveys were carried out in intervention and control villages to document changes in treatments received for diarrhoea in under-five children.

Results

Among surveyed household with an active or recent acute childhood diarrhoea episode, 69% sought help from a health provider. Among these, 62.8% visited an unlicensed private provider. At baseline, 23.9% vs. 22% of control and intervention group children with diarrhoea had received zinc of any type. At endline (6 months later) this had changed to 15.3% vs. 30.2%, respectively. The change in zinc coverage was significantly higher in the intervention villages (p<0.01). Adherence with giving zinc for 10 days or more was significantly higher in the intervention households (9.2% vs. 2.5%; p<0.01). Child''s age, duration of diarrhoea, type of diarrhoea, parental year of schooling as well as oral rehydration solution (ORS) and antibiotic usage were significant predictors of zinc usage.

Conclusion

Training of unlicensed healthcare providers through NGOs increased zinc coverage in the diarrhoea management of under-five children in rural Bangladesh households.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02143921","term_id":"NCT02143921"}}NCT02143921     

National Trends in Main Causes of Hospitalization: A Multi-Cohort Register Study of the Finnish Working-Age Population, 1976–2010

Background

The health transition theory argues that societal changes produce proportional changes in causes of disability and death. The aim of this study was to identify long-term changes in main causes of hospitalization in working-age population within a nation that has experienced considerable societal change.

Methodology

National trends in all-cause hospitalization and hospitalizations for the five main diagnostic categories were investigated in the data obtained from the Finnish Hospital Discharge Register. The seven-cohort sample covered the period from 1976 to 2010 and consisted of 3,769,356 randomly selected Finnish residents, each cohort representing 25% sample of population aged 18 to 64 years.

Principal Findings

Over the period of 35 years, the risk of hospitalization for cardiovascular diseases and respiratory diseases decreased. Hospitalization for musculoskeletal diseases increased whereas mental and behavioral hospitalizations slightly decreased. The risk of cancer hospitalization decreased marginally in men, whereas in women an upward trend was observed.

Conclusions/Significance

A considerable health transition related to hospitalizations and a shift in the utilization of health care services of working-age men and women took place in Finland between 1976 and 2010.     

Structure-Activity Relationships of Novel Salicylaldehyde Isonicotinoyl Hydrazone (SIH) Analogs: Iron Chelation,Anti-Oxidant and Cytotoxic Properties

Salicylaldehyde isonicotinoyl hydrazone (SIH) is a lipophilic, tridentate iron chelator with marked anti-oxidant and modest cytotoxic activity against neoplastic cells. However, it has poor stability in an aqueous environment due to the rapid hydrolysis of its hydrazone bond. In this study, we synthesized a series of new SIH analogs (based on previously described aromatic ketones with improved hydrolytic stability). Their structure-activity relationships were assessed with respect to their stability in plasma, iron chelation efficacy, redox effects and cytotoxic activity against MCF-7 breast adenocarcinoma cells. Furthermore, studies assessed the cytotoxicity of these chelators and their ability to afford protection against hydrogen peroxide-induced oxidative injury in H9c2 cardiomyoblasts. The ligands with a reduced hydrazone bond, or the presence of bulky alkyl substituents near the hydrazone bond, showed severely limited biological activity. The introduction of a bromine substituent increased ligand-induced cytotoxicity to both cancer cells and H9c2 cardiomyoblasts. A similar effect was observed when the phenolic ring was exchanged with pyridine (i.e., changing the ligating site from O, N, O to N, N, O), which led to pro-oxidative effects. In contrast, compounds with long, flexible alkyl chains adjacent to the hydrazone bond exhibited specific cytotoxic effects against MCF-7 breast adenocarcinoma cells and low toxicity against H9c2 cardiomyoblasts. Hence, this study highlights important structure-activity relationships and provides insight into the further development of aroylhydrazone iron chelators with more potent and selective anti-neoplastic effects.     

P2X7 Receptor Modulates Inflammatory and Functional Pulmonary Changes Induced by Silica

Silicosis is an occupational lung disease, characterized by irreversible and progressive fibrosis. Silica exposure leads to intense lung inflammation, reactive oxygen production, and extracellular ATP (eATP) release by macrophages. The P2X7 purinergic receptor is thought to be an important immunomodulator that responds to eATP in sites of inflammation and tissue damage. The present study investigates the role of P2X7 receptor in a murine model of silicosis. To that end wild-type (C57BL/6) and P2X7 receptor knockout mice received intratracheal injection of saline or silica particles. After 14 days, changes in lung mechanics were determined by the end-inflation occlusion method. Bronchoalveolar lavage and flow cytometry analyzes were performed. Lungs were harvested for histological and immunochemistry analysis of fibers content, inflammatory infiltration, apoptosis, as well as cytokine and oxidative stress expression. Silica particle effects on lung alveolar macrophages and fibroblasts were also evaluated in cell line cultures. Phagocytosis assay was performed in peritoneal macrophages. Silica exposure increased lung mechanical parameters in wild-type but not in P2X7 knockout mice. Inflammatory cell infiltration and collagen deposition in lung parenchyma, apoptosis, TGF-β and NF-κB activation, as well as nitric oxide, reactive oxygen species (ROS) and IL-1β secretion were higher in wild-type than knockout silica-exposed mice. In vitro studies suggested that P2X7 receptor participates in silica particle phagocytosis, IL-1β secretion, as well as reactive oxygen species and nitric oxide production. In conclusion, our data showed a significant role for P2X7 receptor in silica-induced lung changes, modulating lung inflammatory, fibrotic, and functional changes.     

Trehalose Improves Human Fibroblast Deficits in a New CHIP-Mutation Related Ataxia

In this work we investigate the role of CHIP in a new CHIP-mutation related ataxia and the therapeutic potential of trehalose. The patient''s fibroblasts with a new form of hereditary ataxia, related to STUB1 gene (CHIP) mutations, and three age and sex-matched controls were treated with epoxomicin and trehalose. The effects on cell death, protein misfolding and proteostasis were evaluated. Recent studies have revealed that mutations in STUB-1 gene lead to a growing list of molecular defects as deregulation of protein quality, inhibition of proteasome, cell death, decreased autophagy and alteration in CHIP and HSP70 levels. In this CHIP-mutant patient fibroblasts the inhibition of proteasome with epoxomicin induced severe pathophysiological age-associated changes, cell death and protein ubiquitination. Additionally, treatment with epoxomicin produced a dose-dependent increase in the number of cleaved caspase-3 positive cells. However, co-treatment with trehalose, a disaccharide of glucose present in a wide variety of organisms and known as a autophagy enhancer, reduced these pathological events. Trehalose application also increased CHIP and HSP70 expression and GSH free radical levels. Furthermore, trehalose augmented macro and chaperone mediated autophagy (CMA), rising the levels of LC3, LAMP2, CD63 and increasing the expression of Beclin-1 and Atg5-Atg12. Trehalose treatment in addition increased the percentage of immunoreactive cells to HSC70 and LAMP2 and reduced the autophagic substrate, p62. Although this is an individual case based on only one patient and the statistical comparisons are not valid between controls and patient, the low variability among controls and the obvious differences with this patient allow us to conclude that trehalose, through its autophagy activation capacity, anti-aggregation properties, anti-oxidative effects and lack of toxicity, could be very promising for the treatment of CHIP-mutation related ataxia, and possibly a wide spectrum of neurodegenerative disorders related to protein disconformation.     

Molecular,Physiological and Biochemical Responses of Theobroma cacao L. Genotypes to Soil Water Deficit

Six months-old seminal plants of 36 cacao genotypes grown under greenhouse conditions were subjected to two soil water regimes (control and drought) to assess, the effects of water deficit on growth, chemical composition and oxidative stress. In the control, soil moisture was maintained near field capacity with leaf water potentials (ΨWL) ranging from −0.1 to −0.5 MPa. In the drought treatment, the soil moisture was reduced gradually by withholding additional water until ΨWL reached values of between −2.0 to −2.5 MPa. The tolerant genotypes PS-1319, MO-20 and MA-15 recorded significant increases in guaiacol peroxidase activity reflecting a more efficient antioxidant metabolism. In relation to drought tolerance, the most important variables in the distinguishing contrasting groups were: total leaf area per plant; leaf, stem and total dry biomass; relative growth rate; plant shoot biomass and leaf content of N, Ca, and Mg. From the results of these analyses, six genotypes were selected with contrasting characteristics for tolerance to soil water deficit [CC-40, C. SUL-4 and SIC-2 (non-tolerant) and MA-15, MO-20, and PA-13 (tolerant)] for further assessment of the expression of genes NCED5, PP2C, psbA and psbO to water deficit. Increased expression of NCED5, PP2C, psbA and psbO genes were found for non-tolerant genotypes, while in the majority of tolerant genotypes there was repression of these genes, with the exception of PA-13 that showed an increased expression of psbA. Mutivariate analysis showed that growth variables, leaf and total dry biomass, relative growth rate as well as Mg content of the leaves were the most important factor in the classification of the genotypes as tolerant, moderately tolerant and sensitive to water deficit. Therefore these variables are reliable plant traits in the selection of plants tolerant to drought.     

Leishmania-HIV Co-infection: Clinical Presentation and Outcomes in an Urban Area in Brazil

BackgroundVisceral leishmaniasis (VL) is an emerging condition affecting HIV-infected patients living in Latin America, particularly in Brazil. Leishmania-HIV coinfection represents a challenging diagnosis because the clinical picture of VL is similar to that of other disseminated opportunistic diseases. Additionally, coinfection is related to treatment failure, relapse and high mortality.ObjectiveTo assess the clinical-laboratory profile and outcomes of VL-HIV-coinfected patients using a group of non HIV-infected patients diagnosed with VL during the same period as a comparator.MethodsThe study was conducted at a reference center for infectious diseases in Brazil. All patients with suspected VL were evaluated in an ongoing cohort study. Confirmed cases were divided into two groups: with and without HIV coinfection. Patients were treated according to the current guidelines of the Ministry of Health of Brazil, which considers antimony as the first-choice therapy for non HIV-infected patients and recommends amphotericin B for HIV-infected patients. After treatment, all patients with CD4 counts below 350 cells/mm³ received secondary prophylaxis with amphotericin B.ResultsBetween 2011 and 2013, 168 patients with suspected VL were evaluated, of whom 90 were confirmed to have VL. In total, 51% were HIV coinfected patients (46 patients). HIV-infected patients had a lower rate of fever and splenomegaly compared with immunocompetent patients. The VL relapse rate in 6 months was 37% among HIV-infected patients, despite receiving secondary prophylaxis. The overall case-fatality rate was 6.6% (4 deaths in the HIV-infected group versus 2 deaths in the non HIV-infected group). The main risk factors for a poor outcome at 6 months after the end of treatment were HIV infection, bleeding and a previous VL episode.ConclusionAlthough VL mortality rates among HIV-infected individuals are close to those observed among immunocompetent patients treated with amphotericin B, HIV coinfection is related to a low clinical response and high relapse rates within 6 months.     

Paracoccidioides lutzii Plp43 Is an Active Glucanase with Partial Antigenic Identity with P. brasiliensis gp43

Background

Paracoccidioides brasiliensis and P. lutzii cause paracoccidioidomycosis (PCM). P. brasiliensis main diagnostic antigen is glycoprotein gp43, and its peptide sequence is 81% identical with a P. lutzii ortholog here called Plp43. P. lutzii (“Pb01-like”) apparently predominates in Midwestern/Northern Brazil, where high percentages of false-negative reactions using P. brasiliensis antigens have recently been reported. The aim of this work was to produce recombinant Plp43 to study its antigenic identity with gp43.

Methodology

We expressed rPlp43 as a secreted major component in Pichia pastoris and studied its reactivity in immunoblot with PCM patients'' sera from Southwestern and Midwestern Brazil.

Principal Findings

We showed that rPlp43 is not glycosylated and bears glucanase activity. The protein did not react with anti-gp43 monoclonal antibodies in immunoblot, suggesting absence of the corresponding gp43 epitopes. Nevertheless, common epitope(s) might exist, considering that gp43-positive PCM sera recognized rPlp43 in immunoblot, while gp43-negative sera (33 out of 51) from patients resident in Midwestern Brazil were also rPlp43-negative. Two genotyped P. lutzii were from patients with gp43-negative sera, suggesting that non-reactive sera are from patients infected with this species.

Conclusion

Our data suggest that gp43 and Plp43 bear one or only a few common epitopes and that gp43 cannot be used in diagnosis of PCM patients infected with P. lutzii probably because Plp43 is poorly expressed during infection.