Simultaneous hydrolysis of cheese whey and lactulose production catalyzed by β-galactosidase from Kluyveromyces lactis NRRL Y1564

Bioprocess and Biosystems Engineering - β-Galactosidase was produced by the yeast Kluyveromyces lactis NRRL Y1564 in cheese whey supplemented with yeast extract under the optimal temperature...     

Down-Regulation of CTLA-4 by HIV-1 Nef Protein

HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.     

Identification of Strain-Specific B-cell Epitopes in Trypanosoma cruzi Using Genome-Scale Epitope Prediction and High-Throughput Immunoscreening with Peptide Arrays

Background

The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA.

Methodology

By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain.

Findings and Conclusions

A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.     

High Throughput Phenotypic Selection of Mycobacterium tuberculosis Mutants with Impaired Resistance to Reactive Oxygen Species Identifies Genes Important for Intracellular Growth

Mycobacterium tuberculosis has the remarkable capacity to survive within the hostile environment of the macrophage, and to resist potent antibacterial molecules such as reactive oxygen species (ROS). Thus, understanding mycobacterial resistance mechanisms against ROS may contribute to the development of new anti-tuberculosis therapies. Here we identified genes involved in such mechanisms by screening a high-density transposon mutant library, and we show that several of them are involved in the intracellular lifestyle of the pathogen. Many of these genes were found to play a part in cell envelope functions, further strengthening the important role of the mycobacterial cell envelope in protection against aggressions such as the ones caused by ROS inside host cells.     

Famine in the Young and Risk of Later Hospitalization for COPD and Asthma

Background

Undernutrition during critical periods of growth and development may permanently affect lung physiology and function.

Objectives

To investigate whether acute undernutrition in childhood or young adulthood increases the risk of later hospitalization for obstructive airways disease, chronic obstructive pulmonary disease (COPD), or asthma.

Methods

We studied 7,841 women from Prospect-EPIC who experienced the 1944–45 Dutch famine between ages 0 and 21. Pulmonary outcomes were measured by registered hospital admissions and exposure-blinded computed tomography (CT) in a subgroup of 295 women. With Cox proportional hazard regression we explored effects of famine exposure on risk of hospitalization for obstructive airways disease, COPD, and asthma. With logistic regression we explored effects of famine on risk of CT evidence of pulmonary disease.

Results

Risks of hospitalization for obstructive airways disease, COPD, and asthma were increased after moderate famine exposure, and significantly increased after severe famine exposure: hazard ratios for obstructive airways disease were 1.31 (95% CI: 0.97 to 1.77) and 1.57 (95% CI: 1.10 to 2.23) respectively. Associations between famine exposure and hospitalization for COPD were stronger in ever-smokers than in never-smokers.

Conclusions

Acute undernutrition in childhood or young adulthood is associated with an increased risk of later COPD and asthma hospitalization, possibly through increased sensitivity for tobacco smoke.     

Proline Dehydrogenase Regulates Redox State and Respiratory Metabolism in Trypanosoma cruzi

Over the past three decades, L-proline has become recognized as an important metabolite for trypanosomatids. It is involved in a number of key processes, including energy metabolism, resistance to oxidative and nutritional stress and osmoregulation. In addition, this amino acid supports critical parasite life cycle processes by acting as an energy source, thus enabling host-cell invasion by the parasite and subsequent parasite differentiation. In this paper, we demonstrate that L-proline is oxidized to Δ¹-pyrroline-5-carboxylate (P5C) by the enzyme proline dehydrogenase (TcPRODH, E.C. 1.5.99.8) localized in Trypanosoma cruzi mitochondria. When expressed in its active form in Escherichia coli, TcPRODH exhibits a K_m of 16.58±1.69 µM and a V_max of 66±2 nmol/min mg. Furthermore, we demonstrate that TcPRODH is a FAD-dependent dimeric state protein. TcPRODH mRNA and protein expression are strongly upregulated in the intracellular epimastigote, a stage which requires an external supply of proline. In addition, when Saccharomyces cerevisiae null mutants for this gene (PUT1) were complemented with the TcPRODH gene, diminished free intracellular proline levels and an enhanced sensitivity to oxidative stress in comparison to the null mutant were observed, supporting the hypothesis that free proline accumulation constitutes a defense against oxidative imbalance. Finally, we show that proline oxidation increases cytochrome c oxidase activity in mitochondrial vesicles. Overall, these results demonstrate that TcPRODH is involved in proline-dependant cytoprotection during periods of oxidative imbalance and also shed light on the participation of proline in energy metabolism, which drives critical processes of the T. cruzi life cycle.     

Pediatric Post-Discharge Mortality in Resource Poor Countries: A Systematic Review

Objectives

Mortality following hospital discharge is an important and under-recognized contributor to overall child mortality in developing countries. The primary objective of this systematic review was to identify all studies reporting post-discharge mortality in children, estimate likelihood of death, and determine the most important risk factors for death.

Search Strategy

MEDLINE and EMBASE were systematically searched using MeSH terms and keywords from the inception date to October, 2012. Key word searches using Google Scholar™ and hand searching of references of retrieved articles was also performed. Studies from developing countries reporting mortality following hospital discharge among a pediatric population were considered for inclusion.

Results

Thirteen studies that reported mortality rates following discharge were identified. Studies varied significantly according to design, underlying characteristics of study population and duration of follow-up. Mortality rates following discharge varied significantly between studies (1%–18%). When reported, post-discharge mortality rates often exceeded in-hospital mortality rates. The most important baseline variables associated with post-discharge mortality were young age, malnutrition, multiple previous hospitalizations, HIV infection and pneumonia. Most post-discharge deaths occurred early during the post-discharge period. Follow-up care was examined in only one study examining malaria prophylaxis in children discharged following an admission secondary to malaria, which showed no significant benefit on post-discharge mortality.

Conclusions

The months following hospital discharge carry significant risk for morbidity and mortality. While several characteristics are strongly associated with post-discharge mortality, no validated tools are available to aid health workers or policy makers in the systematic identification of children at high risk of post-discharge mortality. Future research must focus on both the creation of tools to aid in defining groups of children most likely to benefit from post-discharge interventions, and formal assessment of the effectiveness of such interventions in reducing morbidity and mortality in the first few months following hospital discharge.