A multigene family that interacts with the amino terminus of plasmodium MSP-1 identified using the yeast two-hybrid system

Merozoite surface protein 1 (MSP-1) is a high-molecular-weight protein expressed on the surface of the malaria merozoite in a noncovalent complex with other protein molecules. MSP-1 undergoes a series of proteolytic processing events, but no precise biological role for the various proteolytic fragments of MSP-1 or for the additional proteins present in the complex is known. Through the use of the yeast two-hybrid system, we have isolated genes encoding proteins that interact with a region of the amino-terminal proteolytic fragment of MSP-1 from the mouse parasite Plasmodium yoelii. This analysis has led to the isolation of two sequence-related molecules, one of which is the P. yoelii homologue of MSP-7 originally described in Plasmodium falciparum. BLAST analysis of the P. falciparum database has revealed that there are six related protein molecules present in this species encoded near each other on chromosome 13. In P. falciparum, we designated these molecules MSRP-1 to -5. Analysis of the P. yoelii database indicates a similar chromosomal organization for the two genes in the mouse parasite species. The three P. falciparum sequences with the highest degree of homology to the P. yoelii sequences isolated in the two-hybrid screen have been characterized at the molecular level (MSRP-1 to -3). Expression analysis indicated that the mRNAs are expressed at various levels in the different asexual stages. Immunofluorescence studies colocalized the expression of the MSRP molecules and the amino-terminal portion of MSP-1 to the surfaces of trophozoites. In vitro binding experiments confirmed the interaction between MSRP-1, MSRP-2, and the amino-terminal region of P. falciparum MSP-1.     

Evaluation of oxygen mass transfer in Aspergillus niger fermentation using data reconciliation

Fermentation experiments using Aspergillus niger result in a very viscous broth due to the growth of filamentous microorganism. For viscous fermentation processes, it is difficult to estimate with confidence the volumetric oxygen mass transfer coefficient (K(L)a), which can be used for scale-up or design of bioreactors. In the present study, four methods based on dynamic and stationary approaches were used to measure K(L)a throughout the fermentation. Data reconciliation was used to obtain a more reliable and consistent K(L)a. The K(L)a value obtained by a data reconciliation technique was found to be more reliable since it takes into consideration both the reliability of all measured variables and the accuracy of all mass balance equations.     

Vacuolar type H+ pumping pyrophosphatases of parasitic protozoa.

Trans-membrane proton pumping is responsible for a myriad of physiological processes including the generation of proton motive force that drives bioenergetics. Among the various proton pumping enzymes, vacuolar pyrophosphatases (V-PPases) form a distinct class of proton pumps, which are characterised by their ability to translocate protons across a membrane by using the potential energy released by hydrolysis of the phosphoanhydride bond of inorganic pyrophosphate. Until recently, V-PPases were known to be the purview of only plant vacuoles and plasma membranes of phototrophic bacteria. Recent discoveries of V-PPases in kinetoplastid and apicomplexan parasites, however, have expanded our view of the evolutionary reach of these enzymes. The lack of V-PPases in the vertebrate hosts of these parasites makes them potentially excellent targets for developing broad-spectrum antiparasitic agents. This review surveys the current understanding of V-PPases in parasitic protozoa with an emphasis on malaria parasites. Topological predictions suggest remarkable similarity of the parasite enzymes to their plant homologues with 15-16 membrane spanning domains and conserved sequences shown to constitute critical catalytic residues. Remarkably, malaria parasites have been shown to possess two V-PPase genes, one is an apparent orthologue of the canonical plant enzyme, whereas the other is a more distantly related paralogue with homology to a recently identified new class of K+-insensitive plant V-PPases. V-PPases appear to localise both to the plasma membrane and cytoplasmic organelles believed to be acidocalcisomes or polyphosphate bodies. Gene transfer experiments suggest that one of the malarial V-PPases is predominantly localised to the surface of intraerythrocytic parasites. We suggest a model in which V-PPase localised to the malaria parasite plasma membrane may serve as an electrogenic pump utilising pyrophosphate as an energy source, thus sparing the more precious ATP. Searching for V-PPase inhibitors could prove fruitful as a novel means of antiparasitic chemotherapy.     

Incidence of HIV and the Prevalence of HIV,Hepatitis B and Syphilis among Youths in Maputo,Mozambique: A Cohort Study

Background

Prevalence of HIV in Mozambique among individuals aged 15–49 years is 11.5%. The HIV prevalence is higher in women than in men across the country, peaking at ages 25–29 years and 35–39 years, respectively. In this study, we aimed at determining the prevalence and incidence of HIV, prevalence of Hepatitis B (HBV), and prevalence of syphilis in youths. We also characterized a cohort of youths for future participation in phase I/II HIV vaccine trials.

Methods

The study was conducted at a youth clinic in Maputo Central Hospital from August 2009 to October 2011. Youths of both genders aged 18–24 years (n = 1380) were screened for HIV using a sequential algorithm of two immunochromatographic assays, HBV using an enzyme linked immunosorbant test, and syphilis using a treponemal immunochromatographic strip test. The HIV seronegative participants (n = 1309) were followed-up for 12 months with quarterly study visits. The clinical and behavioral data were collected using structured questionnaires. The HIV seroconversions were confirmed by a molecular assay.

Results

The study population was female dominant (76.8%). All participants had a formal education, with 44.6% studying for technical or higher education degrees. The mean age at sexual debut was 16.6 years (SD: ±1.74), with 85.6% reporting more than one sexual partner in life. The screening showed the prevalence of HIV, HBV, and syphilis at 5.1% (95% CI: 3.97–6.31), 12.2% (95% CI 10.5%–14.0%), and 0.36% (95% CI 0.15%–0.84%), respectively. The HIV incidence rate was found to be 1.14/100 person years (95% CI: 0.67–1.92). Retention rates were stable throughout the study being 85.1% at the last visit.

Conclusion

Incidence of HIV in this cohort of youths in Maputo was relatively low. Also, the prevalence of HIV and syphilis was lower than the national values in this age group. However, the HBV prevalence was higher than in previous reports in the country.     

Cigarette smoking reduces DNA methylation levels at multiple genomic loci but the effect is partially reversible upon cessation

Smoking is a major risk factor in many diseases. Genome wide association studies have linked genes for nicotine dependence and smoking behavior to increased risk of cardiovascular, pulmonary, and malignant diseases. We conducted an epigenome wide association study in peripheral-blood DNA in 464 individuals (22 current smokers and 263 ex-smokers), using the Human Methylation 450 K array. Upon replication in an independent sample of 356 twins (41 current and 104 ex-smokers), we identified 30 probes in 15 distinct loci, all of which reached genome-wide significance in the combined analysis P < 5 × 10⁻⁸. All but one probe (cg17024919) remained significant after adjusting for blood cell counts. We replicated all 9 known loci and found an independent signal at CPOX near GPR15. In addition, we found 6 new loci at PRSS23, AVPR1B, PSEN2, LINC00299, RPS6KA2, and KIAA0087. Most of the lead probes (13 out of 15) associated with cigarette smoking, overlapped regions of open chromatin (FAIRE and DNaseI hypersensitive sites) or / and H3K27Ac peaks (ENCODE data set), which mark regulatory elements. The effect of smoking on DNA methylation was partially reversible upon smoking cessation for longer than 3 months. We report the first statistically significant interaction between a SNP (rs2697768) and cigarette smoking on DNA methylation (cg03329539). We provide evidence that the metSNP for cg03329539 regulates expression of the CHRND gene located circa 95 Kb downstream of the methylation site. Our findings suggest the existence of dynamic, reversible site-specific methylation changes in response to cigarette smoking , which may contribute to the extended health risks associated with cigarette smoking.     

Evaluating hormonal mechanisms of vitamin D receptor agonist therapy in diabetic kidney disease: the VALIDATE-D study

Studies have shown that group Therapeutic Patient Education (TPE) may empower patients with type 2 diabetes to better manage their disease. The mechanism of these interventions is not fully understood. A reduction in resistance to treatment may explain the mechanism by which TPE empowers participants to improve self-management. The Objective of this study was to examine the effectiveness of diabetes groups in reducing resistance to treatment and the association between reduced resistance and better management of the disease. In a program evaluation study, we administered validated questionnaires to measure resistance to treatment (RTQ) in 3 time periods: before the intervention (T1), immediately after the intervention (T2) and six months later (T3). Clinical measures (HbA1C, blood pressure, HDL, LDL and total cholesterol, Triglycerides and BMI) were retrieved from Maccabi Healthcare Services computerized systems, for T1;T2 and a year post intervention (T3). Linear mixed models were used adjusting for age, gender, social support and family status. 157; 156 and 106 TPE participants completed the RTQ in T1; T2 and T3 respectively. HbA1C and systolic and diastolic blood pressure were significantly reduced in the group which achieved a reduction in three out of the five RTQ components. For the other clinical measurements no significant changes were observed. Our findings suggest that reducing resistance to treatment, through an educational program for patients with diabetes, is associated with a better disease control. Identifying patients with higher resistance to treatment, and including components that reduce resistance in patient education programs, have the potential to increase the effectiveness of these programs.     

Computational exploration of structural hypotheses for an additional sequence in a mammalian mitochondrial protein

Background

Proteins involved in mammalian mitochondrial translation, when compared to analogous bacterial proteins, frequently have additional sequence regions whose structural or functional roles are not always clear. For example, an additional short insert sequence in the bovine mitochondrial initiation factor 2 (IF2_mt) seems sufficient to fulfill the added role of eubacterial initiation factor IF1. Prior to our recent cryo-EM study that showed IF2_mt to structurally occupy both the IF1 and IF2 binding sites, the spatial separation of these sites, and the short length of the insert sequence, posed ambiguity in whether it could perform the role of IF1 through occupation of the IF1 binding site on the ribosome.

Results

The present study probes how well computational structure prediction methods can a priori address hypothesized roles of such additional sequences by creating quasi-atomic models of IF2_mt using bacterial IF2 cryo-EM densities (that lack the insert sequences). How such initial IF2_mt predictions differ from the observed IF2_mt cryo-EM map and how they can be suitably improved using further sequence analysis and flexible fitting are analyzed.

Conclusions

By hypothesizing that the insert sequence occupies the IF1 binding site, continuous IF2_mt models that occupy both the IF2 and IF1 binding sites can be predicted computationally. These models can be improved by flexible fitting into the IF2_mt cryo-EM map to get reasonable quasi-atomic IF2_mt models, but the exact orientation of the insert structure may not be reproduced. Specific eukaryotic insert sequence conservation characteristics can be used to predict alternate IF2_mt models that have minor secondary structure rearrangements but fewer unusually extended linker regions. Computational structure prediction methods can thus be combined with medium-resolution cryo-EM maps to explore structure-function hypotheses for additional sequence regions and to guide further biochemical experiments, especially in mammalian systems where high-resolution structures are difficult to determine.