HIV-1 Matrix Protein Interactions with tRNA: Implications for Membrane Targeting

The N-terminally myristoylated matrix (MA) domain of the HIV-1 Gag polyprotein promotes virus assembly by targeting Gag to the inner leaflet of the plasma membrane. Recent studies indicate that, prior to membrane binding, MA associates with cytoplasmic tRNAs (including tRNA^Lys3), and in vitro studies of tRNA-dependent MA interactions with model membranes have led to proposals that competitive tRNA interactions contribute to membrane discrimination. We have characterized interactions between native, mutant, and unmyristylated (myr-) MA proteins and recombinant tRNA^Lys3 by NMR spectroscopy and isothermal titration calorimetry. NMR experiments confirm that tRNA^Lys3 interacts with a patch of basic residues that are also important for binding to the plasma membrane marker, phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂]. Unexpectedly, the affinity of MA for tRNA^Lys3 (K_d = 0.63 ± 0.03 μM) is approximately 1 order of magnitude greater than its affinity for PI(4,5)P₂-enriched liposomes (K_d(apparent) = 10.2 ± 2.1 μM), and NMR studies indicate that tRNA^Lys3 binding blocks MA association with liposomes, including those enriched with PI(4,5)P₂, phosphatidylserine, and cholesterol. However, the affinity of MA for tRNA^Lys3 is diminished by mutations or sample conditions that promote myristate exposure. Since Gag–Gag interactions are known to promote myristate exposure, our findings support virus assembly models in which membrane targeting and genome binding are mechanistically coupled.     

Isolation and characterization of methanogenic bacteria from rice paddies

Abstract Enrichment cultures for H₂-CO₂, methanol- or acetate-utilizing methanogens were prepared from two rice field soil samples. All the cultures except one acetate enrichment showed significant methane production. Pure cultures of Methanobacterium - and Methanosarcina -like organisms were isolated from H₂-CO₂ and methanol enrichment cultures, respectively, and were characterized for various nutritional and growth conditions. The organisms had an optimal pH range of 6.4–6.6 and a temperature optimum of 37°C. The Methanobacterium isolates were able to utilize H₂-CO₂ but no other substrates as sole energy source, while the Methanosarcina isolates were able to utilize methanol, methylamines or H₂-CO₂ as sole energy sources. Both Methanobacterium isolates and one isolate of Methanosarcina were able to use dinitrogen as the sole source of nitrogen for growth. The isolates used several sulfur compounds as sole sources of sulfur.     

Binding of caffeine with caffeic acid and chlorogenic acid using fluorescence quenching,UV/vis and FTIR spectroscopic techniques

The interactions of caffeine (CF) with chlorogenic acid (CGA) and caffeic acid (CFA) were investigated by fluorescence quenching, UV/vis and Fourier transform infrared (FTIR) spectroscopic techniques. The results of the study indicated that the fluorescence quenching between caffeine and hydroxycinnamic acids could be rationalized in terms of static quenching or the formation of non‐fluorescent CF–CFA and CF–CGA complexes. From fluorescence quenching spectral analysis, the quenching constant (K_SV), quenching rate constant (k_q), number of binding sites (n), thermodynamic properties and conformational changes of the interaction were determined. The quenching constants (K_SV) between CF and CGA, CFA are 1.84 × 10⁴ and 1.04 × 10⁴ L/mol at 298 K and their binding site n is ~ 1. Thermodynamic parameters determined using the Van't Hoff equation indicated that hydrogen bonds and van der Waal's forces have a major role in the reaction of caffeine with caffeic acid and chlorogenic acid. The 3D fluorescence, UV/vis and FTIR spectra also showed that the binding of CF with CFA and CGA induces conformational changes in CFA and CGA. Copyright © 2015 John Wiley & Sons, Ltd.     

Mycobacterium tuberculosis Complex and HIV Co-Infection among Extrapulmonary Tuberculosis Suspected Cases at the University of Gondar Hospital,Northwestern Ethiopia

Background

Extrapulmonary Tuberculosis (EPTB) and Human Immunodeficiency Virus (HIV) infection are interrelated as a result of immune depression. The aim of this study was to determine the prevalence of Mycobacterium tuberculosis complex isolates and the burden of HIV co-infection among EPTB suspected patients.

Method

An institution based cross-sectional study was conducted among EPTB suspected patients at the University of Gondar Hospital. Socio-demographic characteristics and other clinical data were collected using a pretested questionnaire. GeneXpert MTB/RIF assay was performed to diagnosis Mycobacterium tuberculosis complex and Rifampicin resistance. All samples were also investigated by cytology and culture. The HIV statuses of all patients were screened initially by KHB, and all positive cases were further re-tested by STAT-pack. Data was analyzed using SPSS version 20 computer software and a P-value of < 0.05 was taken as statistically significant.

Results

A total of 141 extrapulmonary suspected patients were enrolled in this study. The overall prevalence of culture confirmed extrapulmonary tuberculosis infection was 29.8%, but the GeneXpert result showed a 26.2% prevalence of Mycobacterium tuberculosis complex infection. The 78.4% prevalence of extrapulmonary tuberculosis infection was found to be higher among the adult population. The prevalence of HIV infection among EPTB suspected patients was 14.1%, while it was 32.4% among GeneXpert-confirmed extrapulmonary TB cases (12/37). Tuberculosis lymphadenitis was the predominant (78.4%) type of EPTB infection followed by tuberculosis cold abscess (10.7%). Adult hood, previous history of contact with known pulmonary tuberculosis patients, and HIV co-infection showed a statistically significant association with extrapulmonary tuberculosis infection (P<0.013).

Conclusion

The prevalence of culture confirmed-EPTB infection was high, and a higher EPTB-HIV co-infection was also observed.     

Searching Optimal Designs in the Presence of Serially Correlated Errors

Based on simple updating formulaé, a computer algorithm for searching optimal designs when the errors are believed to be serially correlated in the one-dimensional situation is described. The performance of the designs found by the algorithm is compared with the optimal designs available in the literature.     

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA₂₀ to bioactive GA₁) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₂₀ to GA₂₉), suggesting a concerted regulation to increase levels of bioactive GA₁ following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA₁ to GA₈) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA₁, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [¹⁴C]GA₁₂ to [¹⁴C]GA₁ only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA₁ required for initial fruit set and growth.     

Morbid Obesity as a Risk Factor for Hospitalization and Death Due to 2009 Pandemic Influenza A(H1N1) Disease

Background

Severe illness due to 2009 pandemic A(H1N1) infection has been reported among persons who are obese or morbidly obese. We assessed whether obesity is a risk factor for hospitalization and death due to 2009 pandemic influenza A(H1N1), independent of chronic medical conditions considered by the Advisory Committee on Immunization Practices (ACIP) to increase the risk of influenza-related complications.

Methodology/Principal Findings

We used a case-cohort design to compare cases of hospitalizations and deaths from 2009 pandemic A(H1N1) influenza occurring between April–July, 2009, with a cohort of the U.S. population estimated from the 2003–2006 National Health and Nutrition Examination Survey (NHANES); pregnant women and children <2 years old were excluded. For hospitalizations, we defined categories of relative weight by body mass index (BMI, kg/m²); for deaths, obesity or morbid obesity was recorded on medical charts, and death certificates. Odds ratio (OR) of being in each BMI category was determined; normal weight was the reference category. Overall, 361 hospitalizations and 233 deaths included information to determine BMI category and presence of ACIP-recognized medical conditions. Among ≥20 year olds, hospitalization was associated with being morbidly obese (BMI≥40) for individuals with ACIP-recognized chronic conditions (OR = 4.9, 95% CI 2.4–9.9) and without ACIP-recognized chronic conditions (OR = 4.7, 95%CI 1.3–17.2). Among 2–19 year olds, hospitalization was associated with being underweight (BMI≤5^th percentile) among those with (OR = 12.5, 95%CI 3.4–45.5) and without (OR = 5.5, 95%CI 1.3–22.5) ACIP-recognized chronic conditions. Death was not associated with BMI category among individuals 2–19 years old. Among individuals aged ≥20 years without ACIP-recognized chronic medical conditions death was associated with obesity (OR = 3.1, 95%CI: 1.5–6.6) and morbid obesity (OR = 7.6, 95%CI 2.1–27.9).

Conclusions/Significance

Our findings support observations that morbid obesity may be associated with hospitalization and possibly death due to 2009 pandemic H1N1 infection. These complications could be prevented by early antiviral therapy and vaccination.     

Chronic wasting disease of elk and deer and Creutzfeldt-Jakob disease: comparative analysis of the scrapie prion protein

Chronic wasting disease (CWD), a transmissible prion disease that affects elk and deer, poses new challenges to animal and human health. Although the transmission of CWD to humans has not been proven, it remains a possibility. If this were to occur, it is important to know whether the "acquired" human prion disease would show a phenotype including the scrapie prion protein (PrP(Sc)) features that differ from those associated with human sporadic prion disease. In this study, we have compared the pathological profiles and PrP(Sc) characteristics in brains of CWD-affected elk and deer with those in subjects with sporadic Creutzfeldt-Jakob disease (CJD), as well as CJD-affected subjects who might have been exposed to CWD, using histopathology, immunohistochemistry, immunoblotting, conformation stability assay, and N-terminal protein sequencing. Spongiform changes and intense PrP(Sc) staining were present in several brain regions of CWD-affected animals. Immunoblotting revealed three proteinase K (PK)-resistant bands in CWD, representing different glycoforms of PrP(Sc). The unglycosylated PK-resistant PrP(Sc) of CWD migrated at 21 kDa with an electrophoretic mobility similar to that of type 1 human PrP(Sc) present in sporadic CJD affecting subjects homozygous for methionine at codon 129 (sCJDMM1). N-terminal sequencing showed that the PK cleavage site of PrP(Sc) in CWD occurred at residues 82 and 78, similar to that of PrP(Sc) in sCJDMM1. Conformation stability assay also showed no significant difference between elk CWD PrP(Sc) and the PrP(Sc) species associated with sCJDMM1. However, there was a major difference in glycoform ratio of PrP(Sc) between CWD and sCJDMM1 affecting both subjects potentially exposed to CWD and non-exposed subjects. Moreover, PrP(Sc) of CWD exhibited a distinct constellation of glycoforms distinguishable from that of sCJDMM1 in two-dimensional immunoblots. These findings underline the importance of detailed PrP(Sc) characterization in trying to detect novel forms of acquired prion disease.     

Life cycle assessment of wind farms in Ethiopia

Purpose

The overall aim of this study is to contribute to the creation of LCA database on electricity generation systems in Ethiopia. This study specifically estimates the environmental impacts associated with wind power systems supplying high voltage electricity to the national grid. The study has regional significance as the Ethiopian electric system is already supplying electricity to Sudan and Djibouti and envisioned to supply to other countries in the region.

Materials and methods

Three different grid-connected wind power systems consisting of four different models of wind turbines with power rates between 1 and 1.67 MW were analyzed for the situation in Ethiopia. The assessment takes into account all the life cycle stages of the total system, cradle to grave, considering all the processes related to the wind farms: raw material acquisition, manufacturing of main components, transporting to the wind farm, construction, operation and maintenance, and the final dismantling and waste treatment. The study has been developed in line with the main principles of the ISO 14040 and ISO 14044 standard procedures. The analysis is done using SimaPro software 8.0.3.14 multi-user, Ecoinvent database version 3.01, and ReCiPe 2008 impact assessment method. The assumed operational lifetime as a baseline is 20 years.

Results and discussion

The average midpoint environmental impact of Ethiopian wind power system per kWh electricity generated is for climate change: 33.6 g CO₂ eq., fossil depletion: 8 g oil eq., freshwater ecotoxicity: 0.023 g 1,4-DCB eq., freshwater eutrophication: 0.005 g N eq., human toxicity: 9.9 g 1,4-DCB eq., metal depletion: 18.7 g Fe eq., marine ecotoxicity: 0.098 g 1,4-DCB eq., particulate matter formation: 0.097 g PM10 eq., photochemical oxidant formation: 0.144 g NMVOC, and terrestrial acidification: 0.21 g SO₂ eq. The pre-operation phase that includes the upstream life cycle stage is the largest contributor to all the environmental impacts, with shares ranging between 82 and 96%. The values of cumulative energy demand (CED) and energy return on investment (EROI) for the wind power system are 0.393 MJ and 9.2, respectively.

Conclusion

The pre-operation phase is the largest contributor to all the environmental impact categories. The sensitivity and scenario analyses indicate that changes in wind turbine lifespans, capacity factors, exchange rates for parts, transport routes, and treatment activities would result in significant changes in the LCA results.