Microarray compound screening (microARCS) to identify inhibitors of HIV integrase

A novel high-throughput strand transfer assay has been developed, using Microarray Compound Screening (microARCS) technology, to identify inhibitors of human immunodeficiency virus (HIV) integrase. This technology utilizes agarose matrices to introduce a majority of the reagents throughout the assay. Integration of biotinylated donor DNA with fluorescein isothiocyanate (FITC)-labeled target DNA occurs on a SAM membrane in the presence of integrase. An anti-FITC antibody conjugated to alkaline phosphatase (AP) was used to do an enzyme-linked immunosorbent assay with the SAM. An agarose gel containing AttoPhos, a substrate of AP, was used for detection of the integrase reactions on the SAM. For detection, the AttoPhos gel was separated from the SAM after incubation and then the gel was imaged using an Eagle Eye II closed-circuit device camera system. Potential integrase inhibitors appear as dark spots on the gel image. A library of approximately 250,000 compounds was screened using this HIV integrase strand transfer assay in microARCS format. Compounds from different structural classes were identified in this assay as novel integrase inhibitors.     

Activity of yeast d-amino acid oxidase on aromatic unnatural amino acids

d-Amino acid oxidase is a FAD-dependent enzyme that catalyses the conversion of the d-enantiomer of amino acids into the corresponding α-keto acid. Substrate specificity of the enzyme from the yeast Rhodotorula gracilis was investigated towards aromatic amino acids, and particularly synthetic α-amino acids.A significant improvement of the activity (V_max,app) and of the specificity constant (the V_max,app/K_m,app ratio) on a number of the substrates tested was obtained using a single-point mutant enzyme designed by a rational approach. With R. gracilis d-amino acid oxidase the complete resolution of d,l-homo-phenylalanine was obtained with the aim to produce the corresponding pure l-isomer and to use the corresponding α-keto acid as a precursor of the amino acid in the l-form.     

On the mechanism of Rhodotorula gracilis D-amino acid oxidase: role of the active site serine 335

Serine 335 at the active site of D-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO) is not conserved in other DAAO sequences. To assess its role in catalysis, it was mutated to Gly, the residue present in mammalian DAAO, an enzyme with a 35-fold lower turnover number with D-alanine. The spectral and ligand binding properties of the S335G mutant are similar to those of wild-type enzyme, suggesting an active site with minimally altered electrostatic properties. The S335G mutant is catalytically active, excluding an essential role of S335 in catalysis. However, S335-OH contributes to the high efficiency of the mutant enzyme since the catalytic activity of the latter is lower due to a decreased rate of flavin reduction relative to wild-type RgDAAO. Catalytic rates are pH-dependent and appear to converge to very low, but finite and similar values at low pH for both wild-type and S335G RgDAAO. While this dependence exhibits two apparent pKs with wild-type RgDAAO, with the S335G mutant a single, apparent pK approximately 8 is observed, which is attributed to the ionization of the alphaNH2 group of the bound substrate. Removal of S335-OH thus suppresses an apparent pK approximately 6. Both wild-type RgDAAO and the S335G mutant exhibit a substantial deuterium solvent kinetic isotope effect (> or =4) at pH<7 that disappears with increasing pH and reflects a pKapp=6.9 +/- 0.4. Interestingly, the substitution suppresses the activity towards d-lactate, suggesting a role of the serine 335 in removal of the substrate alpha-OH hydrogen.     

Understanding sheath blight resistance in rice: the road behind and the road ahead

Rice sheath blight disease, caused by the basidiomycetous necrotroph Rhizoctonia solani, became one of the major threats to the rice cultivation worldwide, especially after the adoption of high‐yielding varieties. The pathogen is challenging to manage because of its extensively broad host range and high genetic variability and also due to the inability to find any satisfactory level of natural resistance from the available rice germplasm. It is high time to find remedies to combat the pathogen for reducing rice yield losses and subsequently to minimize the threat to global food security. The development of genetic resistance is one of the alternative means to avoid the use of hazardous chemical fungicides. This review mainly focuses on the effort of better understanding the host–pathogen relationship, finding the gene loci/markers imparting resistance response and modifying the host genome through transgenic development. The latest development and trend in the R. solani–rice pathosystem research with gap analysis are provided.     

Bioreaction Engineering Leading to Efficient Synthesis of L‐Glyceraldehyd‐3‐Phosphate

Enantiopure L‐glyceraldehyde‐3‐phosphate (L‐GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L‐glyceraldehyde (L‐GA) by ATP is used for the synthesis of L‐GAP. L‐GAP has a half‐life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg²⁺ to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca²⁺, but not by L‐GAP and inorganic orthophosphate. Moreover, equal amount of Ca²⁺ exerts a different degree of inhibition relative to the activity without the addition of Ca²⁺ depending on the Mg²⁺ to ATP molar ratio. If the Mg²⁺ to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred‐tank reactor is the most efficient process for the synthesis of L‐GAP.     

Disordered eating attitude and associated factors among high school adolescents aged 12–19 years in Addis Ababa,Ethiopia: a cross-sectional study

Background

Eating disorders are very complex, frequently developed and have a public health impact on adolescents. Different studies revealed that eating disorders is a pressing public health problem among adolescents. Eating disorders may also lead to mortality due to their physiological sequelae. There is no previous study regarding disordered of eating attitude in Ethiopian adolescents. Therefore, this study aimed to assess prevalence of disordered eating attitude and its associated factors among adolescents in Addis Ababa high schools.

Methods

A school-based cross sectional study was conducted. Data were collected among 836 high school adolescents aged 12–19 years from May to June, 2015 in Addis Ababa city. The data were collected by self-administered questionnaire containing eating attitudes test-26 items (EAT-26) and socio-demographic factors. Binary logistic regression analysis was carried out to identify factors associated with disordered eating attitude. Both crude odds ratio and adjusted odds ratio were calculated to show the strength of association. In multivariable analysis, variables with a P value of <0.05 were considered statistically significant.

Results

The prevalence of disordered eating attitude among adolescents was 8.6% [95% CI 4.9, 12.3]. Being female [AOR = 1.75, 95% CI 1.03, 3.00], Mother’s educational status (Primary [AOR = 0.28, 95% CI 0.11, 0.78], Certificate/diploma [AOR = 0.22, 95% CI 0.07, 0.58] and first degree and above [AOR = 0.16, 95% CI 0.07, 0.40]) were found to be significantly associated with disordered eating attitude.

Conclusion

The finding of this study revealed that a significant number of adolescents were susceptible to developing disordered eating attitude. Being female and Mothers’ education status were significantly associated with disordered eating attitude among adolescents. Provision of screening test for eating disorders focusing on female adolescents is highly recommended.

     

Selection of resistance in protease inhibitor-experienced, human immunodeficiency virus type 1-infected subjects failing lopinavir- and ritonavir-based therapy: mutation patterns and baseline correlates

The selection of in vivo resistance to lopinavir was characterized by analyzing the longitudinal isolates from 54 protease inhibitor-experienced subjects who either experienced incomplete virologic response or viral rebound subsequent to initial response while on treatment with lopinavir-ritonavir in Phase II and III studies. The evolution of incremental resistance to lopinavir (emergence of new mutation[s] and/or at least a twofold increase in phenotypic resistance compared to baseline isolates) was highly dependent on the baseline phenotype and genotype. Among the subjects demonstrating evolution of lopinavir resistance, mutations at positions 82, 54, and 46 in human immunodeficiency virus protease emerged frequently, suggesting that these mutations are important for conferring high-level resistance. Less common mutations, such as L33F, I50V, and V32I together with I47V/A, were also selected; however, new mutations at positions 84, 90, and 71 were not observed. The emergence of incremental resistance contrasts greatly with the low incidence of resistance observed after initiating lopinavir-ritonavir therapy in antiretroviral-naive patients, suggesting that partial resistance accumulated during prior protease inhibitor therapy can compromise the genetic barrier to resistance to lopinavir-ritonavir. The emergence of incremental resistance was uncommon in subjects whose baseline isolates contained eight or more mutations associated with lopinavir resistance and/or displayed >60-fold-reduced susceptibility to lopinavir, providing insight into suitable upper genotypic and phenotypic breakpoints for lopinavir-ritonavir.     

Yeast D-amino acid oxidase: structural basis of its catalytic properties

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.