Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

Background

The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites.

Methods

Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively.

Results

Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite.

Conclusions

Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host.     

Prevalence of Pulmonary Tuberculosis among Adults in a North Indian District

BackgroundRecent population prevalence estimates of pulmonary tuberculosis (PTB) are not available for several areas in India. We conducted a field-based population survey at a north Indian district to estimate point prevalence of bacteriologically positive PTB.MethodsA stratified cluster sampling design was used to conduct the survey in both urban and rural areas within the district. All adults aged more than 15 years, in 18 rural and 12 urban clusters of 3000 subjects each, were interviewed using a symptom card. Two sputum samples were collected from all persons having symptoms suggestive of PTB, or history of antitubercular treatment, for smear microscopy for acid-fast bacilli and mycobacterial culture. Those having at least one sputum specimen positive on microscopy and/or culture were categorized as having PTB. Prevalence was estimated after adjusting for cluster sampling and incomplete data (through individual level analysis with robust standard error).ResultsOf 91,030 eligible adult participants (47,714 men and 43,316 women), 85,770 (94.2%) completed the symptom cards. Of them, 2,898 persons were considered eligible for sputum examination and 2,839 (98.0%) provided at least one sample. Overall, 21 persons had bacteriologically positive PTB, and cluster level prevalence was estimated at 24.5 per 100,000 population (95% CI 12.8–36.2). Individual level analysis with robust standard error yielded a prevalence estimate of 24.1 per 100,000 populations (95% CI 12.8–35.4).ConclusionThe observed prevalence of bacteriologically positive PTB in this district is lower than empiric national estimates, probably as a result of successful implementation of tuberculosis control measures in the area.     

Proteomic Analysis of Anti-Cancerous Scopularide Production by a Marine Microascus brevicaulis Strain and Its UV Mutant

The marine fungus Microascus brevicaulis strain LF580 is a non-model secondary metabolite producer with high yields of the two secondary metabolites scopularides A and B, which exhibit distinct activities against tumour cell lines. A mutant strain was obtained using UV mutagenesis, showing faster growth and differences in pellet formation besides higher production levels. Here, we show the first proteome study of a marine fungus. Comparative proteomics were applied to gain deeper understanding of the regulation of production and of the physiology of the wild type strain and its mutant. For this purpose, an optimised protein extraction protocol was established. In total, 4759 proteins were identified. The central metabolic pathway of strain LF580 was mapped using the KEGG pathway analysis and GO annotation. Employing iTRAQ labelling, 318 proteins were shown to be significantly regulated in the mutant strain: 189 were down- and 129 upregulated. Proteomics are a powerful tool for the understanding of regulatory aspects: The differences on proteome level could be attributed to limited nutrient availability in the wild type strain due to a strong pellet formation. This information can be applied for optimisation on strain and process level. The linkage between nutrient limitation and pellet formation in the non-model fungus M. brevicaulis is in consensus with the knowledge on model organisms like Aspergillus niger and Penicillium chrysogenum.     

Stability Performance of Inductively Coupled Plasma Mass Spectrometry-Phenotyped Kernel Minerals Concentration and Grain Yield in Maize in Different Agro-Climatic Zones

Deficiency of iron and zinc causes micronutrient malnutrition or hidden hunger, which severely affects ~25% of global population. Genetic biofortification of maize has emerged as cost effective and sustainable approach in addressing malnourishment of iron and zinc deficiency. Therefore, understanding the genetic variation and stability of kernel micronutrients and grain yield of the maize inbreds is a prerequisite in breeding micronutrient-rich high yielding hybrids to alleviate micronutrient malnutrition. We report here, the genetic variability and stability of the kernel micronutrients concentration and grain yield in a set of 50 maize inbred panel selected from the national and the international centres that were raised at six different maize growing regions of India. Phenotyping of kernels using inductively coupled plasma mass spectrometry (ICP-MS) revealed considerable variability for kernel minerals concentration (iron: 18.88 to 47.65 mg kg^–1; zinc: 5.41 to 30.85 mg kg^–1; manganese: 3.30 to17.73 mg kg^–1; copper: 0.53 to 5.48 mg kg^–1) and grain yield (826.6 to 5413 kg ha^–1). Significant positive correlation was observed between kernel iron and zinc within (r = 0.37 to r = 0.52, p < 0.05) and across locations (r = 0.44, p < 0.01). Variance components of the additive main effects and multiplicative interactions (AMMI) model showed significant genotype and genotype × environment interaction for kernel minerals concentration and grain yield. Most of the variation was contributed by genotype main effect for kernel iron (39.6%), manganese (41.34%) and copper (41.12%), and environment main effects for both kernel zinc (40.5%) and grain yield (37.0%). Genotype main effect plus genotype-by-environment interaction (GGE) biplot identified several mega environments for kernel minerals and grain yield. Comparison of stability parameters revealed AMMI stability value (ASV) as the better representative of the AMMI stability parameters. Dynamic stability parameter GGE distance (GGED) showed strong and positive correlation with both mean kernel concentrations and grain yield. Inbreds (CM-501, SKV-775, HUZM-185) identified from the present investigation will be useful in developing micronutrient-rich as well as stable maize hybrids without compromising grain yield.     

PON-P2: Prediction Method for Fast and Reliable Identification of Harmful Variants

More reliable and faster prediction methods are needed to interpret enormous amounts of data generated by sequencing and genome projects. We have developed a new computational tool, PON-P2, for classification of amino acid substitutions in human proteins. The method is a machine learning-based classifier and groups the variants into pathogenic, neutral and unknown classes, on the basis of random forest probability score. PON-P2 is trained using pathogenic and neutral variants obtained from VariBench, a database for benchmark variation datasets. PON-P2 utilizes information about evolutionary conservation of sequences, physical and biochemical properties of amino acids, GO annotations and if available, functional annotations of variation sites. Extensive feature selection was performed to identify 8 informative features among altogether 622 features. PON-P2 consistently showed superior performance in comparison to existing state-of-the-art tools. In 10-fold cross-validation test, its accuracy and MCC are 0.90 and 0.80, respectively, and in the independent test, they are 0.86 and 0.71, respectively. The coverage of PON-P2 is 61.7% in the 10-fold cross-validation and 62.1% in the test dataset. PON-P2 is a powerful tool for screening harmful variants and for ranking and prioritizing experimental characterization. It is very fast making it capable of analyzing large variant datasets. PON-P2 is freely available at http://structure.bmc.lu.se/PON-P2/.     

De Novo Assembly and Genome Analyses of the Marine-Derived Scopulariopsis brevicaulis Strain LF580 Unravels Life-Style Traits and Anticancerous Scopularide Biosynthetic Gene Cluster

The marine-derived Scopulariopsis brevicaulis strain LF580 produces scopularides A and B, which have anticancerous properties. We carried out genome sequencing using three next-generation DNA sequencing methods. De novo hybrid assembly yielded 621 scaffolds with a total size of 32.2 Mb and 16298 putative gene models. We identified a large non-ribosomal peptide synthetase gene (nrps1) and supporting pks2 gene in the same biosynthetic gene cluster. This cluster and the genes within the cluster are functionally active as confirmed by RNA-Seq. Characterization of carbohydrate-active enzymes and major facilitator superfamily (MFS)-type transporters lead to postulate S. brevicaulis originated from a soil fungus, which came into contact with the marine sponge Tethya aurantium. This marine sponge seems to provide shelter to this fungus and micro-environment suitable for its survival in the ocean. This study also builds the platform for further investigations of the role of life-style and secondary metabolites from S. brevicaulis.     

Optimization of nutritional supplements for enhanced lactic acid production utilizing sugar refinery by-products

The present study investigated the synergistic effect of nutritional supplements (amino acid and Tween 80) on lactic acid production by Lactobacillus delbruckii utilizing a sugar refinery by product (cane molasses) in a submerged fermentation process. Initially, the effect of individual factors on lactic acid yield was studied by supplementing amino acids and their combinations, Tween 80 and cane molasses at varying concentrations in production medium. A combination of l-phenylalanine and l-lysine gave a maximum lactic acid yield of 47.89?±?0.1 g/L on a dry cell weight basis at individual factor level. Similarly, maximum lactic acid yield was obtained by supplementing the production medium with 40.0 g/L and 2.0 g/L Tween 80 and cane molasses, respectively, at individual factor level. In order to further improve the lactic acid yield, nutritional supplements were optimized by central composite rotatable design (CCRD) using Minitab 15 software. Shake flask cultivation under optimized conditions, i.e., cane molasses (32.40 g/L), Tween 80 (2.0 g/L) and l-phenylalanine and l-lysine (34.0 mg/L) gave a lactic acid yield of 64.86?±?0.2 g/L, corresponding to 95.0 % of the predicted yield of 67.78?±?0.3 g/L. Batch cultivation performed in 7.5 L bioreactor (working volume: 3.0 L) under optimized conditions gave maximum lactic acid yield and productivity of 79.12?±?0.2 g/L and 3.40 g/L·h, which is higher than previous studies with reduced fermentation time. Screening of lactic acid producing bacteria and characterization of lactic acid was also done.     

Effect of nutritional supplements on bio-plastics (PHB) production utilizing sugar refinery waste with potential application in food packaging

Abstract

Polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage reserve material stored by gram-negative bacteria under nutrient limitation. PHAs are best alternative biodegradable plastics (bio-plastics) due to their resemblance to conventional synthetic plastic. The present study investigated the synergistic effect of nutritional supplements (amino acid and vitamin) on the PHA production by Alcaligenes sp. NCIM 5085 utilizing a sugar refinery waste (cane molasses) under submerged fermentation process. Initially, the effect of individual factor on PHA yield was studied by supplementing amino acids (cysteine, isoleucine, and methionine), vitamin (thiamin), and cane molasses at varying concentration in the production medium. Further, the cultivation medium was optimized by varying the levels of cane molasses, methionine and thiamin using response surface methodology to enhance the PHA yield. The maximum PHA yield of 70.89% was obtained under the optimized condition, which was then scaled up on 7.5?L-bioreactor. Batch cultivation in 7.5?L-bioreactor under the optimized condition gave a maximum PHA yield and productivity of 79.26% and 0.312 gL^?1 h^?1, respectively. The PHA produced was subsequently characterized as PHB by FTIR. PHB extracted was of relatively high molecular weight and crystallinity index. DSC analysis gave T_g, T_m, and X_c of 4.2, 179?°C and 66%, respectively. TGA analysis showed thermal stability with maximized degradation occurring at 302?°C, which is above the melting temperature (179?°C) of the purified polymer. The extracted polymer, therefore, possessed desirable material properties to be used in food packaging.