Fine-scale genetic characterization of Plasmodium falciparum chromosome 7 encompassing the antigenic var and the drug-resistant pfcrt genes

The fact that malaria is still an uncontrolled disease is reflected by the genetic organization of the parasite genome. Efforts to curb malaria should begin with proper understanding of the mechanism by which the parasites evade human immune system and evolve resistance to different antimalarial drugs. We have initiated such a study and presented herewith the results from the in silico understanding of a seventh chromosomal region of the malarial parasite Plasmodium falciparum encompassing the antigenic var genes (coding pfemp1) and the drug-resistant gene pfcrt located at a specified region of the chromosome 7. We found 60 genes of various functions and lengths, majority (61.67%) of them were performing known functions. Almost all the genes have orthologs in other four species of Plasmodium, of which P. chabaudi seems to be the closest to P. falciparum. However, only two genes were found to be paralogous. Interestingly, the drug-resistant gene, pfcrt was found to be surrounded by seven genes coding for several CG proteins out of which six were reported to be responsible for providing drug resistance to P. vivax. The intergenic regions, in this specified region were generally large in size, majority (73%) of them were of more than 500 nucleotide bp length. We also designed primers for amplification of 21 noncoding DNA fragments in the whole region for estimating genetic diversity and inferring the evolutionary history of this region of P. falciparum genome.     

Enhanced and Enduring Protection against Tuberculosis by Recombinant BCG-Ag85C and Its Association with Modulation of Cytokine Profile in Lung

Background

The variable efficacy (0–80%) of Mycobacterium bovis Bacille Calmette Guréin (BCG) vaccine against adult tuberculosis (TB) necessitates development of alternative vaccine candidates. Development of recombinant BCG (rBCG) over-expressing promising immunodominant antigens of M. tuberculosis represents one of the potential approaches for the development of vaccines against TB.

Methods/Principal Findings

A recombinant strain of BCG - rBCG85C, over expressing the antigen 85C, a secretory immuno-dominant protein of M. tuberculosis, was evaluated for its protective efficacy in guinea pigs against M. tuberculosis challenge by aerosol route. Immunization with rBCG85C resulted in a substantial reduction in the lung (1.87 log₁₀, p<0.01) and spleen (2.36 log₁₀, p<0.001) bacillary load with a commensurate reduction in pathological damage, when compared to the animals immunized with the parent BCG strain at 10 weeks post-infection. rBCG85C continued to provide superior protection over BCG even when post-challenge period was prolonged to 16 weeks. The cytokine profile of pulmonary granulomas revealed that the superior protection imparted by rBCG85C was associated with the reduced levels of pro-inflammatory cytokines - interleukin (IL)-12, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, moderate levels of anti-inflammatory cytokine - transforming growth factor (TGF)-β along with up-regulation of inducible nitric oxide synthase (iNOS). In addition, the rBCG85C vaccine induced modulation of the cytokine levels was found to be associated with reduced fibrosis and antigen load accompanied by the restoration of normal lung architecture.

Conclusions/Significance

These results clearly indicate the superiority of rBCG85C over BCG as a promising prophylactic vaccine against TB. The enduring protection observed in this study gives enough reason to postulate that if an open-ended study is carried out with low dose of infection, rBCG85C vaccine in all likelihood would show enhanced survival of guinea pigs.     

Valuing mangrove ecosystem services: linking nutrient retention function of mangrove forests to enhanced agroecosystem production

Mangroves are highly productive wetland ecosystems strategically located at the interface between land and sea. They play an important role in the biogeochemical cycles of the coastal environment, acting as sources of nutrients to adjacent marine and terrestrial ecosystems through active and passive transport. We examined the nutrient contents in mangrove and nonmangrove soils in and around the Bhitarkanika National Park, India and assessed whether the local agricultural producers were aware of and placed a value on this contribution of mangrove forests in enhancing agroecosystem productivity. Soil samples from both mangrove and nonmangrove areas were analysed and quantity of organic carbon, total nitrogen, available phosphorus and potassium were derived. The replacement cost method was used to derive the value of nutrients in mangrove soils. We estimated that each hectare of mangrove contains additional nutrients worth US$232.49 in comparison to nonmangrove areas. The difference in nutrient content in mangrove versus nonmangrove areas gave the value of US$3.37 million for the nutrients in 145 km² of mangrove forests. The agricultural producers were aware that mangrove forests act as a source of nutrients and were willing to pay a higher price for the land adjoining mangrove forests. Around 92% of the producers ranked nutrient retention as a secondary function of mangrove forests. Despite crop depredation from wild ungulates and conflict with salt water crocodiles the agriculturist finds the benefit to cost ratio of mangrove forests high and more than 76% were in favour of mangrove restoration. This study provides an opportunity to highlight the importance of mangrove ecosystems to the livelihoods of the local people and the urgent need to sustain these through proper policy and market interventions.     

Immobilization of Xylan-Degrading Enzymes from Scytalidium thermophilum on Eudragit L-100

Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared to 6.5 in case of free enzyme. Immobilization increased the t_1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The K_m and V_max values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol) as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification of xylan was also improved by xylanase immobilization.     

The ORF3 protein of hepatitis E virus interacts with hemopexin by means of its 26 amino acid N-terminal hydrophobic domain II

Hepatitis E virus (HEV) is a nonenveloped plus-stranded RNA virus that is a major cause of acute hepatitis in many developing countries. Recent work has shown HEV may be endemic in developed countries also. The 5' two-thirds of the 7.2 kb single-stranded RNA genome of HEV encodes ORF1, and the 3' end encodes the structural proteins ORF2 and ORF3. ORF1 is the nonstructural protein involved in viral RNA synthesis, and ORF2 is the major capsid protein, whereas ORF3 is a very small protein of only 123 amino acids. The precise cellular functions of ORF3 protein remain obscure, although it has been postulated to be a viral regulatory protein. To elucidate the role of ORF3 in viral pathogenesis, the yeast two-hybrid system was used to screen a human liver cDNA library for proteins interacting with ORF3. One of the ORF3-interacting partners thus isolated and identified was hemopexin, a 60 kDa acute-phase plasma glycoprotein with a high binding affinity to heme. The two-hybrid result was validated by in vitro pull-down and co-immunoprecipitation assays and finally by intracellular fluorescence resonance energy transfer. Using a deletion mapping approach, the hydrophobic domain II of ORF3 (spanning amino acids 37 to 62) was found to be responsible for binding to Hpx, with amino acids 63 to 77 possibly contributing to the strength of the interaction. The biological significance of this interaction in the virus life cycle has been discussed.     

Characterization and wash performance analysis of an SDS-stable alkaline protease from a Bacillus sp.

An alkaline, SDS-stable protease optimally active at pH 11 from a Bacillus sp. RGR-14 was produced in a complex medium containing soybean meal, starch and calcium carbonate. The protease was active over a wide temperature range of 20–80 °C with major activity between 45 and 70 °C. The protease was completely stable for 1 h in 0.1% SDS and retained 70% of its activity in the presence of 0.5% SDS after 1 h of incubation. The enzyme was active in presence of surfactants (ionic and non-ionic) with 29% enhancement in activity in Tween-85 and was also stable in various oxidizing agents with 100 and 60% activity in presence of 1% sodium perborate and 1% H₂O₂, respectively. The enzyme was also compatible with commercial detergents (1% w/v) such as Surf, Ariel, Wheel, Fena and Nirma, retaining more than 70% activity in all the detergents after 1 h. Wash performance analysis of grass and blood stains on cotton fabric showed an increase in reflectance (14 and 25% with grass and blood stains, respectively) after enzyme treatment. However, enzyme in conjunction with detergent proved best, with a maximum reflectance change of 46 and 34% for grass and blood stain removal, respectively, at 45 °C. Stain removal was also effective after protease treatment at 25 and 60 °C.     

Circadian rhythm of airways caliber and its autonomic modulation

ABSTRACT

The autonomic nervous system (ANS) is one of the effector pathways for circadian variation of many physiological parameters. Autonomic tone and airways caliber have been reported to exhibit circadian variation in separate studies. A simultaneous investigation of heart rate variability (HRV) and airway caliber might ascertain how airway caliber is modulated by autonomic tone. This study was planned to identify the variations in airway caliber and autonomic function tone during a 24-hour span. A total of 56 healthy male subjects with almost similar daily routines were studied. Time domain, frequency domain and nonlinear analysis of R-R interval from 5 min electrocardiogram (ECG) was done seven times during the daytime wake span at 3-hour intervals starting at 05:00 h in the morning until 23:00 h in the night. Simultaneously peak expiratory flow rate (PEFR) was determined using a mini Wright’s peak flow meter. Rhythmometric analysis was done for PEFR and HRV parameters. Significant circadian variation in low frequency (LF) and high frequency (HF) variance was identified in this group of healthy subjects. The circadian rhythm of LF variance was characterized by a gradual increase and corresponding reciprocal change in HF variance from morning until night. The LF/HF ratio and SD2/SD1 ratio reflecting sympatho-vagal balance showed low to high values from morning to evening. The acrophase of the PEFR temporal pattern is similar to that of LF power and almost opposite in phase to that of HF power. PEFR is positively correlated with LF power. The circadian rhythm of airway caliber co-varies with cardiac autonomic tone. It appears that the temporal pattern of cardiac autonomic tone precedes in time that of airways caliber, thereby suggesting the latter operates under the modulatory effect of the 24-hour pattern in sympatho-vagal balance.     

Versatility and commercial status of microbial keratinases: a review

The world’s increasing population and shortage of food and feed is creating an urgently for us to look for new protein sources from waste products like keratinous waste. Poor management of these wastes has made them one of the major recalcitrant pollutants in nature. Microbial keratinases offers an economic and eco-friendly alternative for degrading and recycling keratinous waste into valuable byproducts. Diverse groups of microorganisms viz., bacteria, fungi and actinomycetes have the ability to degrade recalcitrant keratin by producing keratinase enzyme. Microbial keratinases exhibits great diversity in its biochemical properties with respect to activity and stability in various pH and temperature ranges as well as in the range of recalcitrant proteins it degrades like those present in feathers, hairs, nails, hooves etc. Owing to diverse properties and multifarious biotechnological implications, keratinases can be considered as promising biocatalysts for preparation of animal nutrients, protein supplements, leather processing, fiber modification, detergent formulation, feather meal processing for feed and fertilizer, the pharmaceutical, cosmetic and biomedical industries, and waste management. This review article presents an overview of keratin structure and composition, mechanism of microbial keratinolysis, diversity of keratinolytic microorganisms, and their potential applications in various fields.     

Solid-State Stability Issues of Drugs in Transdermal Patch Formulations

The transdermal patch formulation has many advantages, including noninvasiveness, an ability to bypass the first-pass metabolism, low dosage requirements, and prolonged drug delivery. However, the instability of solid-state drugs is one of the most critical problems observed in transdermal patch products. Therefore, a well-characterized approach for counteracting stability problems in solid-state drugs is crucial for improving the performance of transdermal patch products. This review provides insight into the solid-state stability of drugs associated with transdermal patch products and offers a comprehensive update on the various approaches being used for improving the stability of the active pharmaceutical ingredients currently being used.