Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6–9) and temperature (30–65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 μM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

The four overlapping cosmids from the rubradirin producer, Streptomyces achromogenes var rubradiris NRRL 3061, have 58 ORFs within a 105.6 kb fragment. These ORFs harbored essential genes responsible for the formation and attachment of four distinct moieties, along with the genes associated with regulatory, resistance, and transport functions. The PKS (rubA) and glycosyltransferase (rubG2) genes were disrupted in order to demonstrate a complete elimination of rubradirin production. The rubradirin biosynthetic pathway was proposed based on the putative functions of the gene products, the functional identification of sugar genes, and the mutant strains.

Background Dengue cases have been continuously reported in Nepal, including some large outbreaks, since its first introduction in 2004. The disease is now expanding towards newer locations above 1400 m high, especially the country's capital city, Kathmandu. In 2019, >14,000 dengue cases including six deaths were reported. This study was aimed at the detection and molecular characterization of dengue virus (DENV) in dengue patients. Methods A total of 451 patients were enrolled in this study. Demographic, clinical and laboratory information was collected from dengue patients. Dengue infection was confirmed by antibody/antigen detection assays followed by RT-PCR analysis. Results The DENV patients showed fever, body ache, headache, myalgia, retro-orbital pain and arthralgia. The platelets were decreased, serum liver enzymes were increased and leucopenia was seen. Out of 195 patients, 111 (57.0%) were positive for DENV RNA by consensus PCR. We found DENV-2, 70 (63.1%) as the predominant serotype responsible for the 2019 outbreak, while DENV-3 was detected in two patients. Conclusion Our findings suggest that DENV-2 was the major serotype causing the 2019 massive outbreak in Nepal. This information will help in disease control programs to understand the molecular epidemiology and its changing trend.

Pectinase enzymes are one of the commercially important enzymes having great potential in various industries especially in food industry. Pectinases accounts for 25 % of global food enzymes produced and their market is increasing day by day. Therefore, the exploration of microorganism with novel characteristics has always been the focus of the research. Microorganism dwelling in unique habitat may possess unique characteristics. As such, a pectinase producing fungus Aspergillus niger strain MCAS2 was isolated from soil of Manaslu Conservation Area (MCA), Gorkha, Nepal. The optimum production of pectinase enzyme was observed at 48 h of fermentation. The pectinase enzyme was partially purified by cold acetone treatment followed by Sephadex G-75 gel filtration chromatography. The partially purified enzyme exhibited maximum activity 60 U/mg which was almost 8.5-fold higher than the crude pectinase. The approximate molecular weight of the enzyme was found to be 66 kDa as observed from SDS-PAGE. The pectinase enzyme was active at broad range of temperature (30–70 °C) and pH (6.2–9.2). Optimum temperature and pH of the pectinase enzyme were 50 °C and 8.2 respectively. The enzyme was stable up to 70 °C and about 82 % of pectinase activity was still observed at 100 °C. The thermostable and alkaline nature of this pectinase can meet the demand of various industrial processes like paper and pulp industry, in textile industry, fruit juice industry, plant tissue maceration and wastewater treatment. In addition, the effect of different metal ions on pectinase activity was also studied.

Visceral leishmaniasis (VL) is endemic to the southern plains of Nepal. Here, we report the first case of VL from a non-endemic Himalayan region of Nepal. The patient presented with a history of high-grade fever, splenomegaly, and anemia but had not traveled to a VL-endemic region. Visceral leishmaniasis was diagnosed following microscopic detection of the Leishmania species amastigote in a bone marrow aspirate, positive result for the rK39 test, and further validation by nested polymerase chain reaction (PCR). The patient was treated with 5 mg/kg liposomal amphotericin B and was clinically improved upon discharge. Our result suggests that VL is expanding towards non-endemic regions of Nepal, and it should therefore be considered that VL surveillance systems be strengthened, particularly for non-program districts and VL be included as a differential diagnosis in febrile illnesses.

Abstract:Crude polygalacturonase was obtained from Aspergillus flavus cultured in Glucose Free Medium (GFM) by precipitating cell free broth with 66% cold acetone. Effect of different parameters such as temperature, pH incubation time and enzyme concentration on this polygalacturonase preparation was studied. Furthermore, some inhibition studies on this enzyme preparation were conducted.

Background Visceral Leishmaniasis (VL) is caused by a protozoan parasite Leishmania donovani that is transmitted to humans by an infected female sandfly, Phlebotomus argentipes . VL is common in the Indian sub-continent including Nepal and efforts for its elimination are ongoing. However, expansion of disease towards the higher altitude areas, previously considered as VL free in Nepal, may impact the ability to achieve the elimination target by 2020. Methods This was an exploratory study, where VL suspected patients living exclusively in the non-program districts of Nepal and presenting with fever > 2 weeks and splenomegaly was included. The patients’ blood samples were collected, and DNA was extracted. DNA was subjected to PCR amplification and subsequent sequencing. Additionally, past 10 years data of VL cases from the national databases were analysed to see the trends of the disease in program and non program districts. Results Analysis of the past 10 years data revealed that trend of VL cases significantly decreased in the program districts ( p = 0.001) while it increased in the non-program districts ( p = 0.002). The national trend for overall incidence of VL also significantly decreased over this time period. Limited number of patients’ samples ( n = 14) were subjected to molecular investigation, and four patients were found to be positive for Leishmania species by PCR. Interestingly, these cases in non-program districts were indeed also L. donovoni complex . All four patients were male with age ranges from 10 to 68 years. GenBank BLAST of the obtained DNA sequences confirmed identified specimens as L. donovani complex . We identified additional VL cases from non-program districts (including the high lands) of Nepal, indicating that the infection could be an emerging threat for the non-program areas of Nepal. Conclusion The demonstration of VL cases in areas initially considered non-endemic has raised concern about on-going transmission in those regions and may trigger subsequent government plan and action to include those areas in the elimination program. Thus, the government should consider revising the disease control programs to accommodate non-program districts for achieving the VL elimination goal set for 2020.

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