Deubiquitinases Maintain Protein Homeostasis and Survival of Cancer Cells upon Glutathione Depletion

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Development of a genus specific primer set for detection of Leishmania parasites by polymerase chain reaction

Abstract We have compared the sequences of a major class of kinetoplast DNA (kDNA) minicircle (pLURkE3) of Leishmania strain UR6 with other minicircle sequences from different Leishmania species. Alignment of these sequences allowed the selection of a pair of oligonucleotides suitable as primers in polymerase chain reaction (PCR) which is specific for Leishmania parasites. PCR with this genus-specific primer set is capable of detecting 1 femtogram of kDNA. These primers have been tested with kDNAs from both old world and new world Leishmania species. The results indicate that the primers may be suitable for detection of any kind of leishmaniasis.     

Community-wide prevalence and intensity of soil-transmitted helminthiasis and Schistosoma mansoni in two districts of Sierra Leone

In Sierra Leone, nationally powered school-based surveys have documented significant progress in the control of soil-transmitted helminthiasis (STH) and schistosomiasis. In order to assess the district-level prevalence and intensity of infection among key at-risk groups outside of school age children (SAC), we conducted a multi-stage, cluster-sample household survey in Bo and Kenema districts in May 2018. From both districts, we examined 1,282 pre-school age children (PSAC), 730 school age children (SAC), and 517 adults over 14 years (including 387 women of reproductive age, or WRA) for STH and Schistosoma mansoni infection using Kato Katz technique. In Bo, STH prevalence was 8.0% (95% Upper Confidence Limit 10.2%) in PSAC, 6.4% (95% Upper Confidence Limit 9.0%) in SAC, 14.1% (95% Upper Confidence Limit 17.4%) in all adults and 11.9% (95% Upper Confidence Limit 17.4%) in WRA. In Kenema, STH prevalence was 18.1% (95% Upper Confidence Limit 20.5%) in PSAC, 17.3% (95% Upper Confidence Limit 20.7%) in SAC, and 16.9% (95% Upper Confidence Limit 20.5%) in all adults and 16.9% (95% Upper Confidence Limit 22.6%) in WRA. Hookworm species were the most prevalent of STH in both districts overall. The overall prevalence of S. mansoni was <10% in Bo and <20% in Kenema, and was similar across age groups. No moderate or heavy intensity STH infections or heavy intensity S. mansoni infections, as per World Health Organization (WHO) classification, were detected in either district. Sanitation variables, such as toilet access and quality, were independently associated with STH and S. mansoni infection. In Kenema, STH prevalence in SAC was within the WHO-defined range for annual treatment, whereas a previous nationally-powered survey estimated it to lie within the range of treatment once per two years. By utilizing community-based sampling, we were able to assess prevalence among WRA and make recommendations based on current guidance from WHO. To continue toward elimination of STH and S. mansoni as a public health problem, resources should be mobilized to increase access to and uptake of improved sanitation at community and household levels.     

Soybean (Glycine max L. Merr.) Sprouts Germinated under Red Light Irradiation Induce Disease Resistance against Bacterial Rotting Disease

Specific wavelengths of light can exert various physiological changes in plants, including effects on responses to disease incidence. To determine whether specific light wavelength had effects on rotting disease caused by Pseudomonas putida 229, soybean sprouts were germinated under a narrow range of wavelengths from light emitting diodes (LEDs), including red (650–660), far red (720–730) and blue (440–450 nm) or broad range of wavelength from daylight fluorescence bulbs. The controls were composed of soybean sprouts germinated in darkness. After germination under different conditions for 5 days, the soybean sprouts were inoculated with P. putida 229 and the disease incidence was observed for 5 days. The sprouts exposed to red light showed increased resistance against P. putida 229 relative to those grown under other conditions. Soybean sprouts germinated under red light accumulated high levels of salicylic acid (SA) accompanied with up-regulation of the biosynthetic gene ICS and the pathogenesis- related (PR) gene PR-1, indicating that the resistance was induced by the action of SA via de novo synthesis of SA in the soybean sprouts by red light irradiation. Taken together, these data suggest that only the narrow range of red light can induce disease resistance in soybean sprouts, regulated by the SA-dependent pathway via the de novo synthesis of SA and up-regulation of PR genes.     

Studies on a Novel Serine Protease of a ΔhapAΔprtV Vibrio cholerae O1 Strain and Its Role in Hemorrhagic Response in the Rabbit Ileal Loop Model

Background

Two well-characterized proteases secreted by Vibrio cholerae O1 strains are hemagglutinin protease (HAP) and V. cholerae protease (PrtV). The hapA and prtV knock out mutant, V. cholerae O1 strain CHA6.8ΔprtV, still retains residual protease activity. We initiated this study to characterize the protease present in CHA6.8ΔprtV strain and study its role in pathogenesis in rabbit ileal loop model (RIL).

Methodology/Principal Findings

We partially purified the residual protease secreted by strain CHA6.8ΔprtV from culture supernatant by anion-exchange chromatography. The major protein band in native PAGE was identified by MS peptide mapping and sequence analysis showed homology with a 59-kDa trypsin-like serine protease encoded by VC1649. The protease activity was partially inhibited by 25 mM PMSF and 10 mM EDTA and completely inhibited by EDTA and PMSF together. RIL assay with culture supernatants of strains C6709 (FA ratio 1.1+/−0.3 n = 3), CHA6.8 (FA ratio 1.08+/−0.2 n = 3), CHA6.8ΔprtV (FA ratio 1.02+/−0.2 n = 3) and partially purified serine protease from CHA6.8ΔprtV (FA ratio 1.2+/−0.3 n = 3) induced fluid accumulation and histopathological studies on rabbit ileum showed destruction of the villus structure with hemorrhage in all layers of the mucosa. RIL assay with culture supernatant of CHA6.8ΔprtVΔVC1649 strain (FA ratio 0.11+/−0.005 n = 3) and with protease incubated with PMSF and EDTA (FA ratio 0.3+/−0.05 n = 3) induced a significantly reduced FA ratio with almost complete normal villus structure.

Conclusion

Our results show the presence of a novel 59-kDa serine protease in a ΔhapAΔprtV V. cholerae O1 strain and its role in hemorrhagic response in RIL model.     

'LeishMan' topoisomerase I: an ideal chimera for unraveling the role of the small subunit of unusual bi-subunit topoisomerase I from Leishmania donovani

The active site tyrosine residue of all monomeric type IB topoisomerases resides in the C-terminal domain of the enzyme. Leishmania donovani, possesses unusual heterodimeric type IB topoisomerase. The small subunit harbors the catalytic tyrosine within the SKXXY motif. To explore the functional relationship between the two subunits, we have replaced the small subunit of L.donovani topoisomerase I with a C-terminal fragment of human topoisomerase I (HTOP14). The purified LdTOP1L (large subunit of L.donovani topoisomerase I) and HTOP14 were able to reconstitute topoisomerase I activity when mixed in vitro. This unusual enzyme, ‘LeishMan’ topoisomerase I (Leish for Leishmania and Man for human) exhibits less efficiency in DNA binding and strand passage compared with LdTOP1L/S. Fusion of LdTOP1L with HTOP14 yielded a more efficient enzyme with greater affinity for DNA and faster strand passage ability. Both the chimeric enzymes are less sensitive to camptothecin than LdTOP1L/S. Restoration of topoisomerase I activity by LdTOP1L and HTOP14 suggests that the small subunit of L.donovani topoisomerase I is primarily required for supplying the catalytic tyrosine. Moreover, changes in the enzyme properties due to substitution of LdTOP1S with HTOP14 indicate that the small subunit contributes to subunit interaction and catalytic efficiency of the enzyme.     

Efficient enzymatic systems for synthesis of novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria

Two enzymatic systems were developed for the efficient synthesis of glycoside products of α-mangostin, a natural xanthonoid exhibiting anti-oxidant, antibacterial, anti-inflammatory, and anticancer activities. In these systems, one-pot reactions for the synthesis of UDP-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with a glycosyltransferase (GT) from Bacillus licheniformis DSM-13 to afford C-3 and C-6 position modified glucose and 2-deoxyglucose conjugated novel α-mangostin derivatives. α-Mangostin 3-O-β-D-glucopyranoside, α-mangostin 6-O-β-D-glucopyranoside, α-mangostin 3,6-di-O-β-D-glucopyranoside, α-mangostin 3-O-β-D-2-deoxyglucopyranoside, α-mangostin 6-O-β-D-2-deoxyglucopyranoside, and α-mangostin 3,6-di-O-β-D-2-deoxyglucopyranoside were successfully produced in practical quantities and characterized by high-resolution quadruple time-of-flight electrospray ionization-mass spectrometry (HR-QTOF ESI/MS), ¹H and ¹³C NMR analyses. In excess of the substrate, the maximum productions of three α-mangostin glucopyranosides (4.8 mg/mL, 86.5 % overall conversion of α-mangostin) and three α-mangostin 2-deoxyglucopyronosides (4.0 mg/mL, 79 % overall conversion of α-mangostin) were achieved at 4-h incubation period. All the α-mangostin glycosides exhibited improved water solubility, and their antibacterial activity against three Gram-positive bacteria Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus was drastically enhanced by the glucosylation at C-3 position. In this study, diverse glycosylated α-mangostin were produced in significant quantities by using inexpensive starting materials and recycling co-factors within a reaction vessel without use of expensive NDP-sugars in the glycosylation reactions.     

Dry Season Diet of the Sloth Bear (Melursus ursinus) in Hadagarh Wildlife Sanctuary,Eastern India

Dry season food habit of sloth bear (Melursus ursinus) in Hadagarh Wildlife Sanctuary was characterized by scat analysis. Importance value index (IVI) of contribution of different food items in the diet of sloth bear was estimated from the undigested food remnants. Percentage of dry weight and IVI score of Ziziphus fruits was highest, termites were the most frequently occurring food item in the diet and percentage of frequency occurrence was equal for both fruits and insects. The present study, as compared to previous studies, suggests that the dietary preference of sloth bear varies according to availability of food and the habitat quality.     

Anti-Inflammatory Activity of Odina wodier Roxb,an Indian Folk Remedy,through Inhibition of Toll-Like Receptor 4 Signaling Pathway

Inflammation is part of self-limiting non-specific immune response, which occurs during bodily injury. In some disorders the inflammatory process becomes continuous, leading to the development of chronic inflammatory diseases including cardiovascular diseases, diabetes, cancer etc. Several Indian tribes used the bark of Odina wodier (OWB) for treating inflammatory disorders. Thus, we have evaluated the immunotherapeutic potential of OWB methanol extract and its major constituent chlorogenic acid (CA), using three popular in vivo antiinflammatory models: Carrageenan- and Dextran-induced paw edema, Cotton pellet granuloma, and Acetic acid-induced vascular permeability. To elucidate the possible anti-inflammatory mechanism of action we determine the level of major inflammatory mediators (NO, iNOS, COX-2-dependent prostaglandin E2 or PGE2), and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-12). Further, we determine the toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88), c-Jun N-terminal kinases (JNK), nuclear factor kappa-B cells (NF-κB), and NF-kB inhibitor alpha (IK-Bα) by protein and mRNA expression, and Western blot analysis in drug treated LPS-induced murine macrophage model. Moreover, we determined the acute and sub-acute toxicity of OWB extract in BALB/c mice. Our study demonstrated a significant anti-inflammatory activity of OWB extract and CA along with the inhibition of TNF-α, IL-1β, IL-6 and IL-12 expressions. Further, the expression of TLR4, NF-κBp65, MyD88, iNOS and COX-2 molecules were reduced in drug-treated groups, but not in the LPS-stimulated untreated or control groups, Thus, our results collectively indicated that the OWB extract and CA can efficiently inhibit inflammation through the down regulation of TLR4/MyD88/NF-kB signaling pathway.