Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin

The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the alpha4-alpha5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr(178), Gln(180), Asn(183), Asn(185), and Asn(195)) in the hydrophobic helix-alpha5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in alpha5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn(183) located in the middle of the transmembrane alpha5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes.     

Genetic Diversity of the Giant Tiger Shrimp (Penaeus monodon) in Thailand Revealed by PCR-SSCP of Polymorphic EST-Derived Markers

A total of 90 ESTs from normal and 157 from subtractive ovarian cDNA libraries of the giant tiger shrimp (Penaeus monodon) were sequenced. SSCP analysis of disulfide isomerase (DSl), zinc finger protein (ZFP), PMO920, and PMT1700 was carried out for population genetic studies of P. monodon in Thai waters. The number of codominant alleles per locus for overall samples was 6 for PMO920, 5 for PMT1700, and 12 for ZFP, and there were 19 dominant alleles for DSI. The observed heterozygosity of each geographic sample was 0.3043–0.5128 for PMO920, 0.3462–0.4643 for PMT1700, and 0.5000–0.8108 for ZFP. Linkage disequilibrium analysis indicated that genotypes of these loci segregate randomly (P > 0.05). Low genetic distance was found between pairs of geographic samples (0.0077–0.0178). The neighbor-joining tree constructed from the average genetic distance of overall loci allocated the Andaman samples (Satun, Trang, and Phangnga) into one cluster, and Chumphon and Trat into other clusters. Geographic differentiation between Satun-Trat and Satun-Phangnga was found only at the ZFP locus (P < 0.05), suggesting low degrees of genetic subdivision of Thai P. monodon.     

Loop residues of the receptor binding domain of Bacillus thuringiensis Cry11Ba toxin are important for mosquitocidal activity

Using a Cry11Ba toxin model, predicted loops in domain II were analyzed for their role in receptor binding and toxicity. Peptides corresponding to loops α8, 1 and 3, but not loop 2, competed with toxin binding to Aedes midgut membranes. Mutagenesis data reveal loops α8, 1 and 3 are involved in toxicity. Loops 1 and 3 are of greater significance in toxicity to Aedes and Culex larvae than to Anopheles. Cry11Ba binds the apical membrane of larval caecae and posterior midgut, and binding can be competed by loop 1 but not by loop 2 peptides. Cry11Ba binds the same regions to which anti-cadherin antibody binds, and this antibody competes with Cry11Ba binding suggesting a possible role of cadherin in toxication.     

Targeted functional investigations guided by integrative proteome network analysis revealed significant perturbations of renal tubular cell functions induced by high glucose

Recently, several studies employed various proteomic approaches to define diabetes‐induced changes in renal proteins. However, functional significance of those datasets in diabetic nephropathy remained unclear. We thus performed integrative proteome network analysis of such datasets followed by various targeted functional studies in distal renal tubular cells treated with high glucose (HG) (25 mM) compared to normal glucose (NG) (5.5 mM) and NG + mannitol (M) (5.5 + 19.5 mM). The data showed that at 96 h when cell proliferation/death, tight junction protein and β‐/F‐actin expression and organization, and transepithelial resistance remained unchanged, only HG caused increased levels of HSP90, HSP70, and HSP60, and increased accumulation of intracellular protein aggregates. In addition, HG also induced overproduction of intracellular ROS, decreased catalase level, increased level of oxidatively modified proteins, increased intracellular ATP level, and defective transepithelial Ca²⁺ transport. However, both HG and M increased the levels of ubiquitinated proteins. Taken together, this study demonstrated significant perturbations of distal renal tubular cells induced by HG based on targeted functional studies guided by integrative proteome network analysis. These data may, at least in part, lead to better understanding of the pathogenic mechanisms of diabetic nephropathy.     

Control of ion transport in mouse proximal and distal colon by prolactin.

The lactogenic hormone prolactin (PRL) has been known to affect Ca(2+) and electrolyte transport in the intestinal epithelium. In the present study we analyzed ion transport in mouse proximal and distal colon, and acute changes induced by PRL. In the proximal colon, carbachol activated a Ca(2+) dependent Cl(-) secretion that was sensitive to DIDS and NFA. In the distal colon, both ATP and carbachol activated K(+) secretion. Ca(2+) -activated KCl transport in proximal and distal colon was inhibited by PRL (200 ng/ml), while amiloride sensitive Na(+) absorption and cAMP induced Cl(-) secretion remained unaffected. Luminal large conductance Ca(2+) -activated K(+) (BK) channels were largely responsible for Ca(2+) -activated K(+) secretion in the distal colon, and basolateral BK channels supported Ca(2+) -activated Cl(-) secretion in the proximal colon. Ca(2+) chelating by BAPTA-AM attenuated effects of carbachol and abolished effects of PRL. Both inhibition of PI3 kinase with wortmannin and blockage of MAP kinases with SB 203580 or U 0126, interfered with the acute inhibitory effect of PRL on ion transport, while blocking of Jak/Stat kinases with AG 490 was without effects. PRL attenuated the increase in intracellular Ca(2+) that was caused by stimulation of isolated colonic crypts with carbachol. Thus PRL inhibits Ca(2+) dependent Cl(-) and K(+) secretion by interfering with intracellular Ca(2+) signaling and probably by activating PI3 kinase and MAP kinase pathways.     

Optimization of a two-step biodiesel production process comprised of lipid extraction from blended sewage sludge and subsequent lipid transesterification

As a preliminary research for the development of feasible and economical biodiesel production using blended sewage sludge (BSS), a sustainable and non-edible feedstock, the two-step process comprised of lipid extraction (first step) and subsequent transesterification of the lipid with methanol (second step) was optimized. The total lipid content of the free fatty acid (FFA) containing BSS was determined to be 14.5% using the Blight and Dyer method with ultrasonication pretreatment, where 40.8% of the total lipid content was FFAs. The highest lipid yield of 13.5% (g-lipid/g-dry sludge), corresponding to 92.9% extraction efficiency, was obtained using 20 mL-solvent/g-dry sludge of the total solvent mixture with a 2/1 (v/v) ratio of chloroform and methanol. In the transesterification step, an acidic catalyst (H₂SO₄) exhibited significantly higher performance than an alkaline catalyst (NaOH). Thus, the optimal reaction conditions were 0.2% (g/g-lipid) H₂SO₄, 20 mL-methanol/g-lipid, 70°C and 8 h, respectively. Although the reaction temperature was increased from 50 to 70°C, we could save H₂SO₄, methanol, and a reaction time by 75, 50 and 66.7%, respectively compared with previous optimal conditions suggest by others’ research. Under our optimal conditions, a biodiesel yield of 39.0% (g-biodiesel/g-lipid) and an overall yield (i.e., extraction and transesterification) of 5.3% (g-biodiesel/g-BSS) were achieved, which are substantially higher than those from others’ research.     

Mapping Risk of Nipah Virus Transmission from Bats to Humans in Thailand

Nipah virus (NiV) is a zoonotic virus that can pose a serious threat to human and livestock health. Old-world fruit bats (Pteropus spp.) are the natural reservoir hosts for NiV, and Pteropus lylei, Lyle’s flying fox, is an important host of NiV in mainland Southeast Asia. NiV can be transmitted from bats to humans directly via bat-contaminated foods (i.e., date palm sap or fruit) or indirectly via livestock or other intermediate animal hosts. Here we construct risk maps for NiV spillover and transmission by combining ecological niche models for the P. lylei bat reservoir with other spatial data related to direct or indirect NiV transmission (livestock density, foodborne sources including fruit production, and human population). We predict the current and future (2050 and 2070) distribution of P. lylei across Thailand, Cambodia, and Vietnam. Our best-fit model predicted that central and western regions of Thailand and small areas in Cambodia are currently the most suitable habitats for P. lylei. However, due to climate change, the species range is predicted to expand to include lower northern, northeastern, eastern, and upper southern Thailand and almost all of Cambodia and lower southern Vietnam. This expansion will create additional risk areas for human infection from P. lylei in Thailand. Our combined predictive risk maps showed that central Thailand, inhabited by 2.3 million people, is considered highly suitable for the zoonotic transmission of NiV from P. lylei. These current and future NiV transmission risk maps can be used to prioritize sites for active virus surveillance and developing awareness and prevention programs to reduce the risk of NiV spillover and spread in Thailand.