“Protein aggregates” contain RNA and DNA,entrapped by misfolded proteins but largely rescued by slowing translational elongation

All neurodegenerative diseases feature aggregates, which usually contain disease‐specific diagnostic proteins; non‐protein constituents, however, have rarely been explored. Aggregates from SY5Y‐APP_Sw neuroblastoma, a cell model of familial Alzheimer''s disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized “contactome” comprising 11 subnetworks, centered on 24 high‐connectivity hubs. Remarkably, all 24 are nucleic acid‐binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer''s and control aggregates. RNA fragments were mapped to the human genome by RNA‐seq and DNA by ChIP‐seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5‐to 2.5‐fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y‐APP_Sw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E‐box/CLEAR motifs. We identified many G‐quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid‐binding proteins. After RNA‐interference knockdown of the translational‐procession factor EEF2 to suppress translation in SY5Y‐APP_Sw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content.     

Exploring insights for virulent gene inhibition of multidrug resistant Salmonella typhi,Vibrio cholerae,and Staphylococcus areus by potential phytoligands via in silico screening

In our recent studies on prevalence of multidrug resistant pathogens in Byramangala reservoir, Karnataka, India, we identified Salmonella typhi, Staphylococcus aureus, and Vibrio cholerae which had acquired multiple drug resistance (MDR) and emerged as superbugs. Hence, there is a pressing demand to identify alternative therapeutic remedies. Our study focused on the screening of herbal leads by structure-based virtual screening. The virulent gene products of these pathogens towards Kanamycin(aph), Trimethoprim(dfrA1), Methicillin (mecI), and Vancomycin (vanH) were identified as the probable drug targets and their 3D structures were predicted by homology modeling. The predicted models showed good stereochemical validity. By extensive literature survey, we selected 58 phytoligands and their drug likeliness and pharmacokinetic properties were computationally predicted. The inhibitory properties of these ligands against drug targets were studied by molecular docking. Our studies revealed that Baicalein from S. baicalensis (baikal skullcap) and Luteolin from Taraxacum officinale (dandelion) were identified as potential inhibitors against aph of S. typhi. Resveratrol from Vitis vinifera (grape vine) and Wogonin from S. baicalensis were identified as potential inhibitors against dfrA1 of S. typhi. Herniarin from Herniaria glabra (rupture worts) and Pyrocide from Daucus carota (Carrot) were identified as the best leads against dfrA1 of V. cholerae. Taraxacin of T. officinale (weber) and Luteolin were identified as potential inhibitors against Mec1. Apigenin from Coffee arabica (coffee) and Luteolin were identified as the best leads against vanH of S. aureus. Our findings pave crucial insights for exploring alternative therapeutics against MDR pathogens.     

Isolation and characterization of ethanol tolerant yeast strains

Yeast strains are commonly associated with sugar rich environments. Various fruit samples were selected as source for isolating yeast cells. The isolated cultures were identified at Genus level by colony morphology, biochemical characteristics and cell morphological characters. An attempt has been made to check the viability of yeast cells under different concentrations of ethanol. Ethanol tolerance of each strain was studied by allowing the yeast to grow in liquid YEPD (Yeast Extract Peptone Dextrose) medium having different concentrations of ethanol. A total of fifteen yeast strains isolated from different samples were used for the study. Seven strains of Saccharomyces cerevisiae obtained from different fruit sources were screened for ethanol tolerance. The results obtained in this study show a range of tolerance levels between 7%-12% in all the stains. Further, the cluster analysis based on 22 RAPD (Random Amplified polymorphic DNA) bands revealed polymorphisms in these seven Saccharomyces strains.     

Predicting stable functional peptides from the intergenic space of E. coli

Expression of synthetic proteins from intergenic regions of E. coli and their functional association was recently demonstrated (Dhar et al. in J Biol Eng 3:2, 2009. doi:10.1186/1754-1611-3-2). This gave birth to the question: if one can make ‘user-defined’ genes from non-coding genome—how big is the artificially translatable genome? (Dinger et al. in PLoS Comput Biol 4, 2008; Frith et al. in RNA Biol 3(1):40–48, 2006a; Frith et al. in PLoS Genet 2(4):e52, 2006b). To answer this question, we performed a bioinformatics study of all reported E. coli intergenic sequences, in search of novel peptides and proteins, unexpressed by nature. Overall, 2500 E. coli intergenic sequences were computationally translated into ‘protein sequence equivalents’ and matched against all known proteins. Sequences that did not show any resemblance were used for building a comprehensive profile in terms of their structure, function, localization, interactions, stability so on. A total of 362 protein sequences showed evidence of stable tertiary conformations encoded by the intergenic sequences of E. coli genome. Experimental studies are underway to confirm some of the key predictions. This study points to a vast untapped repository of functional molecules lying undiscovered in the non-expressed genome of various organisms.     

Mechanisms of killing of spores of Bacillus subtilis by dimethyldioxirane

AIMS: To determine the mechanisms of Bacillus subtilis spore resistance to and killing by a novel sporicide, dimethyldioxirane (DMDO) that was generated in situ from acetone and potassium peroxymonosulfate at neutral pH. METHODS AND RESULTS: Spores of B. subtilis were effectively killed by DMDO. Rates of killing by DMDO of spores lacking most DNA protective alpha/beta-type small, acid-soluble spore proteins (alpha- beta- spores) or the major DNA repair protein, RecA, were very similar to that of wild-type spore killing. Survivors of wild-type and alpha- beta- spores treated with DMDO also exhibited no increase in mutations. Spores lacking much coat protein due either to mutation or chemical decoating were much more sensitive to DMDO than were wild-type spores, but were more resistant than growing cells. Wild-type spores killed with this reagent retained their large pool of dipicolinic acid (DPA), and the survivors of spores treated with DMDO were sensitized to wet heat. The DMDO-killed spores germinated with nutrients, albeit more slowly than untreated spores, but germinated faster than untreated spores with dodecylamine. The killed spores were also germinated by very high pressures and by lysozyme treatment in hypertonic medium, but many of these spores lysed shortly after their germination, and none of these treatments were able to revive the DMDO-killed spores. CONCLUSIONS: DMDO is an effective reagent for killing B. subtilis spores. The spore coat is a major factor in spore resistance to DMDO, which does not kill spores by DNA damage or by inactivating some component needed for spore germination. Rather, this reagent appears to kill spores by damaging the spore's inner membrane in some fashion. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that DMDO is an effective decontaminant for spores of Bacillus species that can work under mild conditions, and the killed spores cannot be revived. Evidence has also been obtained on the mechanisms of spore resistance to and killing by this reagent.     

Effect of Dielectric and Liquid on Plasma Sterilization Using Dielectric Barrier Discharge Plasma

Plasma sterilization offers a faster, less toxic and versatile alternative to conventional sterilization methods. Using a relatively small, low temperature, atmospheric, dielectric barrier discharge surface plasma generator, we achieved ≥6 log reduction in concentration of vegetative bacterial and yeast cells within 4 minutes and ≥6 log reduction of Geobacillus stearothermophilus spores within 20 minutes. Plasma sterilization is influenced by a wide variety of factors. Two factors studied in this particular paper are the effect of using different dielectric substrates and the significance of the amount of liquid on the dielectric surface. Of the two dielectric substrates tested (FR4 and semi-ceramic (SC)), it is noted that the FR4 is more efficient in terms of time taken for complete inactivation. FR4 is more efficient at generating plasma as shown by the intensity of spectral peaks, amount of ozone generated, the power used and the speed of killing vegetative cells. The surface temperature during plasma generation is also higher in the case of FR4. An inoculated FR4 or SC device produces less ozone than the respective clean devices. Temperature studies show that the surface temperatures reached during plasma generation are in the range of 30°C–66°C (for FR4) and 20°C–49°C (for SC). Surface temperatures during plasma generation of inoculated devices are lower than the corresponding temperatures of clean devices. pH studies indicate a slight reduction in pH value due to plasma generation, which implies that while temperature and acidification may play a minor role in DBD plasma sterilization, the presence of the liquid on the dielectric surface hampers sterilization and as the liquid evaporates, sterilization improves.     

Advantage of using PSIRB over PSRB and IRB to improve plant health of tomato

Twenty-one isolates of phosphate solubilizing-indole acetic acid producing rhizobacteria (PSIRB), 20 isolates of phosphate solubilizing rhizobacteria (PSRB) and 42 isolates of indole acetic acid producing rhizobacteria (IRB) were isolated from 49 rhizospheric soil samples of tomato (Lycopersicon esculentum Mill.) collected from tomato growing regions of Karnataka. A method combining Pikovskaya’s and Bric’s technique was developed to isolate PSRIB, PSRB and IRB’s. The selected isolates were further analyzed for their ability to solubilize calcium phytate. Based on the root colonization assays and the abilities of bacterial isolates to increase the seed germination and seedling vigor under laboratory conditions, five isolates were selected from each group for further studies. Under greenhouse conditions, all the selected rhizobacteria isolates significantly increased root length, shoot length, fresh weight, dry weight and total phosphorus content of 30-day-old-seedlings with respect to control. Isolate PSIRB1 and IRB36 significantly reduced the Fusarium wilt incidence over other isolates of same and other group, and the control. On the basis of results from laboratory and greenhouse studies, one bacterial isolate from each group was selected for plant growth and yield analysis studies. Isolate PSIRB2 showed increased plant height, fresh weight, number of fruits per plant and average weight of fruit over PSRB9, IRB36 and untreated controls. Studies on the nature of protection offered by these bacterial isolates following split-root technique revealed that the isolates PSIRB2 and PSRB9 had the ability to induce systemic resistance. One isolate, IRB36 appeared to protect the tomato seedlings through direct antagonism.