Gain-Assisted Transition Metal Ternary Nitrides (Ti1−xZrxN) Core–Shell Based Sensing of Waterborne Bacteria in Drinking Water

We propose gain-assisted Ti_1 − xZr_xN-based multilayered core–shell nanoparticles (MCSNPs) as refractive index (RI) sensor for real-time and label-free detection of waterborne bacteria. RI sensor designs optimized for diseases caused by pathogen Escherichia coli (E. coli), Serratia marcescens (S. marcescens), and Mierococcus lysodeikticus (M. lysodeikticus) are presented. Mie theory–based analysis shows that the precise incorporation of optical gain can compensate plasmonic losses at resonant wavelength and enhance sensor’s figure of merit (FOM) for bacteria detection up to ~ 10⁶–10⁷. The use of Ti_1 − xZr_xN ternary alloy as plasmonic material is advantageous as (I) Ti_1 − xZr_xN offers an alternative to the conventional plasmonic materials (e.g., Au, Ag, Cu, etc.); (II) specific Ti/Zr fractions in ternary Ti_1 − xZr_xN reduces the critical gain requirement compared to binary TiN; and (III) spectrum can be fine-tuned by controlling Ti/Zr fraction. The present work can led to the development of ultrasensitive plasmonic sensors capable of single bacteria detection as well as level of concentration of bacteria in water.

     

Downregulation of yidC in Escherichia coli by Antisense RNA Expression Results in Sensitization to Antibacterial Essential Oils Eugenol and Carvacrol

Background

The rising drug resistance in pathogenic bacteria and inefficiency of current antibiotics to meet clinical requirements has augmented the need to establish new and innovative approaches for antibacterial drug discovery involving identification of novel antibacterial targets and inhibitors. Being obligatory for bacterial growth, essential gene products are considered vital as drug targets. The bacterial protein YidC is highly conserved among pathogens and is essential for membrane protein insertion due to which it holds immense potential as a promising target for antibacterial therapy.

Methods/Principal Findings

The aim of this study was to explore the feasibility and efficacy of expressed antisense-mediated gene silencing for specific downregulation of yidC in Escherichia coli. We induced RNA silencing of yidC which resulted in impaired growth of the host cells. This was followed by a search for antibacterial compounds sensitizing the YidC depleted cells as they may act as inhibitors of the essential protein or its products. The present findings affirm that reduction of YidC synthesis results in bacterial growth retardation, which warrants the use of this enzyme as a viable target in search of novel antibacterial agents. Moreover, yidC antisense expression in E. coli resulted in sensitization to antibacterial essential oils eugenol and carvacrol. Fractional Inhibitory Concentration Indices (FICIs) point towards high level of synergy between yidC silencing and eugenol/carvacrol treatment. Finally, as there are no known YidC inhibitors, the RNA silencing approach applied in this study put forward rapid means to screen novel potential YidC inhibitors.

Conclusions/Significance

The present results suggest that YidC is a promising candidate target for screening antibacterial agents. High level of synergy reported here between yidC silencing and eugenol/carvacrol treatment is indicative of a potential antibacterial therapy. This is the first report indicating that the essential gene yidC is a therapeutic target of the antibacterial essential oils eugenol and carvacrol in E. coli.     

Promotion of Growth in Mungbean (Phaseolus aureus Roxb.) by Selenium is Associated with Stimulation of Carbohydrate Metabolism

The mungbean plants were grown hydroponically in the absence (control) or presence of 0.1, 0.25, 0.50 and 0.75 ppm selenium (as sodium selenate) for 10 days. The growth of shoots and roots increased with application of selenium with greater extent in shoots. With 0.5 and 0.75 ppm Se levels, the shoot growth was stimulated by 24% to 27% over control, respectively, while the roots showed a corresponding increase of 18-19%, respectively. The shoot-to-root ratio was enhanced significantly with Se application and maximum effects occurred at 0.75 ppm Se. A significant increase was observed in chlorophyll and cellular respiration ability with 0.5 and 0.75 ppm selenium. The increase in growth by selenium was accompanied by elevation of starch, sucrose and reducing sugars. The activity of starch hydrolysing enzymes--amylases and sucrose hydrolysing enzyme--invertase was stimulated significantly with selenium. This was associated with elevation of activities of sucrose synthesising enzymes--sucrose synthase and sucrose phosphate synthase. It was concluded that increase in growth of shoots and roots by application of Se was possibly the result of up-regulation of enzymes of carbohydrate metabolism thus providing energy substrates for enhanced growth.     

Design and discovery of novel quinazolinedione-based redox modulators as therapies for pancreatic cancer

Background

Altered cellular bioenergetics and oxidative stress are emerging hallmarks of most cancers including pancreatic cancer. Elevated levels of intrinsic reactive oxygen species (ROS) in tumors make them more susceptible to exogenously induced oxidative stress. Excessive oxidative insults overwhelm their adaptive antioxidant capacity and trigger ROS-mediated cell death. Recently, we have discovered a novel class of quinazolinediones that exert their cytotoxic effects by modulating ROS-mediated signaling.

Methods

Cytotoxic potential was determined by colorimetric and colony formation assays. An XF24 Extracellular Flux Analyzer, and colorimetric and fluorescent techniques were used to assess the bioenergetics and oxidative stress effects, respectively. Mechanism was determined by Western blots.

Results

Compound 3a (6-[(2-acetylphenyl)amino]quinazoline-5,8-dione) was identified through a medium throughput screen of ~ 1000 highly diverse in-house compounds and chemotherapeutic agents for their ability to alter cellular bioenergetics. Further structural optimizations led to the discovery of a more potent analog, 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) that displayed anti-proliferative activities in low micromolar range in both drug-sensitive and drug-resistant cancer cells. Treatment with 3b causes Akt activation resulting in increased cellular oxygen consumption and oxidative stress in pancreatic cancer cells. Moreover, oxidative stress induced by 3b promoted activation of stress kinases (p38/JNK) resulting in cancer cell death. Treatment with antioxidants was able to reduce cell death confirming ROS-mediated cytotoxicity.

Conclusion

In conclusion, our novel quinazolinediones are promising lead compounds that selectively induce ROS-mediated cell death in cancer cells and warrant further preclinical studies.

General significance

Since 3b (6-[(3-acetylphenyl)amino]quinazoline-5,8-dione) exerts Akt-dependent ROS-mediated cell death, it might provide potential therapeutic options for chemoresistant and Akt-overexpressing cancers.     

Chimeric Vitreoscilla hemoglobin (VHb) carrying a flavoreductase domain relieves nitrosative stress in Escherichia coli: new insight into the functional role of VHb.

Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 microM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.     

Prospecting for Novel Plant-Derived Molecules of Rauvolfia serpentina as Inhibitors of Aldose Reductase,a Potent Drug Target for Diabetes and Its Complications

Aldose Reductase (AR) is implicated in the development of secondary complications of diabetes, providing an interesting target for therapeutic intervention. Extracts of Rauvolfia serpentina, a medicinal plant endemic to the Himalayan mountain range, have been known to be effective in alleviating diabetes and its complications. In this study, we aim to prospect for novel plant-derived inhibitors from R. serpentina and to understand structural basis of their interactions. An extensive library of R. serpentina molecules was compiled and computationally screened for inhibitory action against AR. The stability of complexes, with docked leads, was verified using molecular dynamics simulations. Two structurally distinct plant-derived leads were identified as inhibitors: indobine and indobinine. Further, using these two leads as templates, 16 more leads were identified through ligand-based screening of their structural analogs, from a small molecules database. Thus, we obtained plant-derived indole alkaloids, and their structural analogs, as potential AR inhibitors from a manually curated dataset of R. serpentina molecules. Indole alkaloids reported herein, as a novel structural class unreported hitherto, may provide better insights for designing potential AR inhibitors with improved efficacy and fewer side effects.     

Morphological, Pathological and Molecular Variability in Colletotrichum capsici, the Cause of Fruit Rot of Chillies in the Subtropical Region of North-western India

Fruit rot of chillies (Capsicum annuum L.), caused by Colletotrichum capsici under tropical and subtropical conditions, results in qualitative and quantitative yield losses. Based on variation in cultural and morphological traits of C. capsici populations, 37 isolates were categorized into five groups designated, respectively, as Cc‐I, Cc‐II, Cc‐III, Cc‐IV and Cc‐V. In culture, most of the isolates produced cottony, fluffy or suppressed colonies. However, no significant differences were noticed in shape and size of conidia. The reaction of the 37 isolates on an indigenously developed differential set of Capsicum cultivars indicated the existence of different virulences in Himachal Pradesh (HP) chilli populations. Fifteen pathotypes of the pathogen were characterized from various chilli‐growing regions of HP. Pathotype CCP‐1 was most virulent and attacked all the differential cultivars. The genetic relationship between five morphological groups recognized within C. capsici was investigated using random amplified polymorphic DNA (RAPD) analysis. Molecular polymorphism generated by RAPD confirmed the variation in virulences of C. capsici and different isolates were grouped into five clusters. However, four isolates (Cc‐5, Cc‐33, Cc‐29 and Cc‐37) exhibited identical RAPD haplotypes. The pathological and RAPD grouping of isolates suggested no correlation among the test isolates.     

Chimeric Vitreoscilla Hemoglobin (VHb) Carrying a Flavoreductase Domain Relieves Nitrosative Stress in Escherichia coli: New Insight into the Functional Role of VHb

Dimeric hemoglobin (VHb) from the bacterium Vitreoscilla sp. strain C1 displays 30 to 53% sequence identity with the heme-binding domain of flavohemoglobins (flavoHbs) and exhibits the presence of potential sites for the interaction with its FAD/NADH reductase partner. The intersubunit contact region of VHb indicates a small interface between two monomers of the homodimer, suggesting that the VHb dimers may dissociate easily. Gel filtration chromatography of VHb exhibited a 25 to 30% monomeric population of VHb, at a low protein concentration (0.05 mg/ml), whereas dimeric VHb remained dominant at a high protein concentration (10 mg/ml). The structural characteristics of VHb suggest that the flavoreductase can also associate and interact with VHb in a manner analogous to flavoHbs and could yield a flavo-VHb complex. To unravel the functional relevance of the VHb-reductase association, the reductase domain of flavoHb from Ralstonia eutropha (formerly Alcaligenes eutrophus) was genetically engineered to generate a VHb-reductase chimera (VHb-R). The physiological implications of VHb and VHb-R were studied in an hmp mutant of Escherichia coli, incapable of producing any flavoHb. Cellular respiration the of the hmp mutant was instantaneously inhibited in the presence of 10 μM nitric oxide (NO) but remained insensitive to NO inhibition when these cells produced VHb-R. In addition, E. coli overproducing VHb-R exhibited NO consumption activity that was two to three times slower in cells overexpressing only VHb and totally undetectable in the control cells. A purified preparation of VHb-R exhibited a three- to fourfold-higher NADH-dependent NO uptake activity than that of VHb alone. Overproduction of VHb-R in the hmp mutant of E. coli conferred relief from the toxicity of sodium nitroprusside, whereas VHb alone provided only partial benefit under similar condition, suggesting that the association of VHb with reductase improves its capability to relieve the deleterious effect of nitrosative stress. Based on these results, it has been proposed that the unique structural features of VHb may allow it to acquire two functional states in vivo, namely, a single-domain homodimer that may participate in facilitated oxygen transfer or a two-domain heterodimer in association with its partner reductase that may be involved in modulating the cellular response under different environmental conditions. Due to this inherent structural flexibility, it may perform multiple functions in the cellular metabolism of its host. Separation of the oxidoreductase domain from VHb may thus provide a physiological advantage to its host.     

Immobilization of co-culture of Saccharomyces cerevisiae and Scheffersomyces stipitis in sodium alginate for bioethanol production using hydrolysate of apple pomace under separate hydrolysis and fermentation

Apple pomace as a substrate for bioethanol production is interesting due to its abundance and sustainable availability in varied states like Himachal Pradesh (H.P.), Jammu and Kashmir, Uttarakhand and Arunachal Pradesh, India. In the current study, apple pomace which is the main fruit industrial waste of H.P. was evaluated as feedstock for bioethanol production by the process of enzymatic saccharification using multiple carbohydrases. Microwave pretreatment of the apple pomace resulted in the efficient removal of lignin and crystalline structure of cellulose fibre. The enzymatic saccharification of the pretreated biomass was done by optimizing parameters for maximal saccharification leads to production of 27.50?mg/g of reduce, ng sugar. An enhanced ethanol yield of 44.46?g/l and fermentation efficiency of 58% by immobilized co-culture of Saccharomyces cerevisiae MTCC 3089 and Scheffersomyces stipitis NCIM 3498 under SHF as compared to fermentation performed with free yeast cells, i.e. 34.46?g/l of ethanol and 45% of fermentation efficiency.