Differential activities of the two closely related withanolides, withaferin a and withanone: bioinformatics and experimental evidences

BACKGROUND AND PURPOSE: Withanolides are naturally occurring chemical compounds. They are secondary metabolites produced via oxidation of steroids and structurally consist of a steroid-backbone bound to a lactone or its derivatives. They are known to protect plants against herbivores and have medicinal value including anti-inflammation, anti-cancer, adaptogenic and anti-oxidant effects. Withaferin A (Wi-A) and Withanone (Wi-N) are two structurally similar withanolides isolated from Withania somnifera, also known as Ashwagandha in Indian Ayurvedic medicine. Ashwagandha alcoholic leaf extract (i-Extract), rich in Wi-N, was shown to kill cancer cells selectively. Furthermore, the two closely related purified phytochemicals, Wi-A and Wi-N, showed differential activity in normal and cancer human cells in vitro and in vivo. We had earlier identified several genes involved in cytotoxicity of i-Extract in human cancer cells by loss-of-function assays using either siRNA or randomized ribozyme library. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we have employed bioinformatics tools on four genes, i.e., mortalin, p53, p21 and Nrf2, identified by loss-of-function screenings. We examined the docking efficacy of Wi-N and Wi-A to each of the four targets and found that the two closely related phytochemicals have differential binding properties to the selected cellular targets that can potentially instigate differential molecular effects. We validated these findings by undertaking parallel experiments on specific gene responses to either Wi-N or Wi-A in human normal and cancer cells. We demonstrate that Wi-A that binds strongly to the selected targets acts as a strong cytotoxic agent both for normal and cancer cells. Wi-N, on the other hand, has a weak binding to the targets; it showed milder cytotoxicity towards cancer cells and was safe for normal cells. The present molecular docking analyses and experimental evidence revealed important insights to the use of Wi-A and Wi-N for cancer treatment and development of new anti-cancer phytochemical cocktails.     

Efficient regeneration from hypocotyl explants in three cotton cultivars

A high frequency in vitro shoot bud differentiation and multiple shoot production protocol from hypocotyl segments of 8 to 10-d-old seedlings of cotton has been developed. Murashige and Skoog (MS) basal medium with Nitsch and Nitsch vitamins was found to be optimal in shoot regeneration. A combination of 2 mg dm⁻³ thidiazuron and 0.05 mg dm⁻³ naphthaleneacetic acid was the most effective for shoot regeneration (76 %) and an average of 10.6 shoots per responding explant. Combination of the cytokinins benzylaminopurine and kinetin induced better regeneration response than their individual treatments. Supplementation of the culture medium with ethylene inhibitor silver nitrate and activated charcoal showed beneficial effects. Optimal rooting was obtained on half-strength MS medium supplemented with 1 mg dm⁻³ indolebutyric acid and activated charcoal. Scanning electron micrographs of in vitro cultured explants revealed that shoot primordia were formed de novo.     

Characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strain DOR4 Toxic to Castor Semilooper <Emphasis Type="Italic">Achaea janata</Emphasis>: Proteolytic Processing and Binding of Toxins to Receptors

We have isolated a strain of Bacillus thuringiensis (Bt) from Indian soil samples that was shown to be toxic to Achaea janata larvae. The isolate, named B. thuringiensis DOR4, serotypically identified with the standard subspecies kurstaki (H3a3b3c) and produced bipyramidal inclusions along with an amorphous type. Although the plasmid pattern of DOR4 was different from that of the reference strain, a crystal protein profile showed the presence of two major bands (130 and 65 kDa) similar to those of Bt subsp. kurstaki HD-1. To verify the cry gene content of DOR4, triplex PCR analysis was performed; it showed amplification of the cry1C gene in addition to cry1Aa, cry1Ac, cry2A, and cry2B genes, but not the cry1Ab gene. RT-PCR analysis showed the expression of cry1Aa and cry1Ac genes. In vitro proteolysis of DOR4 protoxin with midgut extract generated products of different sizes. Zymogram analysis of DOR4 protoxin as substrate pointed to a number of distinct proteases that were responsible for activation of protoxins. Furthermore, toxin overlay analysis revealed the presence of multiple toxin-binding proteins in midgut epithelium. Based on all these characterizations, we suggest that the Bt DOR4 strain can be exploited for an A. janata control program.     

Employing oxygen pulses to modulate <Emphasis Type="Italic">Lachancea thermotolerans</Emphasis>–<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> Chardonnay fermentations

Oxygen is sometimes deliberately introduced in winemaking at various stages to enhance yeast biomass formation and prevent stuck fermentation. However, there is limited information on how such interventions affect the dynamics of yeast populations. Our previous study in synthetic grape juice showed that oxygen supply enhances the persistence of Lachancea thermotolerans, Torulaspora delbrueckii and Metschnikowia pulcherrima. The three non-Saccharomyces yeasts showed differences in growth as a function of oxygen. The present study focused on evaluating the influence of short oxygen pulses on population dynamics and the aroma profile of Chardonnay wine inoculated with L. thermotolerans and Saccharomyces cerevisiae. The results confirmed a positive effect of oxygen on the relative performance of L. thermotolerans. The mixed culture fermentation with L. thermotolerans with S. cerevisiae developed a distinct aroma profile when compared to monoculture S. cerevisiae. Specifically, a high concentration of esters, medium chain fatty acids and higher alcohols was detected in the mixed culture fermentation. The data also showed that the longer persistence of L. thermotolerans due to addition of oxygen pulses influenced the formation of major volatile compounds such as ethyl acetate, ethyl butyrate, ethyl hexanoate, ethyl caprylate, ethyl caprate, ethyl-3-hydroxybutanoate, ethyl phenylacetate, propanol, isobutanol, butanol, isoamyl alcohol, hexanol, isobutyric acid, butyric acid, iso-valeric acid, hexanoic acid, octanoic acid, and decanoic acid.     

The SbcC and SbcD homologs of the cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. strain PCC7120 (Alr3988 and All4463) contribute independently to DNA repair

The ubiquitous SbcCD exonuclease complex has been shown to perform an important role in DNA repair across prokaryotes and eukaryotes. However, they have remained uncharacterized in the ancient and stress-tolerant cyanobacteria. In the cyanobacterium Anabaena sp. strain PCC7120, SbcC and SbcD homologs, defined on the basis of the presence of corresponding functional domains, are annotated as hypothetical proteins, namely Alr3988 and All4463 respectively. Unlike the presence of sbcC and sbcD genes in a bicistronic operon in most organisms, these genes were distantly placed on the chromosome in Anabaena, and found to be negatively regulated by LexA. Both the genes were found to be essential in Anabaena as the individual deletion mutants were non-viable. On the other hand, the proteins could be individually overexpressed in Anabaena with no effect on normal cell physiology. However, they contributed positively to enhance the tolerance to different DNA damage-inducing stresses, such as mitomycin C and UV- and γ-radiation. This indicated that the two proteins, at least when overexpressed, could function independently and mitigate the damage caused due to the formation of DNA adducts and single- and double-strand breaks in Anabaena. This is the first report on possible independent in vivo functioning of SbcC and SbcD homologs in any bacteria, and the first effort to functionally characterize the proteins in any cyanobacteria.     

Environmental trade-offs across cascading lithium-ion battery life cycles

Purpose

The purpose of this study was to analyze the environmental trade-offs of cascading reuse of electric vehicle (EV) lithium-ion batteries (LIBs) in stationary energy storage at automotive end-of-life.

Methods

Two systems were jointly analyzed to address the consideration of stakeholder groups corresponding to both first (EV) and second life (stationary energy storage) battery applications. The environmental feasibility criterion was defined by an equivalent-functionality lead-acid (PbA) battery. A critical methodological challenge addressed was the allocation of environmental impacts associated with producing LIBs across the EV and stationary use systems. The model also tested sensitivity to parameters such as the fraction of battery cells viable for reuse, service life of refurbished cells, and PbA battery efficiency.

Results and discussion

From the perspective of EV applications, cascading reuse of an LIB in stationary energy storage can reduce net cumulative energy demand and global warming potential by 15 % under conservative estimates and by as much as 70 % in ideal refurbishment and reuse conditions. When post-EV LIB cells were compared directly to a new PbA system for stationary energy storage, the reused cells generally had lower environmental impacts, except in scenarios where very few of the initial battery cells and modules could be reused and where reliability was low (e.g., life span of 1 year or less) in the secondary application.

Conclusions

These findings demonstrate that EV LIB reuse in stationary application has the potential for dual benefit—both from the perspective of offsetting initial manufacturing impacts by extending battery life span as well as avoiding production and use of a less-efficient PbA system. It is concluded that reuse decisions and diversion of EV LIBs toward suitable stationary applications can be based on life cycle centric studies. However, technical feasibility of these systems must still be evaluated, particularly with respect to the ability to rapidly analyze the reliability of EV LIB cells, modules, or packs for refurbishment and reuse in secondary applications.

     

Association between the risk of coronary artery disease in South Asians and a deletion polymorphism in glutathione S-transferase M1.

South Asians living in Western societies show a greater risk of coronary artery disease (CAD) than the indigenous Caucasian population, probably related to the change to a Westernised lifestyle and an associated genetic susceptibility. Modulation of DNA damage and mutation caused by polymorphisms in detoxification enzymes, including the glutathione S-transferases (GSTs), is a well-established risk factor for tobacco-related carcinogenesis, and a similar change in cellular damage may be involved in the risk of vascular disease associated with tobacco smoking. In this study we examined whether polymorphisms in GST genes influence the risk of CAD in a case-control group of South Asians, following our recent observation of such an association in Caucasians from the same region of the UK. Blood was obtained from 170 patients of South Asian origin admitted for angiographic investigation of chest pain and from 203 controls. Patients were subdivided into those with and without previous acute myocardial infarction (AMI), and DNA was analysed for deletions in the GSTM1 and GSTT1 genes. An association was found between the prevalence of the GSTM1 null genotype and the risk of developing CAD in this study population. The frequency of the null genotype was 52.7% in healthy controls and 41.2% in patients (odds ratio [OR] 0.63, 95% confidence interval [95% CI] 0.42-0.95, p = 0.029). The effect was similar in subjects with or without a prior history of AMI. The association was also independent of smoking history, with both non-smokers and smokers showing a similar pattern of genotype distribution, the frequency of the null genotype being 51.2% in controls versus 37.0% in patients in 'never' smokers (OR 0.56, 95% CI 0.33-0.94, p = 0.037) and 60.0% in controls versus 46.2% in patients in 'ever' smokers (OR 0.57, 95% CI 0.25-1.28, p = 0.223). The association remained after adjusting for age, sex, body mass index and the presence or absence of stenosis. No significant associations were observed between the GSTT1 genotype and cardiovascular disease (chi(2) test, p > 0.1). The results of this study indicate that the GSTM1 null genotype is protective against both CAD and AMI. However, further study is required in order to elucidate the, as yet unexplained, mechanisms underlying this association.     

Simple and Effective Regeneration of Mungbean (Vigna radiata (L) Wilczek) using Cotyledonary Node Explants

Mungbean (Vigna radiata (L) Wilczek cv ML — 267) is a recalcitrant grain legume species. Direct multiple shoots were developed from the cotyledonary node explants of 2-day-old in vitro grown seedlings of mungbean. Maximum number of shoots (an average 12.1 shoots per explant) was obtained on a medium containing MS salts, B5 vitamins and 5.0 mg l^?1 BAP. A medium with lower BAP concentration appeared suitable for rapid shoot elongation. The elongated shoots were rooted on 0.2 mg l^?1 NAA. The rooted plants were acclimatized under field conditions. The survival of the plants in the greenhouse was 90 %. Plants flowered and set seed normally.     

Whole genome sequence of the rifamycin B-producing strain Amycolatopsis mediterranei S699

Amycolatopsis mediterranei S699 is an actinomycete that produces an important antibiotic, rifamycin B. Semisynthetic derivatives of rifamycin B are used for the treatment of tuberculosis, leprosy, and AIDS-related mycobacterial infections. Here, we report the complete genome sequence (10.2 Mb) of A. mediterranei S699, with 9,575 predicted coding sequences.