Beyond antibiotics: phage-encoded lysins against Gram-negative pathogens.

Antibiotics remain the frontline agents for treating deadly bacterial pathogens. However, the indiscriminate use of these valuable agents has led to an alarming rise in AMR. The antibiotic pipeline is insufficient to tackle the AMR threat, especially with respect to the WHO critical category of priority Gram-negative pathogens, which have become a serious problem as nosocomial and community infections and pose a threat globally. The AMR pandemic requires solutions that provide novel antibacterial agents that are not only effective but against which bacteria are less likely to gain resistance. In this regard, natural or engineered phage-encoded lysins (enzybiotics) armed with numerous features represent an attractive alternative to the currently available antibiotics. Several lysins have exhibited promising efficacy and safety against Gram-positive pathogens, with some in late stages of clinical development and some commercially available. However, in the case of Gram-negative bacteria, the outer membrane acts as a formidable barrier; hence, lysins are often used in combination with OMPs or engineered to overcome the outer membrane barrier. In this review, we have briefly explained AMR and the initiatives taken by different organizations globally to tackle the AMR threat at different levels. We bring forth the promising potential and challenges of lysins, focusing on the WHO critical category of priority Gram-negative bacteria and lysins under investigation for these pathogens, along with the challenges associated with developing them as therapeutics within the existing regulatory framework.

Capacity Building for Vaccine Manufacturing Across Developing Countries: The Way Forward.

Approved vaccines prevent 2 to 3 million deaths per year. There is a lack of equitable access to vaccines in the low- and middle-income developing nations. Challenges in the life cycle of vaccine production include process development, lead time, intellectual property, and local vaccine production. A robust and stable manufacturing process and constant raw material supplies over decades is critical. In a continuously evolving vaccine landscape, the need of the hour for developing nations is to manufacture their own vaccines besides having supply security, control over production scheduling and sustainability, control of costs, socio-economic development, and rapid response to local epidemics. There is a need for capacity building of workforce development, technology transfer, and financial support. Technology transfer has improved vaccine access and reduced prices of vaccines. Capacity building for the manufacturing of vaccines in developing countries has always been an area of paramount importance and more so in a pandemic situation.

Roles of retailers in the peer-to-peer electricity market: A single retailer perspective.

Despite extensive research in the past five years and several successfully completed and on-going pilot projects, regulators are still reluctant to implement peer-to-peer trading at a large scale in today's electricity market. The reason could partly be attributed to the perceived disadvantage of current market participants such as retailers due to their exclusion from market participation-a fundamental property of decentralized peer-to-peer trading. As a consequence, recently, there has been growing pressure from energy service providers in favor of retailers' participation in peer-to-peer trading. However, the role of retailers in the peer-to-peer market is yet to be established, as no existing study has challenged this fundamental circumspection of decentralized trading. In this context, this perspective takes the first step to discuss the feasibility of retailers' involvement in the peer-to-peer market. In doing so, we identify key characteristics of retail-based and peer-to-peer electricity markets and discuss our viewpoint on how to incorporate a single retailer in a peer-to-peer market without compromising the fundamental decision-making characteristics of both markets. Finally, we give an example of a hypothetical business model to demonstrate how a retailer can be a part of a peer-to-peer market with a promise of collective benefits for the participants.

Development and pre-clinical evaluation of a synthetic oligosaccharide-protein conjugate vaccine against Neisseria meningitidis serogroup C.

Significant improvement has been made in the development of vaccines against Neisseria meningitidis infections since the introduction of polysaccharide-protein conjugate vaccines. Conventional bacterial capsular polysaccharide (PS) based conjugate vaccines require unique and expensive manufacturing facilities, complex production processes and extensive quality testing. Synthetic oligosaccharide (OS) based approach is one of the novel technologies that is being developed to simplify production of conjugate vaccines. OSs can be chemically synthesized to a desired length long enough to represent the antigenic epitopes which often present as a homogenous mixture. We prepared OSs corresponding to tetramer and octamer of N. meningitidis serogroup C (MenC) PS by organic synthesis. The MenC tetramer and octamer were further conjugated with tetanus toxoid to produce respective monovalent conjugates having the desired physico-chemical characteristics. The conjugates were evaluated in a mouse model for immunogenicity and compared with a licensed PS conjugate vaccine. Synthetic conjugates could induce anti-MenC PS IgG as well as serum bactericidal titers at levels comparable to those elicited by the licensed vaccine. The increase in length of synthetic oligomers from tetramer to octamer did not appear to increase immunogenicity. The results establish the pre-clinical proof of concept for a synthetic MenC oligosaccharide conjugate vaccine candidate.

Inhibition ELISA as a putative tool for the identification and quantification of meningococcal A and X polysaccharides at various stages of vaccine development.

The multivalent glycoconjugate vaccines against Neisseria meningitidis are extremely high-priced for the developing world. The high cost is due to the manufacturing set-up required to produce an effective vaccine and other inflators like complex production steps including the production and purification of the polysaccharide and consequently its conjugation with a protein and finally formulating the finished multivalent product. There is an urgent need for assays which are simple, precise, can be applicable at multiple steps and contribute in reducing the overall manufacturing cost, thereby making the vaccines more equitable to the developing world. WHO recommends serological tests for polysaccharide identification and quantitation at different stages of conjugate vaccine production. We report development of inhibition ELISAs for the identification and quantification of N. meningitidis serogroup A (MenA) and N. meningitidis serogroup X (MenX) polysaccharides (PSs) in samples from stage of cell banking till production of finished product. The method was qualified on various parameters such as specificity, intermediate precision, sensitivity and accuracy. Our results provide a proof of concept for the use of an inhibition ELISA as a common tool for the identification and quantification of PS at various stages of vaccine development and manufacture.

Evaluation of impact of temperature and pH alterations on the size and antigenicity of meningococcal serogroup A and X polysaccharides and conjugates.

The changes in the recommended storage conditions of the glycoconjugate vaccines against Neisseria meningitidis (Men) serogroup A and serogroup X can affect its activity or potency. Elevated temperature and the change in pH may result in the physical instability leading to the size degradation of the polysaccharide (PS) and subsequent loss of PS epitopes. Moreover, high temperature may also result in protein aggregation and altered tertiary structure of the protein in the conjugate. Consequently, the construction of a potent glycoconjugate is dependent on optimal temperature and pH. The changes in both these conditions can also affect the production of a capsular polysaccharide (PS) and its conjugation to a protein carrier and may also affect the integrity of the vaccine molecule including the maintenance of the protective epitopes. In our study we have used inhibition ELISA as a tool to assess the impact of temperature and pH alterations on the antigenicity of N. meningitidis serogroup A and X, PS and conjugates and their correlation with the size distribution analysis using high pressure size exclusion chromatography. The studies on pH alterations from 5 to 9 led to minimal impact on size and antigenicity of all antigens, however, an elevated temperature adversely impacted the antigen size as well as antigenicity to varying extent. Results indicate the higher stability of MenX PS and conjugate as compared to that for MenA counterparts at elevated temperatures. Furthermore, both the MenA and MenX conjugates appears to be more stable as compared to the corresponding PSs.

Rapid processes for purification of capsular polysaccharides from Neisseria meningitidis serogroups A and C.

The glycoconjugate vaccines against Neisseria meningitidis are highly effective, however most of these vaccines are expensive and still out of reach in the developing world as well as the technical know-how and the set-up required for the consistent production of pure polysaccharide is limited. Our laboratory has developed rapid, efficient and scalable processes for the downstream purification of N. meningitidis serogroup A (MenA) and serogroup C (MenC) capsular polysaccharides (PS). The MenC-PS was purified with a novel 2-step procedure including de-O-acetylation and hydrophobic interaction chromatography whereas, MenA-PS was purified using a rapid method as compared to the prior art. The purified PSs were analyzed by various analytical tests including nuclear magnetic resonance, molecular weight, composition and purity analyses to meet desired specifications. Our results provide a proof of principle for the purification of MenA-PS and MenC-PS with reduced timelines.

Resveratrol-induced apoptosis is enhanced in low pH environments associated with cancer.

Many critical factors such as hypoxia, nutrient deficiency, activation of glycolytic pathway/Warburg effect contribute to the observed low pH in tumors compared to normal tissue. Studies suggest that such tumor specific acidic environment can be exploited for the development of therapeutic strategies against cancer. Independent observations show reduction in pH of mammalian cells undergoing internucleosomal DNA fragmentation and apoptosis. As such, our group has extensively demonstrated that anticancer mechanisms of different plant polyphenols involve mobilization of endogenous copper and consequent internucleosomal DNA breakage. Copper is redox active metal, an essential component of chromatin and is sensitive to subtle pH changes in its microenvironment. Here we explored whether, acidic pH promotes growth inhibition, apoptosis, and DNA damaging capacity of chemopreventive agent resveratrol. Our results reveal that growth inhibition and internucleosomal DNA fragmentation induced apoptosis in Capan-2 and Panc-28 pancreatic cancer cell lines (and not in normal HPDE cells) by resveratrol is enhanced at lower pH. Using comet assay, we further demonstrate that DNA breakage by resveratrol is enhanced with acidification. Membrane permeable copper specific chelator neocuproine (and not iron chelator orthophenanthroline) abrogated growth inhibition and apoptosis by resveratrol. Western blot results show enhanced activation of DNA laddering marker H2.aX by resveratrol at acidic pH that was reversed by neocuproine and not by orthophenanthroline. Our findings provide irrevocable proof that low pH environment can be turned into tumor weakness and assist in eradication of cancer cells by resveratrol.

Cellular DNA breakage by soy isoflavone genistein and its methylated structural analogue biochanin A.

Epidemiological studies have indicated that populations with high isoflavone intake through soy consumption have lower rates of breast, prostate, and colon cancer. The isoflavone polyphenol genistein in soybean is considered to be a potent chemopreventive agent against cancer. In order to explore the chemical basis of chemopreventive activity of genistein, in this paper we have examined the structure-activity relationship between genistein and its structural analogue biochanin A. We show that both genistein and its methylated derivative biochanin A are able to mobilize nuclear copper in human lymphocyte, leading to degradation of cellular DNA. However, the relative rate of DNA breakage was greater in the case of genistein. Further, the cellular DNA degradation was inhibited by copper chelator (neocuproine/bathocuproine) but not by compounds that specifically bind iron and zinc (desferrioxamine mesylate and histidine, respectively). We also compared the antioxidant activity of the two isoflavones against tert-butylhydroperoxide-induced oxidative breakage in lymphocytes. Again genistein was found to be more effective than biochanin A in providing protection against oxidative stress induced by tert-butylhydroperoxide. It would therefore appear that the structural features of isoflavones that are important for antioxidant properties are also the ones that contribute to their pro-oxidant action through a mechanism that involves redox cycling of chromatin-bound nuclear copper.

Plumbagin induces cell death through a copper-redox cycle mechanism in human cancer cells.

Plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica has been shown to exert anticancer and anti-proliferative activities in cells in culture as well as animal tumor models. In our previous paper, we have reported the cytotoxic action of plumbagin in plasmid pBR322 DNA as well as human peripheral blood lymphocytes through a redox mechanism involving copper. Copper has been shown to be capable of mediating the action of several plant-derived compounds through production of reactive oxygen species (ROS). The objective of the present study was to determine whether plumbagin induces apoptosis in human cancer cells through the same mechanism which we proposed earlier. Using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt assay, 3-(4,5-B-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for cell growth inhibition, histone/DNA ELISA, homogeneous caspase-3/7 assay for apoptosis as well as alkaline comet assay for DNA single-strand breaks detection in this report, we confirm that plumbagin causes effective cell growth inhibition, induces apoptosis and generates single-strand breaks in cancer cells. Incubation of cancer cells with scavengers of ROS and neocuproine inhibited the cytotoxic action of plumbagin proving that generation of ROS and Cu(I) are the critical mediators in plumbagin-induced cell growth inhibition. This study is the first to investigate the copper-mediated anticancer mechanism of plumbagin in human cancer cells and these properties of plumbagin could be further explored for the development of anticancer agents with higher therapeutic indices, especially for skin cancer.

Plant polyphenols mobilize nuclear copper in human peripheral lymphocytes leading to oxidatively generated DNA breakage: implications for an anticancer mechanism.

It was earlier proposed that an important anti-cancer mechanism of plant polyphenols may involve mobilization of endogenous copper ions, possibly chromatin-bound copper and the consequent pro-oxidant action. This paper shows that plant polyphenols are able to mobilize nuclear copper in human lymphocytes, leading to degradation of cellular DNA. A cellular system of lymphocytes isolated from human peripheral blood and comet assay was used for this purpose. Incubation of lymphocytes with neocuproine (a cell membrane permeable copper chelator) inhibited DNA degradation in intact lymphocytes. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. This study has further shown that polyphenols are able to degrade DNA in cell nuclei and that such DNA degradation is inhibited by neocuproine as well as bathocuproine (both of which are able to permeate the nuclear pore complex), suggesting that nuclear copper is mobilized in this reaction. Pre-incubation of lymphocyte nuclei with polyphenols indicates that it is capable of traversing the nuclear membrane. This study has also shown that polyphenols generate oxidative stress in lymphocyte nuclei which is inhibited by scavengers of reactive oxygen species (ROS) and neocuproine. These results indicate that the generation of ROS occurs through mobilization of nuclear copper resulting in oxidatively generated DNA breakage.

The anthocyanidin delphinidin mobilizes endogenous copper ions from human lymphocytes leading to oxidative degradation of cellular DNA.

Epidemiological and experimental evidence exists to suggest that pomegranate and its juice possess chemopreventive and anticancer properties. The anthocyanidin delphinidin is a major polyphenol present in pomegranates and has been shown to be responsible for these effects. Plant polyphenols are recognized as naturally occurring antioxidants but also catalyze oxidative DNA degradation of cellular DNA either alone or in the presence of transition metal ions such as copper. In this paper we show that similar to various other classes of polyphenols, delphinidin is also capable of causing oxidative degradation of cellular DNA. Lymphocytes were exposed to various concentrations of delphinidin (10, 20, 50 microM) for 1h and the DNA breakage was assessed using single cell alkaline gel electrophoresis (Comet assay). Inhibition of DNA breakage by several scavengers of reactive oxygen species (ROS) indicated that it is caused by the formation of ROS. Incubation of lymphocytes with neocuproine (a cell membrane permeable Cu(I) chelator) inhibited DNA degradation in intact lymphocytes in a dose dependent manner. Bathocuproine, which is unable to permeate through the cell membrane, did not cause such inhibition. We have further shown that delphinidin is able to degrade DNA in cell nuclei and that such DNA degradation is also inhibited by neocuproine suggesting that nuclear copper is mobilized in this reaction. These results indicate that the generation of ROS possibly occurs through mobilization of endogenous copper ions. The results are in support of our hypothesis that the prooxidant activity of plant polyphenols may be an important mechanism for their anticancer properties.

Oxidative breakage of cellular DNA by plant polyphenols: a putative mechanism for anticancer properties.

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. We have shown that several of these compounds are able to bind both DNA and Cu(II) forming a ternary complex. A redox reaction of the polyphenols and Cu(II) in the ternary complex may occur leading to the reduction of Cu(II) to Cu(I), whose reoxidation generates a variety of reactive oxygen species (ROS). We have further confirmed that the polyphenol-Cu(II) system is indeed capable of causing DNA degradation in cells such as lymphocytes. We have also shown that polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Neocuproine (a Cu(I) sequestering agent) inhibits such DNA degradation. It also inhibits the oxidative stress generated in lymphocytes indicating that the cellular DNA breakage involves the generation of Cu(I) and formation of ROS. It is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to electron transfer between copper ions and polyphenols to generate ROS. Thus, our results are in support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper possibly chromatin bound copper and the consequent prooxidant action.

Ascorbic acid mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties.

Several decades back ascorbic acid was proposed as an effective anticancer agent. However, this idea remained controversial and the mechanism of action unclear. In this paper, we show that ascorbic acid at a concentration reported to be achievable through high doses of oral consumption is capable of cytotoxic action against normal cells. Several antioxidants of both animal as well as plant origin including ascorbic acid also possess prooxidant properties. Copper is an essential component of chromatin and can take part in redox reactions. Previously we have proposed a mechanism for the cytotoxic action of plant antioxidants against cancer cells that involves mobilization of endogenous copper ions and the consequent generation of reactive oxygen species. Using human peripheral lymphocytes and Comet assay we show here that ascorbic acid is able to cause oxidative DNA breakage in normal cells at a concentration of 100-200 microM. Neocuproine, a Cu(I) specific sequestering agent inhibited DNA breakage in a dose dependent manner indicating that Cu(I) is an intermediate in the DNA cleavage reaction. The results are in support of our above hypothesis that involves events that lead to a prooxidant action by antioxidants. The results would support the idea that even a plasma concentration of around 200 microM. would be sufficient to cause pharmacological tumor cell death particularly when copper levels are elevated. This would account for the observation of several decades back by Pauling and co-workers where oral doses of ascorbic acid in gram quantities were found to be effective in treating some cancers.

Plant polyphenols mobilize endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for anticancer properties.

Plant polyphenols are important components of human diet and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring antioxidants but also act as prooxidants catalyzing DNA degradation in the presence of transition metal ions such as copper. Using human peripheral lymphocytes and Comet assay we have previously confirmed that resveratrol-Cu(II) is indeed capable of causing DNA degradation in cells. In this paper we show that the polyphenols alone (in the absence of added copper) are also capable of causing DNA breakage in cells. Incubation of lymphocytes with neocuproine inhibited the DNA degradation confirming that Cu(I) is an intermediate in the DNA cleavage reaction. Further, we have also shown that polyphenols generate oxidative stress in lymphocytes which is inhibited by scavengers of reactive oxygen species and neocuproine. These results are in further support of our hypothesis that anticancer mechanism of plant polyphenols involves mobilization of endogenous copper, possibly chromatin bound copper, and the consequent prooxidant action.