Recent Contributions of Mass Spectrometry-Based "Omics" in the Studies of Breast Cancer.

Breast cancer (BC) is one of the most heterogeneous groups of cancer. As every biotype of BC is unique and presents a particular "omic" signature, they are increasingly characterized nowadays with novel mass spectrometry (MS) strategies. BC therapeutic approaches are primarily based on the two features of human epidermal growth factor receptor 2 (HER2) and estrogen receptor (ER) positivity. Various strategic MS implementations are reported in studies of BC also involving data independent acquisitions (DIAs) of MS which report novel differential proteomic, lipidomic, proteogenomic, phosphoproteomic, and metabolomic characterizations associated with the disease and its therapeutics. Recently many "omic" studies have aimed to identify distinct subsidiary biotypes for diagnosis, prognosis, and targets of treatment. Along with these, drug-induced-resistance phenotypes are characterized by "omic" changes. These identifying aspects of the disease may influence treatment outcomes in the near future. Drug quantifications and characterizations are also done regularly and have implications in therapeutic monitoring and in drug efficacy assessments. We report these studies, mentioning their implications toward the understanding of BC. We briefly provide the MS instrumentation principles that are adopted in such studies as an overview with a brief outlook on DIA-MS strategies. In all of these, we have chosen a model cancer for its revelations through MS-based "omics".

Exploring staphylococcal superantigens to design a potential multi-epitope vaccine against Staphylococcus aureus: an in-silico reverse vaccinology approach.

Staphylococcus aureus is a horrifying bacteria capable of causing millions of deaths yearly across the globe. A major contribution to the success of S. aureus as an ESKAPE pathogen is the abundance of virulence factors that can manipulate the innate and adaptive immune system of the individual. Currently, no vaccine is available to treat S. aureus-mediated infections. In this study, we present in-silico approaches to design a stable, safe and immunogenic vaccine that could help to control the infections associated with the bacteria. Three vital pathogenic secreted toxins of S. aureus, such as staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), Toxic-shock syndrome toxin (TSST-1), were selected using the reverse vaccinology approach to design the multi-epitope vaccine (MEV). Linear B-lymphocyte, cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL) epitopes were predicted from these selected proteins. For designing the multi-epitope vaccine (MEV), B-cell epitopes were joined with the KK linker, CTL epitopes were joined with the AAY linker, and HTL epitopes were joined with the GPGPG linker. Finally, to increase the immune response to the vaccine, a human ß-defensin-3 (hBD-3) adjuvant was added to the N-terminus of the MEV construct. The final MEV was found to be antigenic and non-allergen in nature. In-silico immune simulation and cloning analysis predicted the immune-stimulating potential of the designed MEV construct along with the cloning feasibility in the pET28a(+) vector with the E. coli expression system. This immunoinformatics study provides a platform for designing a suitable, safe and effective vaccine against S. aureus.Communicated by Ramaswamy H. Sarma.

Can We Use mTOR Inhibitors for COVID-19 Therapy?

Infection by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes acute inflammation due to extensive replication of the virus in the epithelial cells of the upper and lower respiratory system. The mammalian target of rapamycin (mTOR) is a l signalling protein with critical functions in cell growth, metabolism, and proliferation. It is known for its regulatory functions in protein synthesis and angiogenesis cascades. The structure of mTOR consists of two distinct complexes (mTORC1 and mTORC2) with diverse functions at different levels of the signalling pathway. By activating mRNA translation, the mTORC1 plays a key role in regulating protein synthesis and cellular growth. On the other hand, the functions of mTORC2 are mainly associated with cell proliferation and survival. By using an appropriate inhibitor at the right time, mTOR modulation could provide immunosuppressive opportunities as antirejection regimens in organ transplantation as well as in the treatment of autoimmune diseases and solid tumours. The mTOR also has an important role in the inflammatory process. Inhibitors of mTOR might indeed be promising agents in the treatment of viral infections. They have further been successfully used in patients with severe influenza A/H1N1 pneumonia and acute respiratory failure. The officially accepted mTOR inhibitors that have undergone clinical testing are sirolimus, everolimus, temsirolimus, and tacrolimus. Thus, further studies on mTOR inhibitors for SARS-CoV-2 infection or COVID-19 therapy are well merited.

Potential Drug Strategies to Target Coronaviruses.

INTRODUCTION: As the world has witnessed three severe coronavirus outbreaks in the past two decades, including the recent pandemic COVID19, caused by SARS-CoV2, it has become of utmost importance to develop drugs and vaccines against coronaviruses. The previous two outbreaks, severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) emerged in China and Saudi Arabia in 2003 and 2012, respectively. COVID19 is considered the worst of all and has taken more than 4 million lives so far and crippled the socioeconomic life of human beings in the entire world. Extensive research is being carried out to find out a solution that will not only help us to fight the current situation but also prepare us to prevent further intervention by similar viruses in the future. Here, we aim to highlight potential drug target sites in coronavirus infection or life cycle in general. METHODS: We have gone through the research papers published on coronavirus, with special emphasis on SARS-CoV, MERS-CoV, and SARS-CoV2, in peer-reviewed journals and tried to identify the possible sites in the coronavirus life cycle which can be used as potential drug targets. RESULTS: Studies showed that there are several unique enzymes and mechanisms involved in the coronavirus life cycle which can be manipulated to develop drugs against it. However, it has been always a challenge to develop drugs or vaccines against viruses as they utilize the host cell machinery and more difficult against RNA viruses because of their high mutation rate. CONCLUSIONS: Effective control of the current (2020) pandemic necessarily depends on the development of either a vaccine or an effective therapeutic agent. In the past, many attempts were taken to develop vaccines after the outbreak of SARS-CoV and MERS-CoV, though no successful vaccine reached to the market as the situation came under control. In the current scenario, many laboratories have developed effective vaccines against SARS-CoV2, which have reduced both the severity of the infection and the rate of mortality considerably. However, world needs to be prepared for similar viral outbreaks in future and research must be continued to develop more effective vaccines and therapeutics against coronaviruses.

Possible Targets and Therapies of SARS-CoV-2 Infection.

The global spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that causes COVID-19 has become a source of grave medical and socioeconomic concern to human society. Since its first appearance in the Wuhan region of China in December 2019, the most effective measures of managing the spread of SARS-CoV-2 infection have been social distancing and lockdown of human activity; the level of which has not been seen in our generations. Effective control of the viral infection and COVID-19 will ultimately depend on the development of either a vaccine or therapeutic agents. This article highlights the progresses made so far in these strategies by assessing key targets associated with the viral replication cycle. The key viral proteins and enzymes that could be targeted by new and repurposed drugs are discussed.

Should We Try SARS-CoV-2 Helicase Inhibitors for COVID-19 Therapy?

The discovery of new drugs for treating the new coronavirus (SARS-CoV-2) or repurposing those already in use for other viral infections is possible through understanding of the viral replication cycle and pathogenicity. This article highlights the advantage of targeting one of the non-structural proteins, helicase (nsp13), over other SARS-CoV-2 proteins. Highlighting the experience gained from targeting Nsp13 in similar coronaviruses (SARS-CoV and MERS) and known inhibitors, the article calls for research on helicase inhibitors as potential COVID-19 therapy.

Cajanus indicus leaf protein: Beneficial role in experimental organ pathophysiology. A review.

The herb, Cajanus indicus L, has been and is popular for its medicinal value in India and other countries for long. The herb is mainly cultivated for the seeds which are used as pulses and are rich in proteins. People of rural India and some neighboring countries use the aqueous extract of the leaves of the herb against poor liver function and recently it has been found that the extract is not only useful against liver damage but also beneficial for renal failure and a number of other pathophysiological conditions. Intraperitoneal administration of the aqueous protein fraction of the leaves has shown hepatoprotective activity in mice. The protein fraction revealed the presence of a 43kDa protein having antioxidant and other protective properties in organ pathophysiology. The purified protein, CI-protein, scavenges free radicals generated by different free radical inducers and helps providing cytoprotection. Amino acid sequence of CI-protein has some structural similarity with plastocyanin, an electron carrier protein in photosynthesis. The protein has also been found to be active against a number of organ dysfunction inducer chemicals and drugs, like carbon tetrachloride, thioacetamide and acetaminophen. Signal transduction studies suggest that CI-protein exerts its protective action by free radical scavenging and antioxidative properties; it activates NF-κB and Akt without any involvement of ERK1/ERK2 and STAT-3 in acetaminophen induced hepatic pathophysiology. Besides, it reduces both drug and toxin induced cytotoxicity by decreasing the formation and/or scavenging of free radicals involving cytochrome P450, taking part in detoxification of xenobiotics.

Attenuation of Acetaminophen-Induced Hepatotoxicity In Vivo and In Vitro by a 43-kD Protein Isolated from the Herb Cajanus indicus L.

ABSTRACT The aim of this study was to evaluate the hepatoprotective role of a 43-kD protein (Hp-P) isolated from the leaves of Cajanus indicus L. against acetaminophen (APAP)-induced toxicity in mouse liver and in isolated hepatocytes. The hepatotoxicity of APAP and the hepatoprotective activity of Hp-P in vivo were determined by measuring the liver-specific serum marker enzymes alanine amino transferase (ALT) and alkaline phosphatase (ALP) in murine sera and observing the histological changes in the mice liver treated with the protein before and after (2 mg/kg body weight for 5 days) APAP (at a dose of 300 mg/kg body weight for 2 days) administration. The cell viability, LDH leakage, GSH level, and lipid peroxidation were measured in isolated hepatocytes to evaluate the cytotoxic effect of APAP and the protective role of Hp-P in vitro. Experimental results showed that APAP induced hepatotoxicity in vivo as revealed from the changes in serum-specific marker enzyme levels and histology of liver. It also induced cytotoxicity in hepatocytes as observed from the changes in cell viability and LDH leakage. Pretreatment with Hp-P prevented the APAP-induced elevation of ALT and ALP in murine sera. In addition, posttreatment with Hp-P significantly altered most of the changes induced by APAP. Although some natural recovery has been observed in toxin controls, the Hp-P-induced recovering process is more rapid than the natural ones. In histological studies, less centrilobular necrosis was found in the liver treated with Hp-P before and after APAP intoxication compared to the liver treated with APAP alone. Radical scavenging experiment showed that Hp-P scavenges DPPH radicals directly. Studies also showed that APAP-induced reduced cell viability and cellular LDH leakage could be prevented by the combinatorial effect of Hp-P. Besides, treatment of hepatocytes with Hp-P and APAP together maintained the normal GSH level. APAP-induced enhanced lipid peroxidation was also decreased when cells were treated with APAP and Hp-P together. Hp-P alone, on the other hand, did not induce any alterations of the studied parameters. Results of this study have been compared with a known antioxidant, alpha-tocopherol. Data from both the in vivo (before and after APAP administration) and in vitro studies suggest that Hp-P has potent hepato- and cytoprotective properties against APAP-induced toxicity.

Purification and characterization of a 43 kD hepatoprotective protein from the herb Cajanus indicus L.

Cajanus indicus L, a herb, is popularly known for its hepatoprotective activity. Aqueous extract of the leaves of this plant contains hepatoprotective and hepatostimulatory molecule(s). Present study was aimed to isolate, purify and characterize the active principle(s) responsible for that activity. A hepatoprotective protein molecule has been purified to homogeneity (approximately 300 fold). Homogeneous preparation of the protein was achieved by homogenization, (NH(4))(2)SO(4) precipitation, ion-exchange chromatography, gel filtration and high performance liquid chromatography. The protein purified is composed of a single polypeptide chain having an apparent molecular mass of 43 kD as determined by SDS-PAGE and gel filtration through sephadex G-75 column. The isoelectric point of the protein determined was 4.8. Loss of biological activity after heat and protease treatment confirmed that the active molecule is a protein. Peptide fragments of the protein generated by trypsin cleavage were subjected to MALDI-TOF as well as LC-MS analyses and among the various fragments, four were very prominent and used for the determination of the amino acid sequence of the hepatoprotective protein. While one of the peptide fragment revealed strong sequence homology with plastocyanin, another fragment showed some similarity with a tomato protein present in the NCBI non-redundant database. The third peptide, on the other hand, is unique as it did not show any sequence homology with any known protein in the database. The protein showed maximum hepatoprotective activity when administered at a dose of 2 mg/kg body weight for five days after CCl(4 )administration. Histopathological studies also supported the hepatoprotective nature of the protein. Along with its curative property, the protein also possesses preventive role against a number of toxin induced hepatic damages.

Protective effect of a 43 kD protein from the leaves of the herb, Cajanus indicus L on chloroform induced hepatic-disorder.

Cajanus indicus is a herb with medicinal properties and is traditionally used to treat various forms of liver disorders. Present study aimed to evaluate the effect of a 43 kD protein isolated from the leaves of this herb against chloroform induced hepatotoxicity. Male albino mice were intraperitoneally treated with 2 mg/kg body weight of the protein for 5 days followed by oral application of chloroform (0.75 ml/kg body weight) for 2 days. Different biochemical parameters related to physiology and pathophysiology of liver, such as, serum glutamate pyruvate transaminase and alkaline phosphatase were determined in the murine sera under various experimental conditions. Direct antioxidant role of the protein was also determined from its reaction with Diphenyl picryl hydroxyl radical, superoxide radical and hydrogen peroxide. To find out the mode of action of this protein against chloroform induced liver damage, levels of antioxidant enzymes catalase, superoxide dismutase and glutathione-S-transferase were measured from liver homogenates. Peroxidation of membrane lipids both in vivo and in vitro were also measured as malonaldialdehyde. Finally, histopathological analyses were done from liver sections of control, toxin treated and protein pre- and post-treated (along with the toxin) mice. Levels of serum glutamate pyruvate transaminase and alkaline phosphatase, which showed an elevation in chloroform induced hepatic damage, were brought down near to the normal levels with the protein pretreatment. On the contrary, the levels of antioxidant enzymes such as catalase, superoxide dismutase and glutathione-S-transferase that had gone down in mice orally fed with chloroform were significantly elevated in protein pretreated ones. Besides, chloroform induced lipid peroxidation was effectively reduced by protein treatment both in vivo and in vitro. In cell free system the protein effectively quenched diphenyl picryl hydroxyl radical and superoxide radical, though it could not catalyse the breakdown of hydrogen peroxide. Post treatment with the protein for 3 days after 2 days of chloroform administration showed similar results. Histopathological studies indicated that chloroform induced extensive tissue damage was less severe in the mice livers treated with the 43 kD protein prior and post to the toxin administration. Results from all these data suggest that the protein possesses both preventive and curative role against chloroform induced hepatotoxicity and probably acts by an anti-oxidative defense mechanism.

Herbal (Phyllanthus niruri) protein isolate protects liver from nimesulide induced oxidative stress.

Present study was conducted to evaluate the role of a protein fraction (PI, protein isolate) of the herb, Phyllanthus niruri (P. niruri) against nimesulide-induced oxidative stress in vivo using a murine model. Mice were intraperitoneally treated with that at a dose of 5mg/kg body weight for 7 days before and separately 1-5 days after nimesulide (at a dose of 10mg/kg body weight for 7 days) administration to evaluate its preventive and curative role. Levels of reduced glutathione (GSH), antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), as well as thiobarbituric acid reactive substances (TBARS) were measured in the liver homogenates of all study groups. Pretreatment with isolated P. niruri protein fraction significantly enhanced nimesulide-induced reduced levels of antioxidant enzymes and GSH as well as reduced the enhanced level of lipid peroxidation. Post-treatment studies showed that the recovery after nimesulide induced oxidative stress was more rapid if PI was administered compared to the spontaneous recovery of liver. Histological studies also suggest that this protein fraction could prevent as well as cure liver from nimesulide induced oxidative stress. DPPH radical scavenging assay showed that it could scavenge free radicals. Its antioxidant property was compared with that of a known potent antioxidant, Vitamin E. Besides, the effect of a non-relevant protein, BSA, was also included in the study. Heat treatment and trypsin digestion destroyed the biological activity of this protein fraction. In conclusion, data obtained suggest that the P. niruri protein fraction may protect liver from nimesulide-induced oxidative stress probably via promotion of antioxidant defense.

A 43 kDa protein from the herb Cajanus indicus L. protects thioacetamide induced cytotoxicity in hepatocytes.

The aim of this study was to investigate the role of a hepatoprotective protein isolated from the herb Cajanus indicus L. on thioacetamide (TAA) induced toxicity in isolated mouse hepatocytes. In vitro cell viability, lactate dehydrogenase (LDH), alanine aminotransferase (ALT) and total protein leakage were measured as the indicator of cell damage. The amount of glutathione (GSH) and lipid peroxidation were also measured to determine the oxidative status of the cells. The reduced cell viability in TAA treated hepatocytes was almost completely recovered upon protein treatment. LDH, ALT and total protein secretion outside the cells after TAA treatment confirmed the cell membrane damage. Incubation of hepatocytes with the protein prior to TAA administration significantly prevented the cell membrane damage as revealed from less LDH, ALT and total protein leakage. TAA depleted endogenous antioxidant GSH and increased membrane lipid peroxidation in hepatocytes. The protein had very prominent effect in altering the GSH level and lipid peroxidation. The protein exhibited all these cytoprotective effects in a dose-dependent manner. Besides, measurement of DPPH radical scavenging activity showed that the protein could scavenge free radicals. In addition, the protein resisted TAA induced alterations of various effects when applied in combination with TAA. The cytoprotective activity of the protein was found to be comparable with alpha-tocopherol, a well-known antioxidant. Results suggest that the protein from C. indicus can act as a hepatoprotector and primary antioxidant against TAA-induced cytotoxicity in mouse hepatocytes.

Preventive and curative role of a 43kD protein from the leaves of the herb Cajanus indicus L on thioacetamide-induced hepatotoxicity in vivo.

An approximately 43kD protein has been isolated and purified from the herb Cajanus indicus L and believed to be the most active principle for its hepatoprotective action. In this study, experiments have been performed to evaluate the effectiveness of that protein for the preventive and curative action against thioacetamide-induced toxicity in vivo using a murine model. Mice were treated with the protein intraperitoneally at a dose of 2mg/kg body weight for 2 and 6 days before and separately 1-5 days after thioacetamide administration to evaluate its preventive and curative role, respectively. Thioacetamide was administered once at a dose of 150mg/kg body weight and after 48h of its application, the animals were sacrificed. Levels of various markers related to physiological and pathological conditions of the liver, e.g., glutamate pyruvate transaminase (GPT), alkaline phosphatase (ALP), etc. were determined in the murine sera under different experimental conditions. In addition, antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase (SOD) and catalase (CAT) as well as thiobarbituric acid reactive substances (TBARS), were measured from the liver homogenates. The antioxidant property of the protein was compared with the potent antioxidant, vitamin E (used as a positive control). The active principle effectively reduced the elevated GPT and ALP levels in serum and lipid peroxidation in the liver tissue. The reduced levels of SOD, CAT and GST by thioacetamide were again brought back to almost normal levels upon pre- and post-treatment with the protein. Histopathological changes in the liver of TAA control and protein-treated groups also prove that the protein possesses hepatoprotective activity. The protein acts dose-dependently and maximum hepatoprotectivity was obtained when administered at a dose of 2mg/kg body weight. Data suggest that the active principle plays an important preventive and curative role against thioacetamide-induced hepatotoxicity.