The Promises of Proteomics and Metabolomics for Unravelling the Mechanism and Side Effect Landscape of Beta-Adrenoceptor Antagonists in Cardiovascular Therapeutics.

Cardiovascular medicine witnessed notable advances for the past decade. Multiomics research offers a new lens for precision/personalized medicine for existing and emerging drugs used in the cardiovascular clinic. Beta-blockers are vital in treating hypertension and chronic heart failure. However, clinical use of beta-blockers is also associated with side effects and person-to-person variations in their pharmacokinetics and pharmacodynamics. A comprehensive understanding of the mechanisms that underpin the side effect landscape of beta-blockers is imperative to optimize their therapeutic value. In addition, current research emphasizes the circadian clock's vital roles in regulating pharmacological parameters. Administration of the beta-blockers at specific dosing times could potentially improve their effectiveness and reduce their toxic effects. The rapid development of mass spectrometry technologies with chemical proteomics and thermal proteome profiling methods has also substantially advanced our understanding of underlying side effects mechanisms by unbiased deconvolution of drug targets and off-targets. Metabolomics is steadily demonstrating its utility for conducting mechanistic and toxicological analyses of pharmacological compounds. This article discusses the promises of cutting-edge proteomics and metabolomics approaches to investigate the molecular targets, mechanism of action, adverse effects, and dosing time dependency of beta-blockers.

X-linked hyper-immunoglobulin M syndrome harboring a novel CD40-ligand gene mutation: a case report.

The X-linked hyper-IgM syndrome (X-HIGM1) is a rare primary immunodeficiency disorder (PID) caused by mutations in the gene encoding the CD154 protein, also known as CD40 ligand (CD40LG). X-HIGM1 is characterized by normal or elevated serum levels of IgM in association with decreased levels of IgG, IgA, and IgE. The CD40LG protein expressed on activated T cells interacts with its receptor protein, CD40, on B lymphocytes and dendritic cells. Mutations in the CD40LG gene lead to the production of an abnormal CD40L protein that fails to attach to its receptor, CD40 on B cells resulting in failure to produce IgG, IgA, and IgE antibodies. In the present study, we investigated the molecular defects underlying such a PID in a patient presenting with clinical history of pneumonia and acute respiratory distress syndrome (ARDS) at 7 months of age and diagnosed as transient hypogammaglobulinemia with decreased levels of IgG and increased levels of IgM. We have identified a novel and yet to be reported frame shift deletion of a single base pair (c.229delA) in exon 2 (p.Arg77AspfsTer6) of the CD40L gene ensuing the premature truncation of the protein by 6 amino acids by targeted gene sequencing. This frame shift mutation identified as a CD40L variant was found to be pathogenic which was also validated by Sanger sequencing. The in-silico analysis of c.229 del A mutation also predicted the change to be pathological affecting the structure and function of the CD40L (CD40L, CD154) protein and its protein-protein interaction properties.

Liquid Biopsy, the hype vs. hope in molecular and clinical oncology.

The molecular landscape of tumors has been traditionally established using a biopsy or resection specimens. These modalities result in sampling bias that offer only a single snapshot of tumor heterogeneity. Over the last decade intensive research towards alleviating such a bias and obtaining an integral yet accurate portrait of the tumors, evolved to the use of established molecular and genetic analysis using blood and several other body fluids, such as urine, saliva, and pleural effusions as liquid biopsies. Genomic profiling of the circulating markers including circulating cell-free tumor DNA (ctDNA), circulating tumor cells (CTCs) or even RNA, proteins, and lipids constituting exosomes, have facilitated the diligent monitoring of response to treatment, allowed one to follow the emergence of drug resistance, and enumerate minimal residual disease. The prevalence of tumor educated platelets (TEPs) and our understanding of how tumor cells influence platelets are beginning to unearth TEPs as a potentially dynamic component of liquid biopsies. Here, we review the biology, methodology, approaches, and clinical applications of biomarkers used to assess liquid biopsies. The current review addresses recent technological advances and different forms of liquid biopsy along with upcoming challenges and how they can be integrated to get the best possible tumor-derived genetic information that can be leveraged to more precise therapies for patient as liquid biopsies become increasingly routine in clinical practice.

A new functional role of mitochondria-anchored protein ligase in peroxisome morphology in mammalian cells.

Mitochondria and peroxisomes are metabolically interconnected and functionally active subcellular organelles. These two dynamic organelles, share a number of common biochemical functions such as ß-oxidation of fatty acids and detoxification of peroxides. The biogenesis and morphology of both these organelles in the mammalian cells is controlled by common transcription factors like PGC1α, and by a common fission machinery comprising of fission proteins like DRP1, Mff, and hFis1, respectively. In addition, the outer membrane mitochondria-anchored protein ligase (MAPL), the first mitochondrial SUMO E3 ligase with a RING-finger domain, also regulates mitochondrial morphology inducing mitochondrial fragmentation upon its overexpression. This fragmentation is dependent on both the RING domain of MAPL and the presence of the mitochondrial fission GTPase dynamin-related protein-1 (DRP1). Earlier studies have demonstrated that mitochondrial-derived vesicles are formed independently of the known mitochondrial fission GTPase, DRP1 are enriched for MAPL and are targeted to peroxisomes. The current study shows that MAPL regulates morphology of peroxisomes in a cell-type specific manner. Fascinatingly, the peroxisome elongation caused either due to silencing of DRP1 or by addition of polyunsaturated fatty acid, docosahexaenoic acid was blocked by overexpressing MAPL in mammalian cell lines. Furthermore, the transfection and colocalisation studies of MAPL with peroxisome membrane marker, PMP70, in different cell lines clearly revealed a cell-type specificity of transport of MAPL to peroxisomes. Previous work has placed the Vps35 (retromer component) as vital for delivery of MAPL to peroxisomes, placing the retromer as critical for the formation of MAPL-positive mitochondrial-derived vesicles. The results of polyethylene glycol-based cell-cell fusion assay signified that the enrichment of MAPL in peroxisomes is through vesicles and a retromer dependent phenomenon. Thus, a novel function for MAPL in peroxisomes is established to regulate peroxisome elongation and morphology under growth conditions and thus possibly modulate peroxisome fission.

COVID-19 and cancer: Sailing through the tides.

The COVID-19 (coronavirus disease) pandemic caused by SARS-CoV-2 with its rapid expansion has led to extraordinary implications in our understanding of viral infections and their management globally. In this current scenario of unusual circumstances and public health emergency, the cancer care per se is facing unprecedented challenges. The peculiarity of the SARS-CoV-2 infections is still being uncovered as the pandemic spreads across the populations than showing signs of its curtailment. The review highlights the significance of idiosyncrasy of the SARS-Cov-2 infection especially putting forth the importance of immunosenescence, both in the COVID-19 specific immune response in the infected lungs of the elderly and in the cancer patients infected with SARS-CoV-2.The focus of the article is directed towards demystifying the unparalleled essence of a proprotein convertase, Furin in the biology of the SARS-Cov-2 infection and its role in facilitating viral transmission through expedited cellular entry into alveolar epithelial cells in COVID-19 infected cancer patients. The risk stratification of the cancer treatment and guidelines shaped up by national and international oncology societies in providing uncompromised patient care during the COVID-19 crisis have also been addressed. The global efforts towards vaccination in developing SARS CoV-2 immunity are also discussed in this article.

Immune checkpoints and their inhibitors: Reappraisal of a novel diagnostic and therapeutic dimension in the urologic malignancies.

Advances in molecular immunology have unveiled some of the complexity of the mechanisms regulating cellular immune responses and led to the successful targeting of immune checkpoints in attempts to enhance antitumor T cell responses. Surgery, chemotherapy, and radiation therapy have been the mainstay of treatment in urologic malignancies. Immune checkpoint molecules such as cytotoxic T-lymphocyte associated protein-4, programmed cell death protein-1, and programmed death-ligand 1 have been shown to play central roles in evading cancer immunity. Thus these molecules have been targeted by inhibitors for the management of cancers forming the basis of immunotherapy. Immunotherapy is now among the first line therapeutic options for metastatic renal cell carcinomas. In advanced bladder cancer, immunotherapy is the standard of care in the second line and the first line for cisplatin ineligible patients. There continues to be ongoing research to identify the role if any of immunotherapy in testicular, prostatic, and penile cancers. The ideal biomarker for response to immunotherapy is still elusive. Although programmed death-ligand 1 immunohistochemical testing has been widely used across the globe as a biomarker for immunotherapy, companion diagnostic tests have inherent issues with testing and reporting and cannot have universal applicability. Additional biomarkers including, tumor mutational burden, deficient mismatch repair, high microsatellite instability, and immune gene expression profiling are being evaluated in various clinical trials. This review appraises the data of immunotherapy in the management of urologic malignancies.

Gut microbiota and Covid-19- possible link and implications.

Covid-19 is a major pandemic facing the world today caused by SARS-CoV-2 which has implications on our understanding of infectious diseases. Although, SARS-Cov-2 primarily causes lung infection through binding of ACE2 receptors present on the alveolar epithelial cells, yet it was recently reported that SARS-CoV-2 RNA was found in the faeces of infected patients. Interestingly, the intestinal epithelial cells particularly the enterocytes of the small intestine also express ACE2 receptors. Role of the gut microbiota in influencing lung diseases has been well articulated. It is also known that respiratory virus infection causes perturbations in the gut microbiota. Diet, environmental factors and genetics play an important role in shaping gut microbiota which can influence immunity. Gut microbiota diversity is decreased in old age and Covid-19 has been mainly fatal in elderly patients which again points to the role the gut microbiota may play in this disease. Improving gut microbiota profile by personalized nutrition and supplementation known to improve immunity can be one of the prophylactic ways by which the impact of this disease can be minimized in old people and immune-compromised patients. More trials may be initiated to see the effect of co-supplementation of personalized functional food including prebiotics/probiotics along with current therapies.

DNA damage response and repair pathway modulation by non-histone protein methylation: implications in neurodegeneration.

Protein post-translational modifications (PTMs) have emerged to be combinatorial, essential mechanisms used by eukaryotic cells to regulate local chromatin structure, diversify and extend their protein functions and dynamically coordinate complex intracellular signalling processes. Most common types of PTMs include enzymatic addition of small chemical groups resulting in phosphorylation, glycosylation, poly(ADP-ribosyl)ation, nitrosylation, methylation, acetylation or covalent attachment of complete proteins such as ubiquitin and SUMO. Protein arginine methyltransferases (PRMTs) and protein lysine methyltransferases (PKMTs) enzymes catalyse the methylation of arginine and lysine residues in target proteins, respectively. Rapid progress in quantitative proteomic analysis and functional assays have not only documented the methylation of histone proteins post-translationally but also identified their occurrence in non-histone proteins which dynamically regulate a plethora of cellular functions including DNA damage response and repair. Emerging advances have now revealed the role of both histone and non-histone methylations in the regulating the DNA damage response (DDR) proteins, thereby modulating the DNA repair pathways both in proliferating and post-mitotic neuronal cells. Defects in many cellular DNA repair processes have been found primarily manifested in neuronal tissues. Moreover, fine tuning of the dynamicity of methylation of non-histone proteins as well as the perturbations in this dynamic methylation processes have recently been implicated in neuronal genomic stability maintenance. Considering the impact of methylation on chromatin associated pathways, in this review we attempt to link the evidences in non-histone protein methylation and DDR with neurodegenerative research.

KMT1 family methyltransferases regulate heterochromatin-nuclear periphery tethering via histone and non-histone protein methylation.

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

Mitochondria: the indispensable players in innate immunity and guardians of the inflammatory response.

Mitochondria, the dynamic organelles and power house of eukaryotic cells function as metabolic hubs of cells undergoing continuous cycles of fusion and fission. Recent findings have made it increasingly apparent that mitochondria essentially involved in energy production have evolved as principal intracellular signaling platforms regulating not only innate immunity but also inflammatory responses. Perturbations in mitochondrial dynamics, including fusion/fission, electron transport chain (ETC) architecture and cristae organization have now been actively correlated to modulate metabolic activity and immune function of innate and adaptive immune cells. Several newly identified mitochondrial proteins in mitochondrial outer membrane such as mitochondrial antiviral signaling protein (MAVS) and with mitochondrial DNA acting as danger-associated molecular pattern (DAMP) and mitochondrial ROS generated from mitochondrial sources have potentially established mitochondria as key signaling platforms in antiviral immunity in vertebrates and thereby orchestrating adaptive immune cell activations respectively. A thorough understanding of emerging and intervening role of mitochondria in toll-like receptor-mediated innate immune responses and NLRP3 inflammasome complex activation has gained lucidity in recent years that advocates the imposing functions of mitochondria in innate immunity. Fascinatingly, also how the signals stemming from the endoplasmic reticulum co-operate with the mitochondria to activate the NLRP3 inflammasome is now looked ahead as a stage to unravel as to how different mitochondrial and associated organelle stress responses co-operate to bring about inflammatory consequences. This has also opened avenues of research for revealing mitochondrial targets that could be exploited for development of novel therapeutics to treat various infectious, inflammatory, and autoimmune disorders. Thus, this review explores our current understanding of intricate interplay between mitochondria and other cellular processes like autophagy in controlling mitochondrial homeostasis and regulation of innate immunity and inflammatory responses.

Role of Akirin1 in the regulation of skeletal muscle fiber-type switch.

Akirin1 is a highly conserved ubiquitously expressed nuclear protein. Owing to its strong nuclear localization signal and protein-protein interaction properties, Akirin1 has been speculated to regulate transcription of target genes as a cofactor. Previous studies have reported Akirin1 as a downstream target of myostatin, a potent negative regulator of myogenesis. Mice lacking myostatin displayed enhanced Akirin1 gene expression. Further, in vitro evidence has shown Akirin1 overexpression leads to hypertrophy in C2 C 12 myotubes. In this study, we used Akirin1 knockout mice as a model system to further elucidate the function of Akirin1 in fully differentiated skeletal muscle. Akirin1 knockout mice did not show any obvious phenotypic difference when compared with wild type. However, promoter-reporter assay suggested that Akirin1 regulated the transcription of muscle-specific RING finger 1 (MuRF-1), an important E3 ubiquitin ligase in skeletal muscle. Furthermore, ablation of Akirin1 resulted in increased type IIa and decreased type I muscle fibers, which was further supported by an increase in Myh2 and decrease in Myh7 gene expression. Also, histochemical studies for succinate dehydrogenase activity revealed a less oxidative muscle in the absence of Akirin1. Together, our study suggests a novel role of Akirin1 in maintaining the muscle fiber type and regulation of the metabolic activity of the skeletal muscle.

Immunohistochemical Identification of Muscle Fiber Types in Mice Tibialis Anterior Sections.

Mammalian skeletal muscle is a metabolically active tissue that is made up of different types of muscle fibers. These myofibers are made up of important contractile proteins that provide force during contraction of the muscle like actin and myosin. Murine myofibers have been classified into 4 types: Type I, Type IIa, Type IIb and Type IIX. Each muscle fiber has been identified with specific type of MyHC expressed, which in turn gives differential contractility to the muscle. There have been well-known methodologies to identify different myofibers: histochemical myosin ATPase staining which uses the differential ATPase activity between slow and fast fibers, quantification of metabolic enzymes like malate dehydrogenase and lactate dehydrogenase on specific fragments of muscle fibers. The drawback of these techniques is that they cannot differentiate the subtypes of myofibers, for example, Type IIa and Type IIb. They should be used in conjunction with other known histochemical staining techniques. Here, we devise a direct and robust immunohistochemical staining methodology that utilizes the differential expression of MyHC isoforms in different myofibers types, thus efficiently distinguishing the heterogeneity of the muscle fibers. We use antibodies that specifically recognize Type I, Type IIa and Type IIb fibers on serially cut frozen mouse tibialis anterior sections that can be quantified by ImageJ software.

Emerging roles of mitochondria in the evolution, biogenesis, and function of peroxisomes.

In the last century peroxisomes were thought to have an endosymbiotic origin. Along with mitochondria and chloroplasts, peroxisomes primarily regulate their numbers through the growth and division of pre-existing organelles, and they house specific machinery for protein import. These features were considered unique to endosymbiotic organelles, prompting the idea that peroxisomes were key cellular elements that helped facilitate the evolution of multicellular organisms. The functional similarities to mitochondria within mammalian systems expanded these ideas, as both organelles scavenge peroxide and reactive oxygen species, both organelles oxidize fatty acids, and at least in higher eukaryotes, the biogenesis of both organelles is controlled by common nuclear transcription factors of the PPAR family. Over the last decade it has been demonstrated that the fission machinery of both organelles is also shared, and that both organelles act as critical signaling platforms for innate immunity and other pathways. Taken together it is clear that the mitochondria and peroxisomes are functionally coupled, regulating cellular metabolism and signaling through a number of common mechanisms. However, recent work has focused primarily on the role of the ER in the biogenesis of peroxisomes, potentially overshadowing the critical importance of the mitochondria as a functional partner. In this review, we explore the mechanisms of functional coupling of the peroxisomes to the mitochondria/ER networks, providing some new perspectives on the potential contribution of the mitochondria to peroxisomal biogenesis.

Vps35 mediates vesicle transport between the mitochondria and peroxisomes.

Mitochondria-derived vesicles (MDVs) have been shown to transport cargo from the mitochondria to the peroxisomes. Mitochondria and peroxisomes share common functions in the oxidation of fatty acids and the reduction of damaging peroxides. Their biogenesis is also linked through both the activation of master transcription factors such as PGC-1alpha and the common use of fission machinery, including DRP1, Mff, and hFis1. We have previously shown that MDVs are formed independently of the known mitochondrial fission GTPase Drp1 and are enriched for a mitochondrial small ubiquitin-like modifier (SUMO) E3 ligase called MAPL (mitochondrial-anchored protein ligase). Here, we demonstrate that the retromer complex, a known component of vesicle transport from the endosome to the Golgi apparatus, regulates the transport of MAPL from mitochondria to peroxisomes. An unbiased screen shows that Vps35 and Vps26 are found in complex with MAPL, and confocal imaging reveals Vps35 recruitment to mitochondrial vesicles. Silencing of Vps35 or Vps26A leads to a significant reduction in the delivery of MAPL to peroxisomes, placing the retromer within a novel intracellular trafficking route and providing insight into the formation of MAPL-positive MDVs.