Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 μM) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ΔΨm dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions.     

Caste-Specific Expression of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase in the Asian Weaver Ant, <Emphasis Type="Italic">Oecophylla smaragdina</Emphasis> (Fabricius, 1775)

Social insect colonies adopt different levels of survival strategies and exhibit well-defined reproductive division of labour. Oecophylla smaragdina (Fabricius, 1775) has physiological and behavioral adaptations that enable them to forage at extreme environmental conditions and are lethal to most other insects. Ion homeostasis is the key process in an organism’s survival mechanism. Among ion pumps, the ATP-dependent sodium–potassium ion pump is essential for maintaining the Na⁺ and K⁺ ionic balance and is well known as the primary consumer of energy. Oecophylla smaragdina plays pivotal role as a model among social insects for understanding ion homeostasis at the organization level of the castes. We have evaluated the expression and activity of Na⁺/K⁺-ATPase among various castes of O. smaragdina (worker subcastes, queen and male). Real-time PCR and immunoblotting analyses revealed the differential expression of Na⁺/K⁺-ATPase in the castes. Significantly higher expression of Na⁺/K⁺-ATPase mRNA and protein were observed in the minor workers, queen, major workers and males respectively. These results suggest that in the weaver ant colony, the castes might have variously adapted and evolved with a well-developed ion transport mechanism which allows them to perform allocated tasks within the nest and could be a key to their adaptive benefits towards division of labour.     

Virus isolation and molecular epidemiology of highly pathogenic avian influenza A(H5N8) from an outbreak in free-ranging wild birds,India 2016

We report the molecular epidemiology of highly pathogenic avian influenza (HPAI) virus involved in an outbreak causing death in free-ranging wild birds at Mysore, Karnataka state of India. The virus was typed as HPAI A(H5N8) by conventional and TaqMan probe based real-time PCR assays. Six isolates of HPAI virus were recovered in 9-day-old embryonated chicken eggs. Haemagglutinin gene-based phylogeny of virus isolates showed >?99.9% nucleotide sequence identity with HPAI A(H5N8) isolates from migratory birds and domestic poultry from China and Korea indicating either these wild birds have routed their migration through Korea and/or eastern China or these dead birds must have directly or indirectly contacted with wild birds migrating from Eastern China and/or Korean regions. The study emphasises the role of migratory wild birds in spread of HPAI across the globe.     

All Trans Retinoic Acid Attenuates Markers of Neuroinflammation in Rat Brain by Modulation of SIRT1 and NFκB

Alcohol abuse affects several neurological pathways and causes significant alterations in the brain. Abstention from alcohol causes only a marginal decrease in oxidative stress and neuro inflammation. Our previous studies had shown that an active metabolite of vitamin A, all trans retinoic acid (ATRA), ameliorates alcohol induced toxicity. Hence in the present study we investigated whether ATRA regressed alcohol induced neuroinflammation. We focused on the role of silent mating type information regulation 2 homolog 1(SIRT1) and nuclear factor kappa-B (NFκB). Animals were administered with ethanol at a daily dose of (4 g/kg body weight) for 90 days. On the 91st day ethanol administration was stopped and animals were divided into ethanol abstention (A) and ATRA supplementation group (ATRA?+?A) (100 µg/kg body weight) and maintained for 30 days. Ethanol exposure increased markers of oxidative stress, inflammation and the activities of alcohol and acetaldehyde dehydrogenases and reduced the expression of SIRT1 in the whole brain.The ethanol induced altered expressions of NFκB and SIRT1 were modulated by supplementation of ATRA. Abstention also reduced toxicity, but to a lower extent in comparison with supplementation of ATRA. Our results seemed to suggest that ATRA regressed the mediators of ethanol induced neuroinflammation by reducing oxidative stress and by regulating the expression of NFκB and SIRT1. The ameliorative potential of ATRA was much higher than abstention.     

Characterization of a divergent non-classical MHC class I gene in sharks

Sharks are the most ancient group of vertebrates known to possess members of the major histocompatibility complex (MHC) gene family. For this reason, sharks provide a unique opportunity to gain insight into the evolution of the vertebrate immune system through comparative analysis. Two genes encoding proteins related to the MHC class I gene family were isolated from splenic cDNA derived from spiny dogfish shark ( Squalus acanthias). The genes have been designated MhcSqac-UAA*01 and MhcSqac-UAA*NC1. Comparative analysis demonstrates that the Sqac-UAA*01 protein sequence clusters with classical MHC class I of several shark species and has structural elements common to most classical MHC class I molecules. In contrast, Sqac-UAA*NC1 is highly divergent from all vertebrate classical MHC class I proteins, including the Sqac-UAA *01 sequence and those of other shark species. Although Sqac-UAA*NC1 is clearly related to the MHC class I gene family, no orthologous genes from other species were identified due to the high degree of sequence divergence. In fact, the Sqac NC1 protein sequence is the most divergent MHC class-I-like protein identified thus far in any shark species. This high degree of divergence is similar in magnitude to some of the MHC class-I-related genes found in mammals, such as MICA or CD1. These data support the existence of a class of highly divergent non-classical MHC class I genes in the most primitive vertebrates known to possess homologues of the MHC and other components of the adaptive immune system.     

Grb7 and Filamin‐a associate and are colocalized to cell membrane ruffles upon EGF stimulation

Grb7 is an adaptor molecule mediating signal transduction from multiple cell surface receptors to diverse downstream pathways. Grb7, along with Grb10 and Grb14, make up the Grb7 protein family. This protein family has been shown to be overexpressed in certain cancers and cancer cell lines. Grb7 and a receptor tyrosine kinase, ErbB2, are overexpressed in 20–30% of breast cancers. Grb7 overexpression has been linked to enhanced cell migration and metastasis, although the participants in these pathways have not been fully determined. In this study, we report the Grb7 protein interacts with Filamin‐a, an actin‐crosslinking component of the cell cytoskeleton. Additionally, we have demonstrated the interaction between Grb7 and Flna is specific to the RA‐PH domains of Grb7, and the immunoglobulin‐like repeat 16–19 domains of Flna. We demonstrate that full‐length Grb7 and Flna interact in the mammalian cellular environment, as well as in vitro. Immunofluorescent microscopy shows potential co‐localization of Grb7 and Flna in membrane ruffles upon epidermal growth factor stimulation. These studies are amongst the first to establish a clear connection between Grb7 signaling and cytoskeletal remodeling. Copyright © 2013 John Wiley & Sons, Ltd.     

A dietary colorant crocin mitigates arthritis and associated secondary complications by modulating cartilage deteriorating enzymes,inflammatory mediators and antioxidant status

Articular cartilage degeneration and inflammation are the hallmark of progressive arthritis and is the leading cause of disability in 10–15% of middle aged individuals across the world. Cartilage and synovium are mainly degraded by either enzymatic or non-enzymatic ways. Matrix metalloproteinases (MMPs), hyaluronidases (HAases) and aggrecanases are the enzymatic mediators and inflammatory cytokines and reactive oxygen species being non-enzymatic mediators. In addition, MMPs and HAases generated end-products act as inflammation inducers via CD44 and TLR-4 receptors involved NF-κB pathway. Although several drugs have been used to treat arthritis, numerous reports describe the side effects of these drugs that may turn fatal. On this account several medicinal plants and their isolated molecules have been involved in modern medicine strategies to fight against arthritis. In view of this, the present study investigated the antiarthritic potentiality of Crocin, a dietary colorant carotenoid isolated from stigma of Crocus sativus. Crocin effectively neutralized the augmented serum levels of enzymatic (MMP-13, MMP-3 and MMP-9 and HAases) and non-enzymatic (TNF-α, IL-1β, NF-κB, IL-6, COX-2, PGE₂ and ROS) inflammatory mediators. Further, Crocin re-established the arthritis altered antioxidant status of the system (GSH, SOD, CAT and GST). It also protected the bone resorption by inhibiting the elevated levels of bone joint exoglycosidases, cathepsin-D and tartrate resistant acid phosphatases. Taken together, Crocin revitalized the arthritis induced cartilage and bone deterioration along with inflammation and oxidative damage that could be accredited to its antioxidant nature. Thus, Crocin could be an effective antiarthritic agent which can equally nullify the arthritis associated secondary complication.     

Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells

Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell-activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.