Venomous snakebites: Rapid action saves lives—A multifaceted community education programme increases awareness about snakes and snakebites among the rural population of Tamil Nadu,India

The lack of public awareness surrounding the dangers of snakebite envenomation (SBE) is one of the most critical factors contributing to SBE-induced complications, and subsequently exacerbating the number of deaths and disabilities resulting from SBE. In this study, we deployed a multifaceted community education programme to educate students, healthcare professionals and members of the public in rural areas of Tamil Nadu, India about the dangers of SBE, appropriate first aid measures and the ‘do’s and don’ts’ following a snakebite. An assessment of prior knowledge within these communities identified several misconceptions concerning snakes and SBE. Using a combination of direct engagement (estimated to reach over 200,000 people), information leaflets (200,000 distributed), posters, video documentaries, media and social media (>2.8 million engagements), over the course of one year (January to December 2019) we reached over 3 million people in rural Tamil Nadu (around 8% of population). Evaluation of community-based assemblies indicated that at least 90% of attendees were able to recall the key messages at the end of the events, and at least 85% were able to recall the key messages even after 12 months. Due to high demand, a one-day symposium was organised to provide clinical knowledge and training on SBE to 250 healthcare professionals in rural Tamil Nadu. Notably, an assessment of patient data (291 victims) collected from a snakebite referral hospital over the same 12-month period (2019) indicated that arrival time at hospital following a snakebite was significantly faster and the effective first aid measures were administered to patients who were aware of our activities compared to those that were not. Overall, our approach provides a framework on how to educate rural communities about the dangers of SBE and thereby, mitigate delayed SBE treatment leading to an overall reduction in SBE-induced mortality, morbidity, treatment costs and other socio-economic ramifications.     

Attenuation of Immune-Mediated Influenza Pneumonia by Targeting the Inducible Co-Stimulator (ICOS) Molecule on T Cells

Inducible Co-stimulator (ICOS) plays a critical role in mediating T cell differentiation and function and is considered a key player in balancing T effector and T regulatory (T_reg) cell responses. Here we show that activation of the ICOS signalling pathway during acute influenza A virus (IAV) infection by application of an agonistic ICOS antibody reduced the frequency of CD8⁺ T cells in the respiratory tract of IAV infected animals and delayed pathogen elimination. In line with this, immune-mediated influenza pneumonia was significantly ameliorated in mice that received ICOS agonist as indicated by significantly reduced alveolar infiltrations and bronchointerstitial pneumonia, while at the same time virus-related pathology remained unaffected. Importantly, ICOS agonist treatment resulted in expansion of CD4⁺Foxp3⁺ T_regs in IAV infected mice, which was associated with elevated levels of the immunosuppressive cytokine IL-10 in the alveolar space. Together, our findings suggest a prominent role of ICOS signaling during acute IAV infection by increasing the T_reg/CD8⁺ T cell ratio with beneficial outcome on immune-mediated pneumonia and underline the suitability of ICOS as potential therapeutic target for immune intervention in those infectious conditions characterized by strong immunopathology rather than virus-mediated cytopathic effects.     

Cardiac Myosin Binding Protein C Phosphorylation Affects Cross-Bridge Cycle's Elementary Steps in a Site-Specific Manner

Based on our recent finding that cardiac myosin binding protein C (cMyBP-C) phosphorylation affects muscle contractility in a site-specific manner, we further studied the force per cross-bridge and the kinetic constants of the elementary steps in the six-state cross-bridge model in cMyBP-C mutated transgenic mice for better understanding of the influence of cMyBP-C phosphorylation on contractile functions. Papillary muscle fibres were dissected from cMyBP-C mutated mice of ADA (Ala273-Asp282-Ala302), DAD (Asp273-Ala282-Asp302), SAS (Ser273-Ala282-Ser302), and t/t (cMyBP-C null) genotypes, and the results were compared to transgenic mice expressing wide-type (WT) cMyBP-C. Sinusoidal analyses were performed with serial concentrations of ATP, phosphate (Pi), and ADP. Both t/t and DAD mutants significantly reduced active tension, force per cross-bridge, apparent rate constant (2πc), and the rate constant of cross-bridge detachment. In contrast to the weakened ATP binding and enhanced Pi and ADP release steps in t/t mice, DAD mice showed a decreased ADP release without affecting the ATP binding and the Pi release. ADA showed decreased ADP release, and slightly increased ATP binding and cross-bridge detachment steps, whereas SAS diminished the ATP binding step and accelerated the ADP release step. t/t has the broadest effects with changes in most elementary steps of the cross-bridge cycle, DAD mimics t/t to a large extent, and ADA and SAS predominantly affect the nucleotide binding steps. We conclude that the reduced tension production in DAD and t/t is the result of reduced force per cross-bridge, instead of the less number of strongly attached cross-bridges. We further conclude that cMyBP-C is an allosteric activator of myosin to increase cross-bridge force, and its phosphorylation status modulates the force, which is regulated by variety of protein kinases.     

Isolation and characterization of Pseudomonas sp. KUMS3 from Asian sea bass (Lates calcarifer) with fin rot

Bacteria were isolated from Asian Sea bass, Lates calcarifer kept in a farm, on the South-east coast of India. During an outbreak of fin rot, the affected fish had hemorrhages at the base of fins, mouth and skin muscles and faded pigments. Pure colonies were isolated on NA and ZMA from internal organs of the fish and the bacterial morphology was identified as gram-negative rod-shaped bacteria. Based on different biochemical tests and sequence of 16S rDNA, the causative bacteria were identified as Pseudomonas sp. KUMS3. Bacterial cells were isolated from liver and kidney of all artificially infected moribund fish and confirmed as Pseudomonas sp. KUMS3 by morphological and biochemical characteristics. During the experimental infection, the first incidence of dead fish was observed on 2nd day after exposure to Pseudomonas sp. KUMS3 and no fish died after 12 days post exposure and the cumulative percent of mortality was 70. Histological lesions were observed in the spleen, liver and kidney of the infected fish. Pseudomonas sp. KUMS3 could be considered as an opportunistic pathogen, which can survive on the fish surface or in water or in the gut and may cause disease when unfavorable conditions develop.     

Recent insights into oceanic dimethylsulfoniopropionate biosynthesis and catabolism

Dimethylsulfoniopropionate (DMSP), a globally important organosulfur compound is produced in prodigious amounts (2.0 Pg sulfur) annually in the marine environment by phytoplankton, macroalgae, heterotrophic bacteria, some corals and certain higher plants. It is an important marine osmolyte and a major precursor molecule for the production of climate-active volatile gas dimethyl sulfide (DMS). DMSP synthesis take place via three pathways: a transamination ‘pathway-’ in some marine bacteria and algae, a Met-methylation ‘pathway-’ in angiosperms and bacteria and a decarboxylation ‘pathway-’ in the dinoflagellate, Crypthecodinium. The enzymes DSYB and TpMMT are involved in the DMSP biosynthesis in eukaryotes while marine heterotrophic bacteria engage key enzymes such as DsyB and MmtN. Several marine bacterial communities import DMSP and degrade it via cleavage or demethylation pathways or oxidation pathway, thereby generating DMS, methanethiol, and dimethylsulfoxonium propionate, respectively. DMSP is cleaved through diverse DMSP lyase enzymes in bacteria and via Alma1 enzyme in phytoplankton. The demethylation pathway involves four different enzymes, namely DmdA, DmdB, DmdC and DmdD/AcuH. However, enzymes involved in the oxidation pathway have not been yet identified. We reviewed the recent advances on the synthesis and catabolism of DMSP and enzymes that are involved in these processes.     

Novel role for p90 ribosomal S6 kinase in the regulation of cardiac myofilament phosphorylation

In myocardium, the 90-kDa ribosomal S6 kinase (RSK) is activated by diverse stimuli and regulates the sarcolemmal Na(+)/H(+) exchanger through direct phosphorylation. Only limited information is available on other cardiac RSK substrates and functions. We evaluated cardiac myosin-binding protein C (cMyBP-C), a sarcomeric regulatory phosphoprotein, as a potential RSK substrate. In rat ventricular myocytes, RSK activation by endothelin 1 (ET1) increased cMyBP-C phosphorylation at Ser(282), which was inhibited by the selective RSK inhibitor D1870. Neither ET1 nor D1870 affected the phosphorylation status of Ser(273) or Ser(302), cMyBP-C residues additionally targeted by cAMP-dependent protein kinase (PKA). Complementary genetic gain- and loss-of-function experiments, through the adenoviral expression of wild-type or kinase-inactive RSK isoforms, confirmed RSK-mediated phosphorylation of cMyBP-C at Ser(282). Kinase assays utilizing as substrate wild-type or mutated (S273A, S282A, S302A) recombinant cMyBP-C fragments revealed direct and selective Ser(282) phosphorylation by RSK. Immunolabeling with a Ser(P)(282) antibody and confocal fluorescence microscopy showed RSK-mediated phosphorylation of cMyBP-C across the C-zones of sarcomeric A-bands. In chemically permeabilized mouse ventricular muscles, active RSK again induced selective Ser(282) phosphorylation in cMyBP-C, accompanied by significant reduction in Ca(2+) sensitivity of force development and significant acceleration of cross-bridge cycle kinetics, independently of troponin I phosphorylation at Ser(22)/Ser(23). The magnitudes of these RSK-induced changes were comparable with those induced by PKA, which phosphorylated cMyBP-C additionally at Ser(273) and Ser(302). We conclude that Ser(282) in cMyBP-C is a novel cardiac RSK substrate and its selective phosphorylation appears to regulate cardiac myofilament function.     

Roles for cardiac MyBP-C in maintaining myofilament lattice rigidity and prolonging myosin cross-bridge lifetime

We investigated the influence of cardiac myosin binding protein-C (cMyBP-C) and its constitutively unphosphorylated status on the radial and longitudinal stiffnesses of the myofilament lattice in chemically skinned myocardial strips of the following mouse models: nontransgenic (NTG), effective null for cMyBP-C (t/t), wild-type cMyBP-C expressed into t/t (WT_t/t), and constitutively unphosphorylated cMyBP-C (AllP-_t/t). We found that the absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-_t/t resulted in a compressible cardiac myofilament lattice induced by rigor not observed in the NTG and WT_t/t. These results suggest that the presence and phosphorylation of the N-terminus of cMyBP-C provides structural support and radial rigidity to the myofilament lattice. Examination of myofilament longitudinal stiffness under rigor conditions demonstrated a significant reduction in cross-bridge-dependent stiffness in the t/t compared with NTG controls, but not in the AllP-_t/t compared with WT_t/t controls. The absence of cMyBP-C in the t/t and the unphosphorylated cMyBP-C in the AllP-_t/t both resulted in a shorter myosin cross-bridge lifetime when myosin isoform was controlled. These data collectively suggest that cMyBP-C provides radial rigidity to the myofilament lattice through the N-terminus, and that disruption of the phosphorylation of cMyBP-C is sufficient to abolish this structural role of the N-terminus and shorten cross-bridge lifetime. Although the presence of cMyBP-C also provides longitudinal rigidity, phosphorylation of the N-terminus is not necessary to maintain longitudinal rigidity of the lattice, in contrast to radial rigidity.     

Protein kinase D increases maximal Ca2+-activated tension of cardiomyocyte contraction by phosphorylation of cMyBP-C-Ser315

Cardiac myosin-binding protein C (cMyBP-C) is involved in the regulation of cardiac myofilament contraction. Recent evidence showed that protein kinase D (PKD) is one of the kinases that phosphorylate cMyBP-C. However, the mechanism by which PKD-induced cMyBP-C phosphorylation affects cardiac contractile responses is not known. Using immunoprecipitation, we showed that, in contracting cardiomyocytes, PKD binds to cMyBP-C and phosphorylates it at Ser(315). The effect of PKD-mediated phosphorylation of cMyBP-C on cardiac myofilament function was investigated in permeabilized ventricular myocytes, isolated from wild-type (WT) and from cMyBP-C knockout (KO) mice, incubated in the presence of full-length active PKD. In WT myocytes, PKD increased both myofilament Ca(2+) sensitivity (pCa(50)) and maximal Ca(2+)-activated tension of contraction (T(max)). In cMyBP-C KO skinned myocytes, PKD increased pCa(50) but did not alter T(max). This suggests that cMyBP-C is not involved in PKD-mediated sensitization of myofilaments to Ca(2+) but is essential for PKD-induced increase in T(max). Furthermore, the phosphorylation of both PKD-Ser(916) and cMyBP-C-Ser(315) was contraction frequency-dependent, suggesting that PKD-mediated cMyBP-C phosphorylation is operational primarily during periods of increased contractile activity. Thus, during high contraction frequency, PKD facilitates contraction of cardiomyocytes by increasing Ca(2+) sensitivity and by an increased T(max) through phosphorylation of cMyBP-C.