Calcineurin and actin dynamics in calcium-mediated microneme exocytosis

Plasmodium falciparum invades host erythrocytes by multiple invasion pathways. The invasion of erythrocytes by P. falciparum merozoites is a complex process that requires multiple interactions between host receptors and parasite ligands. A number of parasite proteins that mediate interaction with host receptors during invasion are localized to membrane-bound apical organelles referred to as micronemes and rhoptries. The timely release of these proteins to the merozoite surface is crucial for receptor engagement and invasion. It has been demonstrated previously that exposure of merozoites to a low potassium (K(+)) ionic environment as found in blood plasma leads to a rise in cytosolic calcium (Ca(2+)), which triggers microneme secretion. The signalling pathways that regulate microneme discharge in response to rise in cytosolic Ca(2+) are not completely understood. Here, we show that a P. falciparum Ca(2+)-dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca(2+)-dependent microneme exocytosis. An increase in PfCN activity was observed in merozoites following exposure to a low K(+) environment. Treatment of merozoites with calcineurin inhibitors such as FK506 and cyclosporin A prior to transfer to a low K(+) environment resulted in inhibition of secretion of microneme protein apical merozoite antigen-1 (PfAMA-1). Inhibition of PfCN was shown to result in reduced dephosphorylation and depolymerization of apical actin, which appears to be criticalfor microneme secretion. PfCN thus serves as an effector of Ca(2+)-dependent microneme exocytosis by regulating depolymerization of apical actin. Inhibitors that target PfCN block microneme exocytosis and limit growth of P. falciparum blood-stage parasites providing a novel approach towards development of new therapeutic strategies against malaria

Peer-to-Peer Conferencing using Blockchain, WebRTC and SIP

  The owner of the centralized video platform has more control over uploaded content than the content producer does. But the other Blockchain-based decentralized video services are attempting to reduce ad pressure and get rid of middlemen. The article suggests a combination of a safe encryption technique and an access control mechanism created "with technology" to create a successful decentralized video streaming platform built on the Blockchain. Peer-to-peer (P2P) overlays are one of the complicated network applications and services that have been migrated to the Web as a result of the increasing support for Web Real-Time Communication (WebRTC) standard in modern browsers for real-time communications. The expansion of access networks’ bandwidth also makes it possible for end users to start their own content businesses. This paper presents a preliminary proposal of metrics and technologies to move toward a decentralized cooperative architecture for large-scale, real-time live stream content de- livery based on WebRTC, without the requirement of a Content Delivery Network (CDN) infrastructure. The paper takes into account the light of the aforementioned aspects [6]

A Prospective Study of Profile of Agricultural Orthopaedic Injuries in North India in a Tertiary Care Centre

Background: Over time agriculture has transformed into an industry, both in scale and mechanization. Growing demand for agricultural yield and increasing mechanization has led to growth in the numbers of agricultural accidents and injuries causing significant morbidity and economical loss. Aim: To study the causes, patterns, outcomes of injuries due to agricultural accidents, their effect on the productivity of the victims, and to suggest possible remedial measures, a prospective study was undertaken. Methods: A total of 106 patients reporting agricultural injuries (AI) over 14 months in 2019-20 were enrolled and data was recorded on a pre-structured proforma. Results: With the preponderance of male victims, educational status, skill levels, lighting & climatic conditions, intoxication and fatigue were found to be major determinants in the causation of injuries with upper limbs being the predominantly involved part (75.47%) and most of the cases ended up requiring some form of surgery (88.68%). Agricultural machinery (56.60%) was the main cause of AI and Chaff Cutter Machine caused the maximum AI (37.73%). Amputations were the most common injuries sustained (47.16%). Agricultural injuries affected the range of motion of body parts (32.07%) and work (58.49%) & household activities (49.05%); thus impairing work efficiency and economy. Conclusion: The present study highlights the need for a robust surveillance and data analysis leading to better design of farming machinery and equipment, more relevant education and training systems, stronger legislations as well as a comprehensive rehabilitative program aimed at reducing the socio-economic burden caused by agricultural injuries

cAMP-Dependent Signaling Pathways as Potential Targets for Inhibition of Plasmodium falciparum Blood Stages

We review the role of signaling pathways in regulation of the key processes of merozoite egress and red blood cell invasion by Plasmodium falciparum and, in particular, the importance of the second messengers, cAMP and Ca2+, and cyclic nucleotide dependent kinases. cAMP-dependent protein kinase (PKA) is comprised of cAMP-binding regulatory, and catalytic subunits. The less well conserved cAMP-binding pockets should make cAMP analogs attractive drug leads, but this approach is compromised by the poor membrane permeability of cyclic nucleotides. We discuss how the conserved nature of ATP-binding pockets makes ATP analogs inherently prone to off-target effects and how ATP analogs and genetic manipulation can be useful research tools to examine this. We suggest that targeting PKA interaction partners as well as substrates, or developing inhibitors based on PKA interaction sites or phosphorylation sites in PKA substrates, may provide viable alternative approaches for the development of anti-malarial drugs. Proximity of PKA to a substrate is necessary for substrate phosphorylation, but the P. falciparum genome encodes few recognizable A-kinase anchor proteins (AKAPs), suggesting the importance of PKA-regulatory subunit myristylation and membrane association in determining substrate preference. We also discuss how Pf14-3-3 assembles a phosphorylation-dependent signaling complex that includes PKA and calcium dependent protein kinase 1 (CDPK1) and how this complex may be critical for merozoite invasion, and a target to block parasite growth. We compare altered phosphorylation levels in intracellular and egressed merozoites to identify potential PKA substrates. Finally, as host PKA may have a critical role in supporting intracellular parasite development, we discuss its role at other stages of the life cycle, as well as in other apicomplexan infections. Throughout our review we propose possible new directions for the therapeutic exploitation of cAMP-PKA-signaling in malaria and other diseases caused by apicomplexan parasites

Meridianin C inhibits the growth of YD-10B human tongue cancer cells through macropinocytosis and the down-regulation of Dickkopf-related protein-3

Meridianin C is a marine natural product known for its anti‐cancer activity. At present, the anti‐tumour effects of meridianin C on oral squamous cell carcinoma are unknown. Here, we investigated the effect of meridianin C on the proliferation of four different human tongue cancer cells, YD‐8, YD‐10B, YD‐38 and HSC‐3. Among the cells tested, meridianin C most strongly reduced the growth of YD‐10B cells; the most aggressive and tumorigenic of the cell lines tested. Strikingly, meridianin C induced a significant accumulation of macropinosomes in the YD‐10B cells; confirmed by the microscopic and TEM analysis as well as the entry of FITC‐dextran, which was sensitive to the macropinocytosis inhibitor amiloride. SEM data also revealed abundant long and thin membrane extensions that resemble lamellipodia on the surface of YD‐10B cells treated with meridianin C, pointing out that meridianin C‐induced macropinosomes was the result of macropinocytosis. In addition, meridianin C reduced cellular levels of Dickkopf‐related protein‐3 (DKK‐3), a known negative regulator of macropinocytosis. A role for DKK‐3 in regulating macropinocytosis in the YD‐10B cells was confirmed by siRNA knockdown of endogenous DKK‐3, which led to a partial accumulation of vacuoles and a reduction in cell proliferation, and by exogenous DKK‐3 overexpression, which resulted in a considerable inhibition of the meridianin C‐induced vacuole formation and decrease in cell survival. In summary, this is the first study reporting meridianin C has novel anti‐proliferative effects via macropinocytosis in the highly tumorigenic YD‐10B cell line and the effects are mediated in part through down‐regulation of DKK‐3

A Fluorogenic Assay: Analysis of Chemical Modification of Lysine and Arginine to Control Proteolytic Activity of Trypsin

The chemical modification of amino acids plays an important role in the modulation of proteins or peptides and has useful applications in the activation and stabilization of enzymes, chemical biology, shotgun proteomics, and the production of peptide-based drugs. Although chemoselective modification of amino acids such as lysine and arginine via the insertion of respective chemical moieties as citraconic anhydride and phenyl glyoxal is important for achieving desired application objectives and has been extensively reported, the extent and chemoselectivity of the chemical modification of specific amino acids using specific chemical agents (blocking or modifying agents) has yet to be sufficiently clarified owing to a lack of suitable assay methodologies. In this study, we examined the utility of a fluorogenic assay method, based on a fluorogenic tripeptide substrate (FP-AA1-AA2-AA3) and the proteolytic enzyme trypsin, in determinations of the extent and chemoselectivity of the chemical modification of lysine or arginine. As substrates, we used two fluorogenic tripeptide probes, MeRho-Lys-Gly-Leu(Ac) (lysine-specific substrate) and MeRho-Arg-Gly-Leu(Ac) (arginine-specific substrate), which were designed, synthesized, and evaluated for chemoselective modification of specific amino acids (lysine and arginine) using the fluorogenic assay. The results are summarized in terms of half-maximal inhibitory concentrations (IC50) for the extent of modification and ratios of IC50 values (IC50arginine/IC50lysine and IC50lysine/IC50arginine) as a measure of the chemoselectivity of chemical modification for amino acids lysine and arginine. This novel fluorogenic assay was found to be rapid, precise, and reproducible for determinations of the extent and chemoselectivity of chemical modification

The Central Role of cAMP in Regulating Plasmodium falciparum Merozoite Invasion of Human Erythrocytes

International audienceAll pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K + environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca 2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca 2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria

Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission

International audienceTransmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission

Phosphorylation-Dependent Assembly of a 14-3-3 Mediated Signaling Complex during Red Blood Cell Invasion by Plasmodium falciparum Merozoites

International audienceRed blood cell (RBC) invasion by Plasmodium merozoites requires multiple steps that are regulated by signaling pathways. Exposure of P. falciparum merozoites to the physiological signal of low K+, as found in blood plasma, leads to a rise in cytosolic Ca2+, which mediates microneme secretion, motility, and invasion. We have used global phosphoproteomic analysis of merozoites to identify signaling pathways that are activated during invasion. Using quantitative phosphoproteomics, we found 394 protein phosphorylation site changes in merozoites subjected to different ionic environments (high K+/low K+), 143 of which were Ca2+ dependent. These included a number of signaling proteins such as catalytic and regulatory subunits of protein kinase A (PfPKAc and PfPKAr) and calcium-dependent protein kinase 1 (PfCDPK1). Proteins of the 14-3-3 family interact with phosphorylated target proteins to assemble signaling complexes. Here, using coimmunoprecipitation and gel filtration chromatography, we demonstrate that Pf14-3-3I binds phosphorylated PfPKAr and PfCDPK1 to mediate the assembly of a multiprotein complex in P. falciparum merozoites. A phospho-peptide, P1, based on the Ca2+-dependent phosphosites of PKAr, binds Pf14-3-3I and disrupts assembly of the Pf14-3-3I-mediated multiprotein complex. Disruption of the multiprotein complex with P1 inhibits microneme secretion and RBC invasion. This study thus identifies a novel signaling complex that plays a key role in merozoite invasion of RBCs. Disruption of this signaling complex could serve as a novel approach to inhibit blood-stage growth of malaria parasites.IMPORTANCE Invasion of red blood cells (RBCs) by Plasmodium falciparum merozoites is a complex process that is regulated by intricate signaling pathways. Here, we used phosphoproteomic profiling to identify the key proteins involved in signaling events during invasion. We found changes in the phosphorylation of various merozoite proteins, including multiple kinases previously implicated in the process of invasion. We also found that a phosphorylation-dependent multiprotein complex including signaling kinases assembles during the process of invasion. Disruption of this multiprotein complex impairs merozoite invasion of RBCs, providing a novel approach for the development of inhibitors to block the growth of blood-stage malaria parasites