The parasite Entamoeba histolytica is the etiological agent of amoebiasis and phagocytosis plays a key role in virulence of this organism. Signaling pathways involved in activation of cytoskeletal dynamics required for phagocytosis remain to be elucidated. Phagocytosis is initiated with sequential recruitment of EhC2PK, EhCaBP1, EhCaBP3 and an atypical kinase EhAK1 after particle attachment. Here we show that EhARPC1, an essential subunit of the actin branching complex Arp 2/3 is recruited to the phagocytic initiation sites by EhAK1. Imaging, expression knockdown of different molecules and pull down experiments suggest that EhARPC1 interacts with EhAK1 and that it is required during initiation of phagocytosis and phagosome formation. Moreover, recruitment of EhARPC2 at the phagocytosis initiation by EhAK1 is also observed, indicating that the Arp 2/3 complex is recruited. In conclusion, these results suggests a novel mechanism of recruitment of Arp 2/3 complex during phagocytosis in E. histolytica. E. histolytica is the causative agent of amoebiasis and leads to morbidity and mortality in developing countries. It is known to phagocytose immune and non-immune cells, epithelial tissue, erythrocytes and commensal bacteria. The high rate of phagocytosis in this protist parasite provides a unique system to study the signaling cascade that is activated after attachment of the particle to the cell surface. The major objective of the signaling pathway is to generate force for uptake of the particle and this is done through stimulating cytoskeleton to form appropriate structures. However, the molecular mechanism of the same is still largely unknown in E. histolytica, though this pathway has been characterized in many other systems. We have been investigating this pathway by using red blood cells as a particle and have identified different molecules required during the initial stages of phagocytosis. In this study we demonstrate the mechanism by which actin cytoskeleton branching complex EhARP2/3 is recruited at the site of erythrophagocytosis and show that the recruitment is through an atypical alpha kinase EhAK1. A number of different approaches, such as pull down assay, conditional suppression of EhAK1 expression and imaging were used to decipher this pathway. Therefore this study provides a mechanism by which actin dynamics couples to the initial signaling system, activated on attachment of RBC to the cell receptors.

Background: The recent increase in the incidence of alcohol-associated hepatitis (AH) coincides with the obesity epidemic in the United States. However, current mouse models do not fully replicate the combined insults of obesity, metabolic dysfunction–associated steatohepatitis, and alcohol. The aim of this study was to develop a new mouse model that recapitulates the robust inflammatory and fibrotic phenotype characteristic of human MetALD. Methods: Eight- to 10-week-old male C57BL/6 mice were fed chow or high fat-cholesterol-sugar diet (metabolic dysfunction–associated steatohepatitis diet) and in each group, some received alcohol in drinking water (ad libitum) and weekly alcohol binges (EtOH) for 3 months. The liver was assessed for features of AH. Results: MetALD mice displayed increased liver damage indicated by highly elevated ALT and bilirubin levels compared to all other groups. Liver steatosis was significantly greater in the MetALD mice compared to all other experimental groups. The inflammatory phenotype of MetALD was also recapitulated, including increased IL-6 and IL-1β protein levels as well as increased CD68+ macrophages and Ly6G+ neutrophils in the liver. Sirius red staining and expression of collagen 1, alpha-smooth muscle actin indicated advanced fibrosis in the livers of MetALD mice. In addition, indicators of epithelial-to-mesenchymal transition markers were increased in MetALD mice compared to all other groups. Furthermore, we found increased ductular reaction, dysregulated hedgehog signaling, and decreased liver synthetic functions, consistent with severe AH. Conclusions: Alcohol administration in mice combined with metabolic dysfunction–associated steatohepatitis diet recapitulates key characteristics of human AH including liver damage, steatosis, robust systemic inflammation, and liver immune cell infiltration. This model results in advanced liver fibrosis, ductular reaction, decreased synthetic function, and hepatocyte dedifferentiation, suggesting a robust model of MetALD in mice. Export

The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin–binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca²⁺ in nonmuscle cells. Here we report the mechanism of Ca²⁺-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca²⁺-free and Ca²⁺-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca²⁺, binding of which can only be regulated in the presence of physiological concentrations of Mg²⁺. Ca²⁺ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin–binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.

Binge drinking in obese patients positively correlates with accelerated liver damage and liver-related death. However, the underlying mechanism and the effect of alcohol use on the progression of metabolic-dysfunction-associated steatotic liver disease (MASLD) remain unexplored. Here, we show that short-term feeding of a metabolic-dysfunction-associated steatohepatitis (MASH) diet plus daily acute alcohol binges for three days induce liver injury and activation of the NLRP3 inflammasome. We identify that a MASH diet plus acute alcohol binges promote liver inflammation via increased infiltration of monocyte-derived macrophages, neutrophil recruitment, and NET release in the liver. Our results suggest that both monocyte-derived macrophages and neutrophils are activated via NLRP3, while the administration of MCC950, an NLRP3 inhibitor, dampens these effects.In this study, we reveal important intercellular communication between hepatocytes and neutrophils. We discover that the MASH diet plus alcohol induces IL-1β via NLRP3 activation and that IL-1β acts on hepatocytes and promotes the production of CXCL1 and LCN2. In turn, the increase in these neutrophils recruits chemokines and causes further infiltration and activation of neutrophils in the liver. In vivo administration of the NLRP3 inhibitor, MCC950, improves the early phase of MetALD by preventing liver damage, steatosis, inflammation, and immune cells recruitment.