The gram-positive bacterium Staphylococcus aureus can invade non-professional phagocytic cells by associating with the plasma protein fibronectin to exploit host cell integrins. Integrin-mediated internalization of these pathogens is facilitated by the local production of phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) via an integrin-associated isoform of phosphatidylinositol-5\' kinase. In this study, we addressed the role of PI-4,5-P2-directed phosphatases on internalization of S. aureus. ShRNA-mediated knockdown of individual phosphoinositide 5-phosphatases revealed that synaptojanin1 (SYNJ1) is counteracting invasion of S. aureus into mammalian cells. Indeed, shRNA-mediated depletion as well as genetic deletion of synaptojanin1 via CRISPR/Cas9 resulted in a gain-of-function phenotype with regard to integrin-mediated uptake. Surprisingly, the surface level of integrins was slightly downregulated in Synj1-KO cells. Nevertheless, these cells showed enhanced local accumulation of PI-4,5-P2 and exhibited increased internalization of S. aureus. While the phosphorylation level of the integrin-associated protein tyrosine kinase FAK was unaltered, the integrin-binding and -activating protein talin was enriched in the vicinity of S. aureus in synaptojanin1 knockout cells. Scanning electron microscopy revealed enlarged membrane invaginations in the absence of synaptojanin1 explaining the increased capability of these cells to internalize integrin-bound microorganisms. Importantly, the enhanced uptake by Synj1-KO cells and the exaggerated morphological features were rescued by the re-expression of the wild-type enzyme but not phosphatase inactive mutants. Accordingly, synaptojanin1 activity limits integrin-mediated invasion of S. aureus, corroborating the important role of PI-4,5-P2 during this process.IMPORTANCEStaphylococcus aureus, an important bacterial pathogen, can invade non-professional phagocytes by capturing host fibronectin and engaging integrin α5β1. Understanding how S. aureus exploits this cell adhesion receptor for efficient cell entry can also shed light on the physiological regulation of integrins by endocytosis. Previous studies have found that a specific membrane lipid, phosphatidylinositol-4,5-bisphosphate (PIP2), supports the internalization process. Here, we extend these findings and report that the local levels of PIP2 are controlled by the activity of the PIP2-directed lipid phosphatase Synaptojanin1. By dephosphorylating PIP2 at bacteria-host cell attachment sites, Synaptojanin1 counteracts the integrin-mediated uptake of the microorganisms. Therefore, our study not only generates new insight into subversion of cellular receptors by pathogenic bacteria but also highlights the role of host cell proteins acting as restriction factors for bacterial invasion at the plasma membrane.

Neferine has long been recognized as a medicinal herbal ingredient with various physiological and pharmacological activities. Although previous studies have reported its antithrombotic effect, the underlying mechanisms have not been thoroughly investigated. Since platelets play a key role in thrombosis, we investigated the effects of neferine on human platelet function and the potential mechanisms. Platelet aggregation, adhesion and spreading were performed to investigate the effect of neferine on inhibition of platelet function. Flow cytometry was used to determine platelet alpha granule secretion and integrin IIb/IIIa activation, as detected by CD62P (P-selectin) expression, PAC-1 and fibrinogen binding. Western blotting was utilized to investigate the effect of neferine on intracellular signaling of activated platelet. We found that neferine significantly suppressed platelet aggregation and remarkably promoted the dissociation of platelet aggregates induced by collagen, thrombin, U46619, ADP and adrenaline in a dose-dependent manner. Flow cytometry analysis showed that neferine inhibited thrombin-induced platelet P-selectin expression, PAC-1 and fibrinogen binding. In addition, neferine reduced the adhesion of human platelets on coated collagen under both static and shearing condition at an arterial shear rate of 40 dyne/cm2. Neferine also inhibited the spreading of human platelets on immobilized fibrinogen. Western blot analysis showed that neferine inhibited PI3K activation, and decreased the levels of phosphorylation of Akt, GSK3β and p38 MAPK in platelets. In summary, neferine has the potential to be an antiplatelet and antithrombotic agent by inhibiting the PI3K-Akt-GSK3β/p38 MAPK signaling pathway.

T-type calcium (Ca2+) channels play important physiological functions in excitable cells including cardiomyocyte. Phosphatidylinositol-4,5-bisphosphate (PIP2) has recently been reported to modulate various ion channels\' function. However the actions of PIP2 on the T-type Ca2+ channel remain unclear. To elucidate possible effects of PIP2 on the T-type Ca2+ channel, we applied patch clamp method to investigate recombinant CaV3.1- and CaV3.2-T-type Ca2+ channels expressed in mammalian cell lines with PIP2 in acute- and long-term potentiation. Short- and long-term potentiation of PIP2 shifted the activation and the steady-state inactivation curve toward the hyperpolarization direction of CaV3.1-ICa.T without affecting the maximum inward current density. Short- and long-term potentiation of PIP2 also shifted the activation curve toward the hyperpolarization direction of CaV3.2-ICa.T without affecting the maximum inward current density. Conversely, long-term but not short-term potentiation of PIP2 shifted the steady-state inactivation curve toward the hyperpolarization direction of CaV3.2-ICa.T. Long-term but not short-term potentiation of PIP2 blunted the voltage-dependency of current decay CaV3.1-ICa.T. PIP2 modulates CaV3.1- and CaV3.2-ICa.T not by their current density but by their channel gating properties possibly through its membrane-delimited actions.

The prognostic value of phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) in patients with esophageal squamous cell carcinoma (ESCC) is controversial. We aimed to investigate the prognostic significance of PIK3CA mutation in patients with ESCC. EMBASE, PubMed, and Web of Science databases were systematically searched from inception through Oct. 3, 2016. The hazard ratios (HRs) and 95% confidence intervals (CI) were calculated using a random effects model for overall survival (OS) and disease-free survival (DFS). Seven studies enrolling 1505 patients were eligible for inclusion of the current meta-analysis. Results revealed that PIK3CA mutation was not significantly associated with OS (HR: 0.90, 95% CI: 0.63-1.30, P=0.591), with a significant heterogeneity (I 2=65.7%, P=0.012). Additionally, subgroup analyses were further conducted according to various variables, such as types of specimen, the sample size, technique and statistical methodology. All results suggested that no significant relationship was found between PIK3CA mutation and OS in patients with ESCC. For DFS, there was no significant association between PIK3CA mutation and DFS in patients with ESCC (HR: 1.00, 95% CI=0.47-2.11, P=0.993, I 2=73.7%). Publication bias was not present and the results of sensitivity analysis were very stable in the current meta-analysis. Our findings suggest that PIK3CA mutation has no significant effects on OS and DFS in ESCC patients. More well-designed prospective studies with better methodology for PIK3CA assessment are required to clarify the prognostic significance of PIK3CA mutation in ESCC patients.