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.

Ebola virus (EBOV) is a filamentous negative-sense RNA virus which causes severe hemorrhagic fever. There are limited vaccines or therapeutics for prevention and treatment of EBOV, so it is important to get a detailed understanding of the virus lifecycle to illuminate new drug targets. EBOV encodes for the matrix protein, VP40, which regulates assembly and budding of new virions from the inner leaflet of the host cell plasma membrane (PM). In this work we determine the effects of VP40 mutations altering electrostatics on PM interactions and subsequent budding. VP40 mutations that modify surface electrostatics affect viral assembly and budding by altering VP40 membrane binding capabilities. Mutations that increase VP40 net positive charge by one (e.g., Gly to Arg or Asp to Ala) increase VP40 affinity for phosphatidylserine (PS) and PI(4,5)P2 in the host cell PM. This increased affinity enhances PM association and budding efficiency leading to more effective formation of virus-like particles (VLPs). In contrast, mutations that decrease net positive charge by one (e.g., Gly to Asp) lead to a decrease in assembly and budding because of decreased interactions with the anionic PM. Taken together our results highlight the sensitivity of slight electrostatic changes on the VP40 surface for assembly and budding. Understanding the effects of single amino acid substitutions on viral budding and assembly will be useful for explaining changes in the infectivity and virulence of different EBOV strains, VP40 variants that occur in nature, and for long-term drug discovery endeavors aimed at EBOV assembly and budding.

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.

Alzheimer\'s disease (AD) and type 2 diabetes (T2D) are both diseases with increasing prevalence in aging populations. T2D, characterized by insulin resistance and defective insulin signaling, is a common co-morbidity and a risk factor for AD, increasing the risk approximately two to fourfold. Insulin exerts a wide variety of effects as a growth factor as well as by regulating glucose, fatty acid, and protein metabolism. Certain lifestyle factors, physical inactivity and typical Western diet (TWD) containing high fat and high sugar are strongly associated with insulin resistance and T2D. The PI3K-Akt signaling pathway is a major mediator of effects of insulin and plays a crucial role in T2D pathogenesis. Decreased levels of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) subunits as well as blunted Akt kinase phosphorylation have been observed in the AD brain, characterized by amyloid-β and tau pathologies. Furthermore, AD mouse models fed with TWD have shown to display altered levels of PI3K subunits. How impaired insulin-PI3K-Akt signaling in peripheral tissues or in the central nervous system (CNS) affects the development or progression of AD is currently poorly understood. Interestingly, enhancement of PI3K-Akt signaling in the CNS by intranasal insulin (IN) treatment has been shown to improve memory in vivo in mice and in human trials. Insulin is known to augment neuronal growth and synapse formation through the PI3K-Akt signaling pathway. However, PI3K-Akt pathway mediates signaling related to different functions also in other cell types, like microglia and astrocytes. In this review, we will discuss the most prominent molecular mechanisms related to the PI3K-Akt pathway in AD and how T2D and altered insulin signaling may affect the pathogenesis of AD.

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.

Background: Class IA phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) is an integral mediator of insulin signaling. The p110 catalytic and p85 regulatory subunits of PI3K are the products of separate genes, and while they come together to make the active heterodimer, they have opposing roles in insulin signaling and action. Deletion of hepatic p110α results in an impaired insulin signal and severe insulin resistance, whereas deletion of hepatic p85α results in improved insulin sensitivity due to sustained levels of phosphatidylinositol (3,4,5)-trisphosphate. Here, we created mice with combined hepatic deletion of p110α and p85α (L-DKO) to study the impact on insulin signaling and whole body glucose homeostasis. Methods: Six-week old male flox control and L-DKO mice were studied over a period of 18 weeks, during which weight and glucose levels were monitored, and glucose tolerance tests, insulin tolerance test and pyruvate tolerance test were performed. Fasting insulin, insulin signaling mediators, PI3K activity and insulin receptor substrate (IRS)1-associated phosphatidylinositol kinase activity were examined at 10 weeks. Liver, muscle and white adipose tissue weight was recorded at 10 weeks and 25 weeks. Results: The L-DKO mice showed a blunted insulin signal downstream of PI3K, developed markedly impaired glucose tolerance, hyperinsulinemia and had decreased liver and adipose tissue weights. Surprisingly, however, these mice displayed normal hepatic glucose production, normal insulin tolerance, and intact IRS1-associated phosphatidylinositol kinase activity without compensatory upregulated signaling of other classes of PI3K. Conclusions: The data demonstrate an unexpectedly overall mild metabolic phenotype of the L-DKO mice, suggesting that lipid kinases other than PI3Ks might partially compensate for the loss of p110α/p85α by signaling through other nodes than Akt/Protein Kinase B.

In today\'s world of modern medicine and novel therapies, cancer still remains to be one of the prime contributor to the death of people worldwide. The modern therapies improve condition of cancer patients and are effective in early stages of cancer but the advanced metastasized stage of cancer remains untreatable. Also most of the cancer therapies are expensive and are associated with adverse side effects. Thus, considering the current status of cancer treatment there is scope to search for efficient therapies which are cost-effective and are associated with lesser and milder side effects. Phytochemicals have been utilized for many decades to prevent and cure various ailments and current evidences indicate use of phytochemicals as an effective treatment for cancer. Hyperactivation of phosphoinositide 3-kinase (PI3K) signaling cascades is a common phenomenon in most types of cancers. Thus, natural substances targeting PI3K pathway can be of great therapeutic potential in the treatment of cancer patients. This chapter summarizes the updated research on plant-derived substances targeting PI3K pathway and the current status of their preclinical studies and clinical trials.

The E6 oncoproteins of high-risk human papillomaviruses (HPV) of genus alpha contain a short peptide sequence at the carboxy-terminus, the PDZ binding domain, with which they interact with the corresponding PDZ domain of cellular proteins. Interestingly, E6 proteins from papillomaviruses of genus beta (betaPV) do not encode a comparable PDZ binding domain. Irrespective of this fact, we previously showed that the E6 protein of HPV8 (betaPV type) could circumvent this deficit by targeting the PDZ protein Syntenin-2 through transcriptional repression (Lazic et al., 2012). Despite its high binding affinity to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), very little is known about Syntenin-2. This study aimed to extend the knowledge on Syntenin-2 and how its expression is controlled. We now identified that Syntenin-2 is expressed at high levels in differentiating and in lower amounts in keratinocytes cultured in serum-free media containing low calcium concentration. HPV8-E6 led to a further reduction of Syntenin-2 expression only in cells cultured in low calcium. In the skin of patients suffering from Epidermodysplasia verruciformis, who are predisposed to betaPV infection, Syntenin-2 was expressed in differentiating keratinocytes of non-lesional skin, but was absent in virus positive squamous tumors. Using 5-Aza-2\'-deoxycytidine, which causes DNA demethylation, Syntenin-2 transcription was profoundly activated and fully restored in the absence and presence of HPV8-E6, implicating that E6 mediated repression of Syntenin-2 transcription is due to promoter hypermethylation. Since Syntenin-2 binds to PI(4,5)P2, we further tested whether the PI(4,5)P2 metabolic pathway might govern Syntenin-2 expression. PI(4,5)P2 is generated by the activity of phosphatidylinositol-4-phosphate-5-kinase type I (PIP5KI) or phosphatidylinositol-5-phosphate-4-kinase type II (PIP4KII) isoforms α, β and γ. Phosphatidylinositide kinases have recently been identified as regulators of gene transcription. Surprisingly, transfection of siRNAs directed against PIP5KI and PIP4KII resulted in higher Syntenin-2 expression with the highest effect mediated by siPIP5KIα. HPV8-E6 was able to counteract siPIP4KIIα, siPIP4KIIβ and siPIP5KIγ mediated Syntenin-2 re-expression but not siPIP5KIα. Finally, we identified Syntenin-2 as a key factor regulating PIP5KIα expression. Collectively, our data demonstrates that Syntenin-2 is regulated through multiple mechanisms and that downregulation of Syntenin-2 expression may contribute to E6 mediated dedifferentiation of infected skin cells.

BACKGROUND: Giant-cell arteritis (GCA) is an inflammatory disease of large/medium-sized arteries, frequently involving the temporal arteries (TA). Inflammation-induced vascular remodelling leads to vaso-occlusive events. Circulating endothelin-1 (ET-1) is increased in patients with GCA with ischaemic complications suggesting a role for ET-1 in vascular occlusion beyond its vasoactive function.
OBJECTIVE: To investigate whether ET-1 induces a migratory myofibroblastic phenotype in human TA-derived vascular smooth muscle cells (VSMC) leading to intimal hyperplasia and vascular occlusion in GCA.
RESULTS: Immunofluorescence/confocal microscopy showed increased ET-1 expression in GCA lesions compared with control arteries. In inflamed arteries, ET-1 was predominantly expressed by infiltrating mononuclear cells whereas ET receptors, particularly ET-1 receptor B (ETBR), were expressed by both mononuclear cells and VSMC. ET-1 increased TA-derived VSMC migration in vitro and α-smooth muscle actin (αSMA) expression and migration from the media to the intima in cultured TA explants. ET-1 promoted VSMC motility by increasing activation of focal adhesion kinase (FAK), a crucial molecule in the turnover of focal adhesions during cell migration. FAK activation resulted in Y397 autophosphorylation creating binding sites for Src kinases and the p85 subunit of PI3kinases which, upon ET-1 exposure, colocalised with FAK at the focal adhesions of migrating VSMC. Accordingly, FAK or PI3K inhibition abrogated ET-1-induced migration in vitro. Consistently, ET-1 receptor A and ETBR antagonists reduced αSMA expression and delayed VSMC outgrowth from cultured GCA-involved artery explants.
CONCLUSIONS: ET-1 is upregulated in GCA lesions and, by promoting VSMC migration towards the intimal layer, may contribute to intimal hyperplasia and vascular occlusion in GCA.

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.