Clostridium perfringens Alpha-Toxin Induces Gm1a Clustering and Trka Phosphorylation in the Host Cell Membrane.

Clostridium perfringens alpha-toxin elicits various immune responses such as the release of cytokines, chemokines, and superoxide via the GM1a/TrkA complex. Alpha-toxin possesses phospholipase C (PLC) hydrolytic activity that contributes to signal transduction in the pathogenesis of gas gangrene. Little is known about the relationship between lipid metabolism and TrkA activation by alpha-toxin. Using live-cell fluorescence microscopy, we monitored transbilayer movement of diacylglycerol (DAG) with the yellow fluorescent protein-tagged C1AB domain of protein kinase C-γ (EYFP-C1AB). DAG accumulated at the marginal region of the plasma membrane in alpha toxin-treated A549 cells, which also exhibited GM1a clustering and TrkA phosphorylation. Annexin V binding assays showed that alpha-toxin induced the exposure of phosphatidylserine on the outer leaflet of the plasma membrane. However, H148G, a variant toxin which binds cell membrane and has no enzymatic activity, did not induce DAG translocation, GM1a clustering, or TrkA phosphorylation. Alpha-toxin also specifically activated endogenous phospholipase Cγ-1 (PLCγ-1), a TrkA adaptor protein, via phosphorylation. U73122, an endogenous PLC inhibitor, and siRNA for PLCγ-1 inhibited the formation of DAG and release of IL-8. GM1a accumulation and TrkA phosphorylation in A549 cells treated with alpha-toxin were also inhibited by U73122. These results suggest that the flip-flop motion of hydrophobic lipids such as DAG leads to the accumulation of GM1a and TrkA. We conclude that the formation of DAG by alpha-toxin itself (first step) and activation of endogenous PLCγ-1 (second step) leads to alterations in membrane dynamics, followed by strong phosphorylation of TrkA

Receptor discordance after nipple-sparing mastectomy

Background: Recent studies have shown that receptor status of breast cancer change between primary tumor and recurrence, which may influence treatment strategy and prognosis, but there are few reports on receptor discordance between primary tumors and local recurrence (LR) after nipple-sparing mastectomy (NSM). Patients and methods: We collected 74 patients who had LR after NSM for newly diagnosed stages 0 to 3 breast cancer between 2008 and 2016 at 14 institutions. We classified into 4 subtypes based on hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2). We evaluated clinicopathological factors that correlate with receptor discordance and assessed the impact of receptor discordance on survival. Results: Discordance rates in estrogen receptor (ER), progesterone receptor (PgR) and HER2 were 9.5, 10.8 and 5.4 %, respectively. The most common change was from HR-/HER2+ to HR+/HER2+, and this pattern of receptor change occurred only in patients with nipple–areolar recurrence. Non-invasive tumors in LR, nipple–areolar recurrence (NAR), HR-/HER2+ primary tumor subtype, and the presence of chemotherapy for primary tumors were significantly associated with receptor discordance. With a median follow-up of 44.5 months (4–153 months), patients in the receptor-discordant group had no disease-free survival (DFS) event after LR resection (5-year DFS; 100 % in the receptor-discordant group vs 85.1 % in the receptor-concordant group; p = 0.2). Conclusion: Our study demonstrates that the presence of chemotherapy for primary tumors, nipple-areolar recurrence, and its related factors (non-invasive tumor in LR, HR-/HER2+ primary tumor subtype) were associated with receptor discordance. However, further studies with longer follow-up periods and larger sample sizes are needed

Possible involvement of zinc transporter ZIP13 in myogenic differentiation

Abstract Ehlers–Danlos syndrome spondylodysplastic type 3 (EDSSPD3, OMIM 612350) is an inherited recessive connective tissue disorder that is caused by loss of function of SLC39A13/ZIP13, a zinc transporter belonging to the Slc39a/ZIP family. We previously reported that patients with EDSSPD3 harboring a homozygous loss of function mutation (c.221G > A, p.G64D) in ZIP13 exon 2 (ZIP13 G64D ) suffer from impaired development of bone and connective tissues, and muscular hypotonia. However, whether ZIP13 participates in the early differentiation of these cell types remains unclear. In the present study, we investigated the role of ZIP13 in myogenic differentiation using a murine myoblast cell line (C2C12) as well as patient-derived induced pluripotent stem cells (iPSCs). We found that ZIP13 gene expression was upregulated by myogenic stimulation in C2C12 cells, and its knockdown disrupted myotubular differentiation. Myocytes differentiated from iPSCs derived from patients with EDSSPD3 (EDSSPD3-iPSCs) also exhibited incomplete myogenic differentiation. Such phenotypic abnormalities of EDSSPD3-iPSC-derived myocytes were corrected by genomic editing of the pathogenic ZIP13 G64D mutation. Collectively, our findings suggest the possible involvement of ZIP13 in myogenic differentiation, and that EDSSPD3-iPSCs established herein may be a promising tool to study the molecular basis underlying the clinical features caused by loss of ZIP13 function

Inhibition of endogenous PLC prevented the alpha-toxin-induced release of IL-8 and formation of diacylglycerol.

<p>(A) A549 cells were pretreated with various amounts of U73122 or U73343 at 37°C for 60 min, and then incubated with or without alpha-toxin (1.0 μg/mL) at 37°C for 3 h. The concentration of IL-8 in culture supernatants was determined by ELISA. (B) A549 cells were pretreated with various amounts of U73122 or U73343 at 37°C for 60 min, and then incubated with or without alpha-toxin (1.0 μg/mL) at 37°C for 60 min and intracellular DAG levels were determined. Values represent mean ± S.E.; <i>n</i> = 4; *, <i>p</i> < 0.01.</p