Clinicopathological and Molecular Features of Penile Melanoma With a Proposed Staging System.

Penile melanomas (PM) are an exceedingly rare subtype of mucosal melanoma (MM), and we reviewed the clinicopathologic features and molecular profile in 8 PMs. The patient ages ranged from 46 to 78 (mean: 62.8) years with involvement on the glans (n=5; 62.5%), penile urethra (n=2; 25%), and foreskin (n=1, 12.5%). Tumor depth ranged from 1.6 to 10.0 (mean: 5.25) mm. Most of the patients underwent partial penectomy (n=6; 75%) and sentinel lymph node (LN) biopsy N=7; 87.5%). Seven patients had metastatic disease at diagnosis, 6 involving LNs and 1 the adrenal gland, and 4 died of disease with a mean follow-up period of 40.5 (2 to 95) months. Five of 7 (71%) cases identified 15 molecular alterations within KIT , CDKN2A , NF1 , PTEN , and APC (n=2 each), and NRAS , MAP3K1 , CDH1 , MSH6 , and TERT (n=1 each). Two cases were not found to harbor genetic aberrations, and 1 case failed testing. In addition, we reviewed the English literature and included 93 cases with a reported depth of invasion and follow-up. A total of 101 PMs were analyzed for prognostic parameters, and the overall survival was significantly worse in patients with LN metastasis (P=0.0008), distant metastasis (P=0.0016), and greater depth of invasion (P=0.0222) based upon T-stage. While T4 conferred substantially worse survival, the delineation of the survival curves between T2 and T3 was less clear, and combining T2+T3 disease had a strong prognostic impact ( P =0.0024). Prognostic parameters used in the staging of cutaneous melanomas may also be used in PMs. An alternative staging system expanding the inclusion criteria for T2 might provide a more accurate prognostic stratification.

Quantification of cyclin D1 and D2 proteins in multiple myeloma identifies different expression patterns from those revealed by gene expression profiling.

Upregulation of a cyclin D gene determined by expression microarrays is an almost universal event in multiple myeloma (MM), but this finding has not been properly confirmed at the protein level. For this reason, we carried out a quantitative analysis of cyclin D proteins using a capillary electrophoresis nanoimmunoassay in newly diagnosed MM patients. Exclusive expression of cyclin D1 and D2 proteins was detected in 54 of 165 (33%) and 30 of 165 (18%) of the MM patients, respectively. Of note, cyclin D1 or D2 proteins were undetectable in 41% of the samples. High levels of cyclin D1 protein were strongly associated with the presence of t(11;14) or 11q gains. Cyclin D2 protein was detected in all the cases bearing t(14;16), but in only 24% of patients with t(4;14). The presence of cyclin D2 was associated with shorter overall survival (hazard ratio =2.14; P=0.017), although patients expressing cyclin D2 protein, but without 1q gains, had a favorable prognosis. In conclusion, although one of the cyclins D is overexpressed at the mRNA level in almost all MM patients, in approximately half of the patients this does not translate into detectable protein. This suggests that cyclins D could not play an oncogenic role in a proportion of patients with MM (clinicaltrials gov. identifier: NCT01916252).

Extubation and Noninvasive Ventilation of Patients Supported by Extracorporeal Life Support for Cardiogenic Shock: A Single-Center Retrospective Observational Cohort Study.

BACKGROUND: Temporary extracorporeal life support (ECLS) by venoarterial extracorporeal membrane oxygenation is an emerging therapy for patients with severe, ongoing cardiogenic shock. After stabilization of the hemodynamic status and end-organ function, sedation weaning, extubation, and noninvasive ventilation (NIV) can be attempted. The goal of this study was to analyze the feasibility of extubation and NIV during versus after ECLS for cardiogenic shock. METHODS: Single-center retrospective observational study of 132 patients undergoing ECLS due to severe cardiogenic shock between January 2015 and December 2016 at a tertiary care university hospital. RESULTS: Patients received ECLS due to acute myocardial infarction (20.6%), ongoing cardiogenic shock (15.2%), postoperative low-cardiac-output syndrome (24.2%), and extracorporeal cardiopulmonary resuscitation (40.2%). Overall, intensive care unit survival was 44.7%. Sixty-nine (52.3%) patients could never be extubated. Forty-three (32.6%) were extubated while on ECLS support (group 1) and 20 (15.1%) were extubated after weaning from ECLS (group 2). Patients extubated during ECLS had a significantly shorter total time on ventilator (P = .003, mean difference: -284 hours [95% confidence limits: -83 to -484]) and more invasive ventilation free days (P = .0018; mean difference 8 days [95%CL: 2-14]). Mortality and NIV failure rates were similar between groups. CONCLUSIONS: Extubation and NIV are feasible in patients who stabilize during ECLS therapy. Further studies need to address whether extubation has the potential to improve patients outcome or if the feasibility to extubate is a surrogate for disease severeness.

To stage or not to stage: determining the true clinical significance of the biopsy tract through perinephric fat in assessing renal cell carcinoma.

AIMS: Perinephric fat invasion (PFI) is a key component of renal cell carcinoma (RCC) staging, but there are limited data pertaining to biopsy tract seeding (BTS) resulting in perirenal tissue involvement [BTS with perinephric fat invasion (BTS-P)].The aim is to correlate clinical outcomes with pathologic stage to determine whether the presence of BTS-P should be considered a criterion to stage RCC as part of the pT3a category in the absence of any other upstaging variables. MATERIALS AND RESULTS: We identified 304 renal biopsies from patients with subsequent nephrectomies for RCC; 33 of the tumours contained PFI. Each case was reviewed to determine the presence of BTS-P and other forms of invasion [e.g. non-BTS-P PFI, sinus fat invasion (SFI), and/or renal vein invasion (RVI)], and these findings were compared with survival outcomes. Ten (30%) of 33 tumours with PFI showed BTS-P as the only finding, and were otherwise pT1 tumours; six (60%) patients were alive without disease (AWOD) (mean, 77.5 months), three were lost to follow-up (LTF), and one died of other disease (DOOD). Two patients showed true PFI plus BTS-P; one was LTF and one is AWOD at 107 months. Ten (43%) of 23 patients with tumours with true invasion (PFI ± SFI and/or RVI) are AWOD (mean, 97.7 months), eight (35%) died of disease (DOD), four were LTF, and one DOOD. Kaplan-Meier survival curves showed that the cancer-specific survival was significantly worse in patients with true invasion (P = 0.044) than in those with BTS-P as the sole finding. CONCLUSION: Patients with tumours showing BTS-P only appear to have better outcomes than those with other non-PFI invasion, suggesting that this finding should not be upstaged to pT3a. Additional studies are needed to corroborate the significance of our observations.

Update on Renal Neoplasms: Clinicopathologic-Radiologic Correlation With Case-Based Examples.

OBJECTIVE. This article provides a brief overview of the clinicopathologic and radiologic correlation of 12 renal neoplasms, encompassing the conventional subtypes of renal cell carcinoma and a few of the newly recognized subtypes from the 2016 World Health Organization classification of renal tumors. In addition, we touch upon infrequent neoplasms that may enter the differential diagnosis of a renal mass, with corresponding radiologic and gross images and histologic findings of case-based examples. CONCLUSION. Familiarity with the radiologic and pathologic characteristics of renal cell carcinoma and other renal neoplasms is important to correctly identify and treat these masses.

RAC1-Dependent ORAI1 Translocation to the Leading Edge Supports Lamellipodia Formation and Directional Persistence.

Tumor invasion requires efficient cell migration, which is achieved by the generation of persistent and polarized lamellipodia. The generation of lamellipodia is supported by actin dynamics at the leading edge where a complex of proteins known as the WAVE regulatory complex (WRC) promotes the required assembly of actin filaments to push the front of the cell ahead. By using an U2OS osteosarcoma cell line with high metastatic potential, proven by a xenotransplant in zebrafish larvae, we have studied the role of the plasma membrane Ca2+ channel ORAI1 in this process. We have found that epidermal growth factor (EGF) triggered an enrichment of ORAI1 at the leading edge, where colocalized with cortactin (CTTN) and other members of the WRC, such as CYFIP1 and ARP2/3. ORAI1-CTTN co-precipitation was sensitive to the inhibition of the small GTPase RAC1, an upstream activator of the WRC. RAC1 potentiated ORAI1 translocation to the leading edge, increasing the availability of surface ORAI1 and increasing the plasma membrane ruffling. The role of ORAI1 at the leading edge was studied in genetically engineered U2OS cells lacking ORAI1 expression that helped us to prove the key role of this Ca2+ channel on lamellipodia formation, lamellipodial persistence, and cell directness, which are required for tumor cell invasiveness in vivo.

Updates in Histologic Grading of Urologic Neoplasms.

CONTEXT.­: Tumor histology offers a composite view of the genetic, epigenetic, proteomic, and microenvironmental determinants of tumor biology. As a marker of tumor histology, histologic grading has persisted as a highly relevant factor in risk stratification and management of urologic neoplasms (ie, renal cell carcinoma, prostatic adenocarcinoma, and urothelial carcinoma). Ongoing research and consensus meetings have attempted to improve the accuracy, consistency, and biologic relevance of histologic grading, as well as provide guidance for many challenging scenarios. OBJECTIVE.­: To review the most recent updates to the grading system of urologic neoplasms, including those in the 2016 4th edition of the World Health Organization (WHO) Bluebook, with emphasis on issues encountered in routine practice. DATA SOURCES.­: Peer-reviewed publications and the 4th edition of the WHO Bluebook on the pathology and genetics of the urinary system and male genital organs. CONCLUSIONS.­: This article summarizes the recently updated grading schemes for renal cell carcinoma, prostate adenocarcinomas, and bladder neoplasms of the genitourinary tract.

PNL2: A Useful Adjunct Biomarker to HMB45 in the Diagnosis of Uterine Perivascular Epithelioid Cell Tumor (PEComa).

Perivascular epithelioid cell tumors (PEComa) are rare neoplasms characterized by co-expression of melanocytic and muscle markers. HMB45 and Melan-A are used to confirm a PEComa diagnosis; however, both are often focally expressed and sensitivity for Melan-A is low. PNL2 is a reliable biomarker for epithelioid melanoma and renal angiomyolipoma/PEComa. The objective of this study was to determine PNL2 utility in diagnosing uterine PEComas as well as distinguishing PEComas from uterine smooth muscle tumors (SMTs). Twenty-one uterine PEComas and 45 SMTs were analyzed for PNL2; a subset was also stained for HMB45, Melan-A, Cathepsin-K, Desmin, and h-Caldesmon. Cases were scored as negative (0), focal (<10% of tumor cells), or patchy to diffusely positive (>10% of tumor cells). PEComas were positive for PNL2, HMB45, and Melan-A in 86%, 100%, and 57% of cases, respectively. In PEComas, PNL2 was patchy to diffusely positive more frequently (10/18, 56%) than Melan-A (4/12, 33%). In contrast, 2 of 45 (4%) SMTs were focally PNL2 positive; HMB45 was focally positive in 4 SMTs (11%) and all were negative for Melan-A. Desmin and h-Caldesmon were positive in 90% and 57% of PEComas, and 91% and 82% of SMTs. Cathepsin-K was positive in 100% of PEComas and 93% of SMTs. PNL2 is a useful biomarker for the diagnosis of uterine PEComa, with comparable sensitivity and specificity to HMB45. In contrast, PNL2 stains more PEComas when compared with Melan-A. Cathepsin-K, Desmin, and h-Caldesmon are of little utility for distinguishing PEComas and SMTs; however, lack of Cathepsin-K argues against PEComa. These results suggest that PNL2 should be used in conjunction with HMB45 in the diagnosis of PEComa of the uterine corpus.

Correction to: STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry.

The original publication of this paper contains errors.

Demographic and seasonal characteristics of respiratory pathogens in neonates and infants aged 0 to 12 months in the Central-East region of Tunisia.

BACKGROUND: This study aimed to characterize the epidemiology of pathogenic respiratory agents in patients aged 0 to 12 months and hospitalized for acute respiratory infections in Tunisia between 2013 and 2014. METHODS: A total of 20 pathogens, including viruses, Mycoplasma pneumoniae, and Streptococcus pneumoniae, were detected using molecular sensitive assays, and their associations with the patient's demographic data and season were analyzed. RESULTS: Viral infectious agents were found in 449 (87.2%) of 515 specimens. Dual and multiple infectious agents were detected in 31.4% and 18.6% of the samples, respectively. Viral infection was predominant in the pediatric environment (90.8%, P < 0.001), male patients (88.0%), and spring (93.8%). Rhinovirus was the most detected virus (51.8%) followed by respiratory syncytial virus A/B (34.4%), coronavirus group (18.5%), adenovirus (17.9%), and parainfluenza viruses 1-4 (10.9%). Respiratory Syncytial virus A/B was significantly associated with gender (38.0% male cases vs 28.3% female cases, P = 0.02). Infections by Adenovirus, Bocavirus, and Metapneumovirus A/B increased with increasing age of patients (predominated cases aged 6-12 months, P < 0.001). S. pneumoniae was detected in 30.9% of th tested samples. In 18.2% of the negative viral infections, only S. pneumoniae was identified. CONCLUSION: A predominance of the rhinovirus infection was observed in this study. Coronavirus subtypes were described for the first time in Tunisia. The observed different pathogenic profiles across age groups could be helpful to avoid the misclassification of patients presenting with ARIs at the triage level when no standardized protocol is available. This study will provide clues for physicians informing decisions regarding preventive strategies and medication in Tunisia.

STIM1 deficiency is linked to Alzheimer's disease and triggers cell death in SH-SY5Y cells by upregulation of L-type voltage-operated Ca2+ entry.

STIM1 is an endoplasmic reticulum protein with a role in Ca2+ mobilization and signaling. As a sensor of intraluminal Ca2+ levels, STIM1 modulates plasma membrane Ca2+ channels to regulate Ca2+ entry. In neuroblastoma SH-SY5Y cells and in familial Alzheimer's disease patient skin fibroblasts, STIM1 is cleaved at the transmembrane domain by the presenilin-1-associated γ-secretase, leading to dysregulation of Ca2+ homeostasis. In this report, we investigated expression levels of STIM1 in brain tissues (medium frontal gyrus) of pathologically confirmed Alzheimer's disease patients, and observed that STIM1 protein expression level decreased with the progression of neurodegeneration. To study the role of STIM1 in neurodegeneration, a strategy was designed to knock-out the expression of STIM1 gene in the SH-SY5Y neuroblastoma cell line by CRISPR/Cas9-mediated genome editing, as an in vitro model to examine the phenotype of STIM1-deficient neuronal cells. It was proved that, while STIM1 is not required for the differentiation of SH-SY5Y cells, it is absolutely essential for cell survival in differentiating cells. Differentiated STIM1-KO cells showed a significant decrease of mitochondrial respiratory chain complex I activity, mitochondrial inner membrane depolarization, reduced mitochondrial free Ca2+ concentration, and higher levels of senescence as compared with wild-type cells. In parallel, STIM1-KO cells showed a potentiated Ca2+ entry in response to depolarization, which was sensitive to nifedipine, pointing to L-type voltage-operated Ca2+ channels as mediators of the upregulated Ca2+ entry. The stable knocking-down of CACNA1C transcripts restored mitochondrial function, increased mitochondrial Ca2+ levels, and dropped senescence to basal levels, demonstrating the essential role of the upregulation of voltage-operated Ca2+ entry through Cav1.2 channels in STIM1-deficient SH-SY5Y cell death. KEY MESSAGES: STIM1 protein expression decreases with the progression of neurodegeneration in Alzheimer's disease. STIM1 is essential for cell viability in differentiated SH-SY5Y cells. STIM1 deficiency triggers voltage-regulated Ca2+ entry-dependent cell death. Mitochondrial dysfunction and senescence are features of STIM1-deficient differentiated cells.

Utility of Immunohistochemistry in Distinguishing Primary Adnexal Carcinoma From Metastatic Breast Carcinoma to Skin and Squamous Cell Carcinoma.

The distinction between primary adnexal carcinoma (PAC) from metastatic breast carcinoma (BrCa) to skin and squamous cell carcinoma (SCC), particularly those with ductal differentiation (SCCDD), can be quite challenging, despite adequate history. The aim of the study was to determine the utility of an immunohistochemistry (IHC) panel to differentiate these entities and apply them to ambiguous tumors. Twenty-seven PAC, 7 metastatic BrCa, 28 SCC, and 16 ambiguous cases (SCCDD vs. PAC, n = 13 and metastatic BrCa vs. PAC, n = 3) were analyzed using CD23, PAX5, D2-40, P63, and CD117 immunohistochemistry. A total of 9 (33%) PAC were CD117 positive, whereas all metastatic BrCa and SCC were negative (P = 0.0002). D2-40 was expressed in 16 (59%) PAC and 16 (57%) SCC cases, but none of the metastatic BrCa cases (P = 0.0041). Of the 13 ambiguous tumors with a differential diagnosis of SCCDD versus PAC, all were positive for P63, 10 were positive for D2-40, and 1 was positive for CD117. Of the 3 ambiguous tumors with a differential diagnosis of PAC versus metastatic BrCA, 2 were positive for CD117, whereas none showed reactivity for D2-40 or P63. All cases were negative for CD23 and PAX5. Our study indicates that CD117 reactivity favors a PAC with a sensitivity and specificity of 33% and 100%, respectively. D2-40 and P63 expression highlighted both PAC and SCC and seems to be useful in excluding metastatic BrCa with a sensitivity and specificity of 58% and 100%, and 98% and 100%, respectively. Despite previous reports, CD23 and PAX5 do not seem to be useful.

The Value of Direct Immunofluorescence on Proteinase-Digested Formalin-Fixed Paraffin-Embedded Skin Biopsies.

Direct immunofluorescence (DIF) on frozen tissue (DIF-F) is the method of choice for the identification of immune deposits present in skin and other tissues. DIF can also be performed on formalin-fixed paraffin-embedded tissue (DIF-P) after antigen retrieval with proteases and has proven to be of value in renal pathology. However, its utility in skin biopsies has not been fully examined. In this study, we performed DIF-P on 60 skin biopsies that comprised of bullous pemphigoid (n = 18), pemphigoid gestationis (n = 1), pemphigus (n = 7), linear IgA disease (n = 7), vasculitis (n = 20), lupus erythematosus (n = 3), and dermatitis herpetiformis (n = 4) cases. We compared the results of DIF-P with those of DIF-F from the same patients. The diagnostic features were found in 15 of 19 (79%) pemphigoid (bullous pemphigoid and pemphigoid gestationis), 3 of 7 (43%) pemphigus, 3 of 7 (43%) linear IgA disease, 14 of 20 (70%) vasculitis, 1 of 3 (33%) lupus erythematosus, and none (0%) of the dermatitis herpetiformis cases tested. Overall, DIF-P is less sensitive than DIF-F but seems to be a valuable technique that could aid in the diagnosis of vasculitides, immunobullous, and connective tissue disorders when fresh tissue is unavailable.

Epidemiology and clinical profile of pathogens responsible for the hospitalization of children in Sousse area, Tunisia.

This study aimed to identify a broad spectrum of respiratory pathogens from hospitalized and not-preselected children with acute respiratory tract infections in the Farhat Hached University-hospital of Sousse, Tunisia. Between September 2013 and December 2014, samples from 372 children aged between 1 month and 5 years were collected, and tested using multiplex real-time RT-PCR by a commercial assay for 21 respiratory pathogens. In addition, samples were screened for the presence of Streptococcus pneumoniae 16S rDNA using real-time PCR. The viral distribution and its association with clinical symptoms were statistically analyzed. Viral pathogens were detected in 342 (91.93%) of the samples of which 28.76% were single positive and 63.17% had multiple infections. The most frequent detected viruses were rhinovirus (55.64%), respiratory syncytial virus A/B (33.06%), adenovirus (25.00%), coronavirus NL63, HKU1, OC43, and 229E (21.50%), and metapneumovirus A/B (16.12%). Children in the youngest age group (1-3 months) exhibited the highest frequencies of infection. Related to their frequency of detection, RSV A/B was the most associated pathogen with patient's demographic situation and clinical manifestations (p<0.05). Parainfluenza virus 1-4 and parechovirus were found to increase the risk of death (p<0.05). Adenovirus was statistically associated to the manifestation of gastroenteritis (p = 0.004). Rhinovirus infection increases the duration of oxygen support (p = 0.042). Coronavirus group was statistically associated with the manifestation of bronchiolitis (p = 0.009) and laryngitis (p = 0.017). Streptococcus pneumoniae DNA was detected in 143 (38.44%) of tested samples. However, only 53 samples had a concentration of C-reactive protein from equal to higher than 20 milligrams per liter, and 6 of them were single positive for Streptocuccus pneumoniae. This study confirms the high incidence of respiratory viruses in children hospitalized for acute respiratory tract infections in the Sousse area, Tunisia.

Transcription factor TFEB cell-autonomously modulates susceptibility to intestinal epithelial cell injury in vivo.

Understanding the transcription factors that modulate epithelial resistance to injury is necessary for understanding intestinal homeostasis and injury repair processes. Recently, transcription factor EB (TFEB) was implicated in expression of autophagy and host defense genes in nematodes and mammalian cells. However, the in vivo roles of TFEB in the mammalian intestinal epithelium were not known. Here, we used mice with a conditional deletion of Tfeb in the intestinal epithelium (Tfeb ΔIEC) to examine its importance in defense against injury. Unperturbed Tfeb ΔIEC mice exhibited grossly normal intestinal epithelia, except for a defect in Paneth cell granules. Tfeb ΔIEC mice exhibited lower levels of lipoprotein ApoA1 expression, which is downregulated in Crohn's disease patients and causally linked to colitis susceptibility. Upon environmental epithelial injury using dextran sodium sulfate (DSS), Tfeb ΔIEC mice exhibited exaggerated colitis. Thus, our study reveals that TFEB is critical for resistance to intestinal epithelial cell injury, potentially mediated by APOA1.

Store-operated calcium entry is dispensable for the activation of ERK1/2 pathway in prostate cancer cells.

STIM1, the endoplasmic reticulum Ca2+ sensor that modulates the activity of plasma membrane Ca2+ channels, becomes phosphorylated at ERK1/2 target sites during Ca2+ store depletion triggered by thapsigargin or epidermal growth factor (EGF). This ERK1/2-dependent phosphorylation regulates STIM1 localization and dissociation from microtubules, and it is known that enhances the binding to ORAI1, a store-operated Ca2+ entry (SOCE) channel, leading to the activation of this Ca2+ influx pathway. However, there remained some evidence of a role for SOCE in the activation of ERK1/2, and here we assessed the contribution of SOCE to ERK1/2 activation by generating a STIM1-deficient cell line by CRISPR/Cas9 genome editing of the STIM1 locus in prostate cancer PC3 cells. The genomic modification consisted of a 16 base-pair insertion in exon 5 of both alleles, therefore abrogating STIM1 synthesis. STIM1-KO cells did show a striking decrease in Ca2+ influx in response to thapsigargin or EGF, a result that demonstrates that SOCE mediates Ca2+ entry in PC3 cells during stimulation with EGF. Moreover, identical levels of total ERK1/2 were found in STIM1-KO cells and the parental cell line, and ERK1/2 activation was fully activated in KO cells, both in the presence and in the absence of extracellular Ca2+, a result that supports that STIM1 and SOCE are not required for ERK1/2 activation. This activation was sensitive to Src kinase inhibition, but not to CAMKII nor PKC inhibition, a result that sets STIM1 and SOCE as downstream targets of the axis Src-Raf-MEK-ERK, rather than upstream regulators.

Regulation of membrane ruffling by polarized STIM1 and ORAI1 in cortactin-rich domains.

Cell motility and migration requires the reorganization of the cortical cytoskeleton at the leading edge of cells and extracellular Ca2+ entry is essential for this reorganization. However the molecular nature of the regulators of this pathway is unknown. This work contributes to understanding the role of STIM1 and ORAI1 in the promotion of membrane ruffling by showing that phospho-STIM1 localizes at the leading edge of cells, and that both phospho-STIM1 and ORAI1 co-localize with cortactin (CTTN), a regulator of the cytoskeleton at membrane ruffling areas. STIM1-KO and ORAI1-KO cell lines were generated by CRISPR/Cas9 genome editing in U2OS cells. In both cases, KO cells presented a notable reduction of store-operated Ca2+ entry (SOCE) that was rescued by expression of STIM1-mCherry and ORAI1-mCherry. These results demonstrated that SOCE regulates membrane ruffling at the leading edge of cells. Moreover, endogenous ORAI1 and overexpressed ORAI1-GFP co-immunoprecipitated with endogenous CTTN. This latter result, in addition to the KO cells' phenotype, the preservation of ORAI1-CTTN co-localization during ruffling, and the inhibition of membrane ruffling by the Ca2+-channel inhibitor SKF96365, further supports a functional link between SOCE and membrane ruffling.

Pre-vaccine era cervical human papillomavirus infection among screening population of women in west Austria.

BACKGROUND: In order to evaluate the newly implemented gender-neutral HPV vaccination program, knowledge on the pre-vaccine prevalence of HPV infection is of paramount importance. Data on HPV infection among the women with no known previous cytological abnormalities are inexistent in Austria. This study presents data on the prevalence and distribution of HPV genotypes among women with no known cytological abnormalities in west Austria. METHODS: Women between 18 and 65 years of age attending annual cervical cancer screening examinations were included in the study. Data on socio-demographic and reproductive factors were collected using structured questionnaires. Corresponding cervical swab samples were tested for the presence of HPV DNA and were genotyped. Questionnaire data and HPV status were linked with the corresponding cytological findings. RESULTS: A total of 542 women were included in the study. The mean age of the study participants was 35.9 (SD = 11.5). The prevalence of HPV infection was 20.5 %. HPV 16 (6.5 %), HPV 33 (3.3 %) and HPV 31 (3.0 %) were the dominant genotypes detected. Multivariate analysis showed that women younger than 30 years of age, smokers, women with a higher number of lifetime sexual partners and those living in the eastern districts of the study region were at significantly higher risk of HPV infection. CONCLUSIONS: With this study we present the first data on the prevalence of cervical HPV genotypes among a screening population in Austria. The results not only fill the missing information on HPV infection in this group of women in the country, they also provide baseline data for a future evaluation of the impact of the Austrian gender-neutral HPV immunization program. Moreover, our finding of higher HPV prevalence in the eastern compared to the western district of the study region may - at least partly - explain the east-west gradient in the standardized incidence rate of cervical cancer in the region.

Phospho-STIM1 is a downstream effector that mediates the signaling triggered by IGF-1 in HEK293 cells.

STIM1 is a Ca(2+) sensor of the endoplasmic reticulum (ER) that triggers the activation of plasma membrane Ca(2+) channels upon depletion of Ca(2+) levels within the ER. During thapsigargin-triggered Ca(2+) store depletion, ERK1/2 phosphorylates STIM1 at Ser575, Ser608, and Ser621. This phosphorylation plays a role in the regulation of STIM1 dissociation from the microtubule plus-end binding protein EB1, an essential step for STIM1 activation by thapsigargin. However, little is known regarding the physiological role of this phosphorylation. Because IGF-1 triggers the activation of the RAF-MEK-ERK and the phosphoinositide pathways, the role of STIM1 phosphorylation in IGF-1 stimulation was studied. There was found to be phosphorylation of ERK1/2 in both the presence and the absence of extracellular Ca(2+), demonstrating that Ca(2+) influx is not essential for ERK1/2 activation. In parallel, IGF-1 triggered STIM1 phosphorylation at the aforementioned sites, an effect that was blocked by PD0325901, a MEK1/2 inhibitor used to block ERK1/2 activation. Also, STIM1-GFP was found in clusters upon IGF-1 stimulation, and STIM1-S575A/S608A/S621A-GFP strongly reduced this multimerization. Interestingly, phospho-STIM1 was mainly found in clusters when cells were treated with IGF-1, and IGF-1 triggered the dissociation of STIM1 from EB1, similarly to what has been observed for thapsigargin, suggesting that STIM1 mediates the IGF-1 signaling pathway. A study of IGF-1-stimulated NFAT translocation was therefore performed, finding that STIM1-S575A/S608A/S621A blocked this translocation, as did the fusion protein STIM1-EB1, confirming that both STIM1 phosphorylation and STIM1-EB1 dissociation are required for IGF-1-triggered Ca(2+)-dependent signaling, and demonstrating that STIM1 phosphorylation plays a role as a downstream effector of the RAF-MEK-ERK pathway and an upstream activator of Ca(2+) entry.

STIM1 phosphorylation triggered by epidermal growth factor mediates cell migration.

STIM1 is a key regulator of store-operated calcium entry (SOCE), and therefore a mediator of Ca²âº entry-dependent cellular events. Phosphorylation of STIM1 at ERK1/2 target sites has been described as enhancing STIM1 activation during intracellular Ca²âº emptying triggered by the inhibition of the sarco(endo)plasmic Ca²âº -ATPase with thapsigargin. However, no physiological function is known for this specific phosphorylation. The present study examined the role of STIM1 phosphorylation in cell signaling triggered by EGF. Using a human endometrial adenocarcinoma cell line (Ishikawa cells) EGF or H-Ras(G12V), an active mutant of H-Ras, was found to trigger STIM1 phosphorylation at residues Ser575, Ser608, and Ser621, and this process was sensitive to PD0325901, an inhibitor of ERK1/2. Both, ERK1/2 activation and STIM1 phosphorylation took place in the absence of extracellular Ca²âº, indicating that both events are upstream steps for Ca²âºentry activation. Also, EGF triggered the dissociation of STIM1 from EB1 (a regulator of microtubule plus-ends) in a manner similar to that reported for the activation of STIM1 by thapsigargin. Migration of the Ishikawa cells was impaired when STIM1 phosphorylation was targeted by Ser-to-Ala substitution mutation of ERK1/2 target sites. This effect was also observed with the Ca²âº channel blocker SKF96365. Phosphomimetic mutation of STIM1 restored the migration to levels similar to that found for STIM1-wild type. Finally, the increased vimentin expression and relocalization of E-cadherin triggered by EGF were largely inhibited by targeting STIM1 phosphorylation, while STIM1-S575E/S608E/S621E normalized the profiles of these two EMT markers.