Characterisation and expression of SPLUNC2, the human orthologue of rodent parotid secretory protein

We recently described the Palate Lung Nasal Clone (PLUNC) family of proteins as an extended group of proteins expressed in the upper airways, nose and mouth. Little is known about these proteins, but they are secreted into the airway and nasal lining fluids and saliva where, due to their structural similarity with lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein, they may play a role in the innate immune defence. We now describe the generation and characterisation of novel affinity-purified antibodies to SPLUNC2, and use them to determine the expression of this, the major salivary gland PLUNC. Western blotting showed that the antibodies identified a number of distinct protein bands in saliva, whilst immunohistochemical analysis demonstrated protein expression in serous cells of the major salivary glands and in the ductal lumens as well as in cells of minor mucosal glands. Antibodies directed against distinct epitopes of the protein yielded different staining patterns in both minor and major salivary glands. Using RT-PCR of tissues from the oral cavity, coupled with EST analysis, we showed that the gene undergoes alternative splicing using two 5' non-coding exons, suggesting that the gene is regulated by alternative promoters. Comprehensive RACE analysis using salivary gland RNA as template failed to identify any additional exons. Analysis of saliva showed that SPLUNC2 is subject to N-glycosylation. Thus, our study shows that multiple SPLUNC2 isoforms are found in the oral cavity and suggest that these proteins may be differentially regulated in distinct tissues where they may function in the innate immune response

Modulation of Experimental Herpes Encephalitis-Associated Neurotoxicity through Sulforaphane Treatment

Reactive oxygen species (ROS) produced by brain-infiltrating macrophages and neutrophils, as well as resident microglia, are pivotal to pathogen clearance during viral brain infection. However, unchecked free radical generation is also responsible for damage to and cytotoxicity of critical host tissue bystander to primary infection. These unwanted effects of excessive ROS are combated by local cellular production of antioxidant enzymes, including heme oxygenase-1 (HO-1) and glutathione peroxidase 1 (Gpx1). In this study, we showed that experimental murine herpes encephalitis triggered robust ROS production, as well as an opposing upregulation of the antioxidants HO-1 and Gpx1. This antioxidant response was insufficient to prevent tissue damage, neurotoxicity, and mortality associated with viral brain infection. Previous studies corroborate our data supporting astrocytes as the major antioxidant producer in brain cell cultures exposed to HSV-1 stimulated microglia. We hypothesized that stimulating opposing antioxidative responses in astrocytes, as well as neurons, would mitigate the effects of ROS-mediated neurotoxicity both in vitro and during viral brain infection in vivo. Here, we demonstrate that the addition of sulforaphane, a potent stimulator of antioxidant responses, enhanced HO-1 and Gpx1 expression in astrocytes through the activation of nuclear factor-E2-related factor 2 (Nrf2). Additionally, sulforaphane treatment was found to be effective in reducing neurotoxicity associated with HSV-stimulated microglial ROS production. Finally, intraperitoneal injections of sulforaphane into mice during active HSV infection reduced neuroinflammation via a decrease in brain-infiltrating leukocytes, macrophage- and neutrophil-produced ROS, and MHCII-positive, activated microglia. These data support a key role for astrocyte-produced antioxidants in modulating oxidative stress and neuronal damage in response to viral infection

Effects of interactions between the constituents of chitosan-edible films on their physical properties

The main objective of this work was to evaluate the effect of chitosan and plasticizer concentrations and oil presence on the physical and mechanical properties of edible films. The effect of the film constituents and their in-between interactions were studied through the evaluation of permeability, opacity and mechanical properties. The effects of the studied variables (concentrations of chitosan, plasticizer and oil) were analysed according to a 2 3 factorial design. Pareto charts were used to identify the most significant factors in the studied properties (water vapour, oxygen and carbon dioxide permeability; opacity; tensile strength; elongation at break and Young's modulus). When addressing the influence of the interactions between the films' constituents on the properties above, results show that chitosan and plasticizer concentrations are the most significant factors affecting most of the studied properties, while oil incorporation has shown to be of a great importance in the particular case of transport properties (gas permeability), essentially due to its hydrophobicity. Water vapour permeability values (ranging from 1. 62 × 10 -11 to 4. 24 × 10 -11 g m -1 s -1 Pa -1) were half of those reported for cellophane films. Also the mechanical properties (tensile strength values from 0. 43 to 13. 72 MPa and elongation-at-break values from 58. 62% to 166. 70%) were in the range of those reported for LDPE and HDPE. Based on these results, we recommend the use of 1. 5% (w/w) chitosan concentration to produce films, where the oil and plasticizer proportions will have to be adjusted in a case-by-case basis according to the use intended for the material. This work provides a useful guide to the formulation of chitosan-based film-forming solutions for food packaging applications.The author MA Cerqueira is a recipient of a fellowship from Fundacao para a Ciencia e Tecnologia (FCT, SFRH/BD/23897/2005) and BWS Souza is a recipient of a fellowship from the Coordenacao Aperfeicoamento de Pessoal de Nivel Superior, Brazil (Capes, Brazil)

Performance of the CMS Cathode Strip Chambers with Cosmic Rays

The Cathode Strip Chambers (CSCs) constitute the primary muon tracking device in the CMS endcaps. Their performance has been evaluated using data taken during a cosmic ray run in fall 2008. Measured noise levels are low, with the number of noisy channels well below 1%. Coordinate resolution was measured for all types of chambers, and fall in the range 47 microns to 243 microns. The efficiencies for local charged track triggers, for hit and for segments reconstruction were measured, and are above 99%. The timing resolution per layer is approximately 5 ns

TAT-Mediated Transduction of MafA Protein In Utero Results in Enhanced Pancreatic Insulin Expression and Changes in Islet Morphology

Alongside Pdx1 and Beta2/NeuroD, the transcription factor MafA has been shown to be instrumental in the maintenance of the beta cell phenotype. Indeed, a combination of MafA, Pdx1 and Ngn3 (an upstream regulator of Beta2/NeuroD) was recently reported to lead to the effective reprogramming of acinar cells into insulin-producing beta cells. These experiments set the stage for the development of new strategies to address the impairment of glycemic control in diabetic patients. However, the clinical applicability of reprogramming in this context is deemed to be poor due to the need to use viral vehicles for the delivery of the above factors. Here we describe a recombinant transducible version of the MafA protein (TAT-MafA) that penetrates across cell membranes with an efficiency of 100% and binds to the insulin promoter in vitro. When injected in utero into living mouse embryos, TAT-MafA significantly up-regulates target genes and induces enhanced insulin production as well as cytoarchitectural changes consistent with faster islet maturation. As the latest addition to our armamentarium of transducible proteins (which already includes Pdx1 and Ngn3), the purification and characterization of a functional TAT-MafA protein opens the door to prospective therapeutic uses that circumvent the use of viral delivery. To our knowledge, this is also the first report on the use of protein transduction in utero

RhoE Is Regulated by Cyclic AMP and Promotes Fusion of Human BeWo Choriocarcinoma Cells

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N6-phenyl-cAMP, and a specific inhibitor of PKA (14–22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways

Chromosomal amplifications, 3q gain and deletions of 2q33-q37 are the frequent genetic changes in cervical carcinoma

BACKGROUND: Carcinoma of uterine cervix is the second most common cancers among women worldwide. Combined radiation and chemotherapy is the choice of treatment for advanced stages of the disease. The prognosis is poor, with a five-year survival rate ranging from about 20–65%, depending on stage of the disease. Therefore, genetic characterization is essential for understanding the biology and clinical heterogeneity in cervical cancer (CC). METHODS: We used a genome-wide screening method – comparative genomic hybridization (CGH) to identify DNA copy number changes in 77 patients with cervical cancer. We applied categorical and survival analyses to analyze whether chromosomal changes were related to clinico-pathologic characteristics and patients survival. RESULTS: The CGH analysis revealed a loss of 2q33-q37 (57.1%), gain of 3q (54.5%) and chromosomal amplifications (20.77%) as frequent genetic changes. A total of 15 amplified chromosomal sites were detected in 16 cases that include 1p31, 2q32, 7q22, 8q21.2-q24, 9p22, 10q21, 10q24, 11q13, 11q21, 12q15, 14q12, 17p11.2, 17q22, 18p11.2, and 19q13.1. Recurrent amplified sites were noted at 11q13, 11q21, and 19q13.1. The genomic alterations were further evaluated for prognostic significance in CC patients, and we did not find any correlation with a number of clinical or histological parameters. The tumors harboring HPV18 exhibited higher genomic instability compared to tumors with HPV 16. CONCLUSIONS: This study demonstrated that 2q33-q37 deletions, 3q gains and chromosomal amplifications as characteristic changes in invasive CC. These genetic alterations will aid in the identification of novel tumor suppressor gene(s) at 2q33-q37 and oncogenes at amplified chromosomal sites. Molecular characterization of these chromosomal changes utilizing the current genomic technologies will provide new insights into the biology and clinical behavior of CC

The Sigma-trial protocol: a prospective double-blind multi-centre comparison of laparoscopic versus open elective sigmoid resection in patients with symptomatic diverticulitis

BACKGROUND: Diverticulosis is a common disease in the western society with an incidence of 33-66%. 10-25% of these patients will develop diverticulitis. In order to prevent a high-risk acute operation it is advised to perform elective sigmoid resection after two episodes of diverticulitis in the elderly patient or after one episode in the younger ( 50 years or in case of progressive abdominal complaints due to strictures caused by a previous episode of diverticulits. The diagnosis is confirmed by CT-scan, barium enema and/or coloscopy.It is required that the participating surgeons have performed at least 15 laparoscopic and open sigmoid resections. Open resection is performed by median laparotomy, laparoscopic resection is approached by 4 or 5 cannula. Sigmoid and colon which contain serosal changes or induration are removed and a tension free anastomosis is created. After completion of either surgical procedure an opaque dressing will be used, covering from 10 cm above the umbilicus to the pubic bone. Surgery details will be kept separate from the patient's notes.Primary endpoints are the postoperative morbidity and mortality. We divided morbidity in minor (e.g. wound infection), major (e.g. anastomotic leakage) and late (e.g. incisional hernias) complications, data will be collected during hospital stay and after six weeks and six months postoperative. Secondary endpoints are the operative and the postoperative recovery data. Operative data include duration of the operation, blood loss and conversion to laparotomy. Post operative recovery consists of return to normal diet, pain, analgesics, general health (SF-36 questionnaire) and duration of hospital stay. DISCUSSION: The Sigma-trial is a prospective, multi-center, double-blind, randomized study to define the role of laparoscopic sigmoid resection in patients with symptomatic diverticuliti

Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation

Background: Cyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth. Results: Using the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism. Conclusions: Directional RNA-seq data were obtained that provide comprehensive mapping of transcript boundaries and abundance for all transcribed RNAs in Anabaena filaments during the response to nitrogen deprivation. We have identified genes and noncoding RNAs that are transcriptionally regulated during heterocyst development. These data provide detailed information on the Anabaena transcriptome as filaments undergo heterocyst development and begin nitrogen fixation