Characterization of a disease-associated mutation affecting a putative splicing regulatory element in intron 6b of the cystic fibrosis transmembrane conductance regulator (CFTR) gene

Cystic fibrosis (CF) is a common recessive disorder caused by >1600 mutations in the CF transmembrane conductance regulator (CFTR) gene. About 13% of CFTR mutations are classified as “splicing mutations,” but for almost 40% of these, their role in affecting the pre-mRNA splicing of the gene is not yet defined. In this work, we describe a new splicing mutation detected in three unrelated Italian CF patients. By DNA analyses and mRNA studies, we identified the c.1002–1110_1113delTAAG mutation localized in intron 6b of the CFTR gene. At the mRNA level, this mutation creates an aberrant inclusion of a sequence of 101 nucleotides between exons 6b and 7. This sequence corresponds to a portion of intron 6b and resembles a cryptic exon because it is characterized by an upstream ag and a downstream gt sequence, which are most probably recognized as 5′- and 3′-splice sites by the spliceosome. Through functional analysis of this splicing defect, we show that this mutation abolishes the interaction of the splicing regulatory protein heterogeneous nuclear ribonucleoprotein A2/B1 with an intronic splicing regulatory element and creates a new recognition motif for the SRp75 splicing factor, causing activation of the cryptic exon. Our results show that the c.1002–1110_1113delTAAG mutation creates a new intronic splicing regulatory element in intron 6b of the CFTR gene exclusively recognized by SRp75

Efficacy of periportal infiltration and intraperitoneal instillation of ropivacaine after laparoscopic surgery in children

Postoperative pain is less intense after laparoscopic than after open surgery. However, minimally invasive surgery is not a a pain-free procedure. Many trials have been done in adults using intraperitoneal and/or incisional local anesthetic, but similar studies have not yet been reported in the literature in children. Aim: The aim of this study was to evaluate the analgesic effect of periportal infiltration and intraperitoneal instillation of ropivacaine in children undergoing laparoscopic surgery. Materials and Methods: Thirty patients who underwent laparoscopic surgery were randomly allocated to one of three groups. Group A (n 10) received local infiltration of port sites with 10 mL of ropivacaine. Group B (n 10) received both an infiltration of port sites with 10 mL of ropivacaine and an intraperitoneal instillation of 10 mL of ropivacaine. Group C did not receive any analgesic treatment. The local anesthetic was always administered at the end of surgery. The degree of postoperative abdominal parietal pain, abdominal visceral pain, and shoulder pain was assessed by using a Wong-Baker pain scale and a Visual Analog Scale (VAS) at 3, 6 12, and 24 hours postoperatively. The following parameters were also evaluated: rescue analgesic treatment, length of hospital stay, and time of return to normal activities. Results: Three hours after operation, patients had low pain scores. Six and 12 hours postoperatively, the abdominal parietal pain was significantly higher (P 0.0005) in group C than in the other two groups, both treated with an infiltration at the trocar sites; mean intensity of abdominal visceral pain was significantly lower (P 0.0005) in group B than in groups A and C; the overall incidence of shoulder pain was significantly lower (P 0.0005) in group B patients than in patients of groups A and C. At 20 hours postoperatively, pain scores were significantly reduced of intensity in all groups. Rescue analgesic treatment was significantly higher in group C, if compared to groups A and B 12 hours after the operation. No statistically significant difference was found in length of hospital stay, but children who received analgesic treatment had a more rapid return to normal activities than untreated patients (P 0.0005). Conclusions: Our study demonstrates that the combination of local infiltration and intraperitoneal instillation of ropivacaine is more effective for pain relief in children after laparoscopic surgery than the administration of ropivacaine only at the trocar sites

Examination of the Relationship between In-Store Environmental Factors and Fruit and Vegetable Purchasing among Hispanics.

Retail food environments have received attention for their influence on dietary behaviors and for their nutrition intervention potential. To improve diet-related behaviors, such as fruit and vegetable (FV) purchasing, it is important to examine its relationship with in-store environmental characteristics. This study used baseline data from the "El Valor de Nuestra Salud" study to examine how in-store environmental characteristics, such as product availability, placement and promotion, were associated with FV purchasing among Hispanic customers in San Diego County. Mixed linear regression models indicated that greater availability of fresh FVs was associated with a $0.36 increase in FV purchasing (p = 0.01). Placement variables, specifically each additional square foot of display space dedicated to FVs (p = 0.01) and each additional fresh FV display (p = 0.01), were associated with a$0.02 increase and $0.29 decrease, respectively, in FV purchasing. Introducing FV promotions in the final model was not related to FV purchasing. Exploratory analyses indicated that men reported spending$3.69 fewer dollars on FVs compared to women, controlling for covariates (p = 0.02). These results can help inform interventions targeting in-store environmental characteristics to encourage FV purchasing among Hispanics

Estimulación del sistema mediado por interferón tipo I de lenguado senegalés (Solea senegalensis) en respuesta a infecciones por nodavirus

El lenguado senegalés es susceptible a la infección por el Virus de la Necrosis Nerviosa Viral (VNNV). Los betanodavirus se clasifican en cuatro genotipos, siendo los genotiposSJNNV, RGNNV y virus recombinantes RGNNV-SJNNV los que causan mortalidad en lenguado. En condiciones experimentales es el recombinante el que provoca mayor mortalidad. Las diferencias en la tasa de mortalidad pueden indicar variaciones en la interacción entre los distintos genotipos y el sistema inmune de lenguado. El sistema del interferón tipo I es un componente esencial de la respuesta inmune frente a infecciones virales, induciendo la expresión de genes que codifican proteínas antivíricas, tales como la Mx, la ISG15 y la PKR. El objetivo del presente trabajo ha sido cuantificar la transcripción de Mx, ISG15 y PKR en respuesta a infecciones por SJNNV, RGNNV y un recombinante RG-SJ.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Co-targeting of Bcl-2 and mTOR pathway triggers synergistic apoptosis in BH3 mimetics resistant acute lymphoblastic leukemia

Several chemo-resistance mechanisms including the Bcl-2 protein family overexpression and constitutive activation of the PI3K/Akt/mTOR signaling have been documented in acute lymphoblastic leukemia (ALL), encouraging targeted approaches to circumvent this clinical problem. Here we analyzed the activity of the BH3 mimetic ABT-737 in ALL, exploring the synergistic effects with the mTOR inhibitor CCI-779 on ABT-737 resistant cells. We showed that a low Mcl-1/Bcl-2 plus Bcl-xL protein ratio determined ABT-737 responsiveness. ABT-737 exposure further decreased Mcl-1, inducing apoptosis on sensitive models and primary samples, while not affecting resistant cells. Co-inhibition of Bcl-2 and the mTOR pathway resulted cytotoxic on ABT-737 resistant models, by downregulating mTORC1 activity and Mcl-1 in a proteasome-independent manner. Although Mcl-1 seemed to be critical, ectopic modulation did not correlate with apoptosis changes. Importantly, dual targeting proved effective on ABT-737 resistant samples, showing additive/synergistic effects. Together, our results show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that resistance to ABT-737 mostly correlated with Mcl-1 overexpression. Co-targeting of the Bcl-2 protein family and mTOR pathway enhanced drug-induced cytotoxicity by suppressing Mcl-1, providing a novel therapeutic approach to overcome BH3 mimetics resistance in ALL

Metabolism of phenylpropionic acid in enteropathogenic Escherichia coli belonging to serogroup O111 and its application for diagnosis

We evaluated a biochemical assay based on the ability to metabolise beta -phenylpropionic acid (PPA) as a diagnostic aid in the identification of typical enteropathogenic Escherichia coli (EPEC) strains. A total of 1061 E. coli strains of serogroups O55, O111, and O119 were initially characterised regarding their H types (serotypes) and the presence of EPEC DNA sequences, eae, EAF, and bfpA. in case of the serogroup O111 strains, 84.6% carried the typical EPEC markers, and the great majority of those (98.1%) were PPA-positive. in contrast, only 0.9% of the serogroups O55 and O119 strains carrying the typical EPEC markers (53.6% and 75.4%, respectively) were PPA-positive. We conclude that the PPA test is a useful method to detect typical EPEC strains only among strains of the O111 serogroup. (C) 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.Inst Adolfo Lutz Registro, Secao Bacteriol, BR-01246902 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, SP, BrazilWeb of Scienc

Lower sperm DNA fragmentation after r-FSH administration in functional hypogonadotropic hypogonadism

Abstract PURPOSE: An observational clinical and molecular study was designed to evaluate the effects of the administration of recombinant human FSH on sperm DNA fragmentation in men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. METHODS: In the study were included 53 men with a non-classical form of hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia. In all patients, sperm DNA fragmentation index (DFI), assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) in situ DNA nick end-labelling (TUNEL) assay, was evaluated before starting the treatment with 150 IU of recombinant human FSH, given three times a week for at least 3 months. Patients' semen analysis and DNA fragmentation index were re-evaluated after the 3-month treatment period. RESULTS: After recombinant human FSH therapy, we did not find any differences in terms of sperm count, motility and morphology. The average DNA fragmentation index was significantly reduced (21.15 vs 15.2, p 15 %), while no significant variation occurred in the patients with DFI values ≤15 %. CONCLUSIONS: Recombinant human FSH administration improves sperm DNA integrity in hypogonadotropic hypogonadism and idiopathic oligoasthenoteratozoospermia men with DNA fragmentation index value >15 %

Comparison of Short-Term Estrogenicity Tests for Identification of Hormone-Disrupting Chemicals

The aim of this study was to compare results obtained by eight different short-term assays of estrogenlike actions of chemicals conducted in 10 different laboratories in five countries. Twenty chemicals were selected to represent direct-acting estrogens, compounds with estrogenic metabolites, estrogenic antagonists, and a known cytotoxic agent. Also included in the test panel were 17β-estradiol as a positive control and ethanol as solvent control. The test compounds were coded before distribution. Test methods included direct binding to the estrogen receptor (ER), proliferation of MCF-7 cells, transient reporter gene expression in MCF-7 cells, reporter gene expression in yeast strains stably transfected with the human ER and an estrogen-responsive reporter gene, and vitellogenin production in juvenile rainbow trout. 17β-Estradiol, 17α-ethynyl estradiol, and diethylstilbestrol induced a strong estrogenic response in all test systems. Colchicine caused cytotoxicity only. Bisphenol A induced an estrogenic response in all assays. The results obtained for the remaining test compounds—tamoxifen, ICI 182.780, testosterone, bisphenol A dimethacrylate, 4-n-octylphenol, 4-n-nonylphenol, nonylphenol dodecylethoxylate, butylbenzylphthalate, dibutylphthalate, methoxychlor, o,p′-DDT, p,p′-DDE, endosulfan, chlomequat chloride, and ethanol—varied among the assays. The results demonstrate that careful standardization is necessary to obtain a reasonable degree of reproducibility. Also, similar methods vary in their sensitivity to estrogenic compounds. Thus, short-term tests are useful for screening purposes, but the methods must be further validated by additional interlaboratory and interassay comparisons to document the reliability of the methods