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

Listeria monocytogenes in Five Sardinian Swine Slaughterhouses: Prevalence, Serotype, and Genotype Characterization

In a 3-year study (2008 to 2011) to estimate the prevalence and the contamination sources of Listeria monocytogenes in pork meat in Sardinia, Italy, 211 samples were collected from five Sardinian swine slaughterhouses: 171 samples from slaughtered pigs and 40 from the slaughterhouse environment. Fifty L. monocytogenes isolates were characterized by PCR-based serotyping, presence of virulence-associated genes, and pulsed-field gel electrophoresis restriction analysis. The overall prevalence of L. monocytogenes was 33% in swine carcasses, 7% in cecal material, 23% on meat contact surfaces, and 25% on noncontact surfaces. Only two serotypes were detected: 1/2c (78%) and 1/2a (22%). In all, based on the presence of virulence-associated genes, eight pathogenic profiles were detected. Only 42% of all isolates carried the full complement of virulence-associated genes and were allotted to profile 1. Six pulsed-field gel electrophoresis profiles persisted in the slaughterhouses; restriction profiles appeared to be specific to each plant

Vitamin E–loaded dialyzer resets PBMC-operated cytokine network in dialysis patients

Vitamin E–loaded dialyzer resets PBMC-operated cytokine network in dialysis patients.BackgroundIn hemodialysis patients the activity of stimulated Th1 lymphocytes is depressed, while Th2 cells are constitutively primed. Such phenomena may depend on monocyte activation and altered release of interleukin (IL)-12 and IL-18, which regulate Th cell differentiation. Reactive oxygen species (ROS) activate monocytes; therefore, a hemodialyzer with antioxidant activity would contrast ROS, prevent monocyte activation, reset IL-12 and IL-18 release, and restore Th1/Th2 balance.MethodsTen patients on regular dialysis treatment (RDT) with cellulosic membrane (CM) were shifted to vitamin E–coated dialyzer (VE). During treatment with CM and after 3, 6, and 12months of treatment with VE, peripheral blood mononuclear cells (PBMC) and purified CD4+ cells were isolated, and cultured, resting, mitogen-stimulated, and interferon γ (IFNγ), IL-4, IL-10, IL-12, and IL-18 release was measured. Vitamin E and A plasma levels and the effects of a single dialysis session on peripheral blood NO levels were assayed.ResultsThe constitutive release of IL-4 and IL-10 by CD4+ cells was abated significantly by treatment with VE (nadir -77.8% and -55.3%, respectively, at 12months). INFγ release by mitogen-stimulated CD4+ recovered with VE (zenith +501% at 12months). PBMC constitutive production of IL-12 and IL-18 was significantly reduced by VE (nadir at 12months -64.7% and -51.3%, respectively). VE increased plasma levels of vitamins E and A. NO plasma levels fell after a single dialysis treatment with VE (-17%, P < 0.05) in contrast with CU (+27.1%, P < 0.05).ConclusionThe network of cytokines released by monocytes and Th cells is reset toward normality by treatment with vitamin E–coated dialyzer

AIRE acetylation and deacetylation: effect on protein stability and transactivation activity

The AIRE protein plays a remarkable role as a regulator of central tolerance by controlling the promiscuous expression of tissue-specific antigens in thymic medullary epithelial cells. Defects in AIRE gene cause the autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy, a rare disease frequent in Iranian Jews, Finns, and Sardinian population. AIRE protein is primarily known as a transcriptional regulator and is capable of interacting with numerous proteins. The first characterized partner of AIRE is the ubiquitous transcription factor CREB-binding protein (CBP), which regulates DNA transcription through the acetylation and deacetylation of histones. More recently, the role of p300 in AIRE acetylation, which could influence the selection of AIRE activated genes, has been described. Results: In this study, we have precisely mapped, by mass spectrometry experiments, the sites of protein acetylation and, by mutagenesis assays, we have described a set of acetylated lysines as being crucial in influencing the subcellular localization of AIRE. Furthermore, we have also determined that the de-acetyltransferase enzymes HDAC1-2 are involved in the lysine de-acetylation of AIRE. Conclusions: On the basis of our results and those reported in literature, we propose a model in which lysines acetylation increases the stability of AIRE in the nucleus. In addition, we observed that the interaction of AIRE with deacetylases complexes inhibits its transcriptional activity and is probably responsible for the instability of AIRE, which becomes more susceptible to degradation in the proteasome

Quark mixing in the discrete dark matter model

We consider a model in which dark matter is stable as it is charged under a Z2 symmetry that is residual after an A4 flavour symmetry is broken. We consider the possibility to generate the quark masses by charging the quarks appropriately under A4. We find that it is possible to generate the CKM mixing matrix by an interplay of renormalisable and dimension-six operators. In this set-up, we predict the third neutrino mixing angle to be large and the dark matter relic density to be in the correct range. However, low energy observables - in particular meson-antimeson oscillations - strongly limit the available parameter space.Comment: 10 pages, 4 figures; version 3 as appeared in Nuclear Physics

COVID-19 rehabilitation units are twice as expensive as regular rehabilitation units

Objective: The COVID-19 pandemic has caused significant motor, cognitive, psychological, neurological and cardiological disabilities in many infected patients. Functional rehabilitation of infectious COVID-19 patients has been implemented in the acute care wards and in appropriate, ad hoc, multidisciplinary COVID-19 rehabilitation units. However, because COVID-19 rehabilitation units are a clinical novelty, clinical and organizational benchmarks are not yet available. The aim of this study is to describe the organizational needs and operational costs of such a unit, by comparing its activity, organization, and costs with 2 other functional rehabilitation units, in San Raffaele Hospital, Milan, Italy. Methods: The 2-month activity of the COVID-19 Rehabilitation Unit at San Raffaele Hospital, Milan, Italy, which was created in response to the emergency need for rehabilitation of COVID-19 patients, was compared with the previous year's activity of the Cardiac Rehabilitation and Motor Rehabilitation Units of the same institute. Results: The COVID-19 Rehabilitation Unit had the same number of care beds as the other units, but required twice the amount of staff and instrumental equipment, leading to a deficit in costs. Discussion: The COVID-19 Rehabilitation Unit was twice as expensive as the 2 other units studied. World health systems are organizing to respond to the pandemic by expanding capacity in acute intensive care and sub-intensive care units. This study shows that COVID-19 rehabilitation units must be organized and equiped according to the clinical and rehabilitative needs of patients, following specific measures to prevent the spread of infection amongs patients and workers

Gender differences and cardiometabolic risk. the importance of the risk factors

Metabolic syndrome (Mets) is a clinical condition characterized by a cluster of major risk factors for cardiovascular disease (CVD) and type 2 diabetes: proatherogenic dyslipidemia, elevated blood pressure, dysglycemia, and abdominal obesity. Each risk factor has an independent effect, but, when aggregated, they become synergistic, doubling the risk of developing cardiovascular diseases and causing a 1.5-fold increase in all-cause mortality. We will highlight gender differences in the epidemiology, etiology, pathophysiology, and clinical expression of the aforementioned Mets components. Moreover, we will discuss gender differences in new biochemical markers of metabolic syndrome and cardiovascular risk

Shelf-life of Halal fresh and minced beef meat packaged under modified atmosphere

The shelf-life of Halal fresh cut and minced beef meat, packaged under modified atmosphere (MAP) was evaluated. The microbial profile of the carcasses intended for cutting and mincing was investigated by detecting spoilage and pathogenic bacteria. Samples of diced meat (DM), marrowbones (MB), steaks (S) and minced meat (MM) were packaged in MAP (66.0% O2, 25.0% CO2 and 9.0% N2) and stored at +2 and +8°C. At 0, 7 and 14 days, gas composition of headspace was measured. Moreover, in all the samples colony count at 30°C, Enterobacteriaceae, lactic acid bacteria (LAB) and Pseudomonas spp. were determined. The carcasses contamination was in compliance with the criteria fixed by EC Reg. 2073/2005. Gas composition of the headspace changed significantly during the storage, mainly at +8°C, where a significant decrease of O2 (until 0.1-0.6%) and an increase of CO2 (until 81.0-89.0%) were recorded. This could be related to the level of LAB and Pseudomonas spp. Less significant changes were observed at +2°C. At 7 days of storage colony count, mean values were higher than >107 CFU/g in the samples at +8°C, and also at 14 days at +2°C, presumably due to the high levels of Pseudomonas spp., that was dominant at the end of the test. Overall, the microbial mean counts were higher than those detected in similar products packaged under vacuum. In order to extend the shelf-life of the fresh meat and meat preparations, differentiated gas mixtures, and particularly a higher percentage of CO2, could be employed

Anti-Ro52 antibodies positivity in antisynthetase syndrome: a single centre cohort study

Objectives: Although antisynthetase antibodies (ARS) are the established markers of the so-called antisynthetase syndrome (ASSD), in these patients the concomitant positivity of anti-Ro52 antibodies, reported in up to the 50% of cases, is not rare. Several studies focused on the effect of different ARS specificities on the evolution of ASSD, the most recent showing no effects. On the contrary, the role of co-occurring anti-Ro52 antibodies in ASSD is still debated. We investigated the potential of anti-Ro52 antibodies in identifying a clinical phenotype of ASSD or influencing prognosis, irrespectively to the underlying ARS specificity. Methods: Retrospective analysis of clinical, imaging and laboratory characteristics, therapeutic approaches and outcome at baseline and at last follow-up, of 60 ASSD patients progressively enrolled at our Hospital. Results: We identified 34 anti-Ro+ and 26 anti-Ro- ASSD patients. Classic triad prevalence at baseline was similar between the two groups, whereas interstitial lung disease (ILD) (p value=0.01) and myositis (p value=0.03) were significantly more prevalent in anti-Ro52+ and in anti-Ro52- patients at last follow up, respectively. No differences in therapeutic approaches, oxygen need and ILD patterns were observed. Overall mortality was 25% (15 subjects). No differences in mortality, overall and disease related, between anti-Ro52+ and anti-Ro52- patients were observed (p value=0.764), despite the more frequent ILD occurrence in anti-Ro52+ patients. Survival curves were not different at any time point (Log-rank test, p value 0.98). Conclusions: Anti-Ro52 antibodies affect time course and clinical characteristics of ASSD. Although ILD is significantly more associated to anti-Ro52 antibodies, no difference in mortality was observed compared to anti-Ro52 negative patients