A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population

530. Development of New Lentiviral Vectors With a Reduced Splicing Interference Potential and a Safer In Vivo Genotoxic Profile

The excellent therapeutic potential of self-inactivating (SIN) lentiviral vectors (LV) has been demonstrated in pre-clinical studies and clinical trials. However, weaker mechanisms of insertional mutagenesis could endanger their clinical applications. Systemic vector injection into newborn tumor-prone Cdkn2a-/- and Cdkn2a+/-mice, conducted in our previous work, demonstrated that SINLVs harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset with respect to control mice. Integration sites analyses of vector-induced tumor showed that oncogene activations or tumor suppressor inactivation by LV integrations occur by combining mechanisms of transcript truncation, induction of aberrant splicing and/or enhancer-mediated overexpression of cellular transcription units. Although oncogene activation may be reduced by the use of self-inactivating design, moderate cellular promoters and insulator sequences how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear.From this and a previous study, we identified the LV sequences most frequently involved in chimeric transcript formation. In our rationale, these LV sequences could be tagged by sequences complementary to microRNAs (mirT sequence) active in hematopoietic cells in order to allow selective degradation, through the miRNA pathway, of vector-mediated aberrantly spliced transcripts. Hence, we specifically designed SIN LVs harboring mirT sequences recognized by mir223 and mir142-3p (that are expressed in hematopoietic lineages) within the SIN LTR (mirsT-LTR LV) or in the vector backbone and outside the gene expression cassette (mirT LV). We then assessed the genotoxicity of the SIN LVs harboring mirT sequences by taking advantage of our in vivo models. Interestingly, injection of mirsT-LTR LV (N=73) and mirT LV (N=73) in Cdkn2a-/- mice did not caused any significant acceleration in hematopoietic tumor onset compared to control un-injected mice (N=40). Similar results have been obtained after injection in Cdkn2a+/- mice (N=28 for mirsT-LTR LV, N=26 for mirT LV and N=34 un-injected mice). We are currently performing integration site analyses in Cdkn2a-/- and Cdkn2a+/- treated mice to dissect if and how the integrated mirsT-LTR LV and mirT LV proviral genome interacts with the surrounding cellular genome.Overall, these studies show that this new advanced design lentiviral vectors completely abrogated residual vector genotoxicity in highly sensitive mouse models and could represent the vector design of choice in future gene therapy applications

529. Lentiviral Vectors with a Reduced Splicing Interference Potential Have a Significantly Improved Safety Profile In Vivo

Genotoxicity assays based on systemic vector injection into newborn tumor-prone Cdkn2a−/− and Cdkn2a+/− mice has shown that self-inactivating (SIN) lentiviral vector (LV) harboring strong or moderate enhancer/promoters in internal position caused acceleration in hematopoietic tumor onset compared to control mice. Integration site (IS) analysis in vector-induced tumors showed that oncogene activation or tumor suppressor inactivation occurs by mechanisms of aberrant splicing and/or enhancer-mediated overexpression of cellular genes. Although oncogene activation may be reduced by the use of SIN design, moderate cellular promoters and insulator sequences, how to reduce genotoxic splicing-capture events and aberrant transcript formation triggered by vector integration is still unclear. Here, we specifically designed SINLVs harboring sequences complementary to microRNAs (mirT sequence) which are active in hematopoietic cells (mir223 and mir142-3p) within the SIN LTR (mirsT-LTR.LV) or in the vector backbone and outside the gene expression cassette (mirT-LV). In our rationale, the mirT sequences when incorporated in an aberrantly generated mRNA would be selectively degraded through the miRNA pathway. Thus, by taking advantage of our in vivo models, we assessed the genotoxicity of these LVs with mirT sequences. Systemic injection of mirsT-LTR.LV (N=34) and mirT-LV (N=39) in Cdkn2a−/− mice did not cause any significant acceleration in hematopoietic tumor onset compared to un-injected mice (N=37) or mice injected with a SINLV that does not harbor mirT sequences (N=24). Similar results have been obtained after injection of the same vectors in Cdkn2a+/− mice (N=29 mirsT-LTR.LV, N=25 mirT-LV, N=40 un-injected and N=15 injected control mice). To gain additional information on the safety profile of these vectors, we performed IS analysis (N>10,000) in tumor-derived DNA. By this analysis, we previously found that Map3k8 activation by LV insertions was the major mechanism of genotoxicity when prototypical SINLVs were injected into Cdkn2a−/− mice. Now, we found that mice treated with mirsT-LTR.LV and mirT-LV did not show any Map3k8 activating insertions, suggesting that the new vectors are efficient in preventing its activation and confirming their superior safety profile. Furthermore, as expected, Pten was the most frequently targeted gene in tumors derived from Cdkn2a−/− mice injected with the LVs harboring mirT sequences. Pten insertions mainly targeted exons, suggesting the potential inactivation of its transcription unit. Finally, we found that Sfi1 was the major Common Insertion Site (CIS) in Cdkn2a+/− mice injected with LVs harboring mirT sequences. This CIS gene however appears to be the product of an intrinsic bias of LV integration, rather than the result of a selection process. Overall, our studies showed that these new advanced design LVs have a significantly improved safety profile and could represent the vector design of choice in future gene therapy applications

Depression and cardiovascular disease: The deep blue sea of women's heart

Abstract Cardiovascular disease (CVD) constitutes a leading worldwide health problem, with increasing evidence of differences between women and men both in epidemiology, pathophysiology, clinical management, and outcomes. Data from the literature suggest that women experience a doubled incidence of CVD related deaths, while angina, heart failure and stroke are increasingly prevalent in females. About 20–25% of women go through depression during their life, and depressive symptoms have been considered a relevant emergent, non-traditional risk factor for CVD in this part of the general population. Underlying mechanisms explaining the link between depression and CVD may range from behavioral to biological risk factors, including sympathetic nervous system hyperactivity and impairment in hypothalamic-pituitary-adrenal function. However, the neuroendocrine-driven background could only partially explain the differences mentioned above for chronic systemic inflammation, altered hemostasis and modulation of cardiac autonomic control. In addition, some evidence also suggests the existence of gender-specific differences in biological responses to mental stress. Given these premises, we here summarize the current knowledge about depression and CVD relationship in women, highlighting the sex differences in physiopathology, clinical presentation and treatments

Effects of turmeric powder on intestinal and biliary functions: The influence of curcuminoids concentration on spontaneous contractility

Two turmeric food powders (C1 and C2) were studied for curcuminoid content and their effects on the guinea pig intestinal tract in vitro. C1 contained a higher curcuminoid content than C2 (5.22% vs 2.31%). C1 and C2 increased gallbladder (similar to 10%) and biliary smooth muscle tone (similar to 15%), without affecting the sphincter of Oddi smooth muscle contractility. C2 was more effective than C1 in lowering ileum tone (-22% vs -37%), whereas the reverse occurred in the colon (-50% vs 20%). Standard Fast Fourier transforms and absolute powers analysis of the frequency bands highlighted that, in the bile duct, C2 induced contractions of higher variability and ampler oscillations of low-frequency waves. At the Oddi sphincter, C1 had a biphasic effect, increasing and then drastically decreasing the oscillations. The same occurred with C2 in the ileum, while both samples reduced the fluctuations in the colon

Voices and views : English language issues from different perspectives

The articles in this collection were produced within the framework of the MA program “Maestría en Inglés”, Facultad de Lenguas, Universidad Nacional de Córdoba, Argentina. They are based on selected MA theses and dissertations on Applied Linguistics and Literary Studies. The collection focuses on EFL teacher training, pronunciation assessment, academic writing, Critical Discourse Analysis and Literature

One Health Evaluation: A Case Study at the University of Bologna

The level of One Health (OH), or \u201cOne Health-ness,\u201d of health interventions has been defined as the capacity to operate according to six dimensions concerning OH operations and OH infrastructures, respectively (thinking, planning, and working; and information sharing, reciprocal learning, and systemic organization). Although health initiatives and research increasingly claim their orientation toward OH, such a capacity is rarely assessed. The objective of this study is to evaluate the One Health-ness of the academic team of the University of Bologna (UNIBO Team) working in the \u201cELEPHANT\u201d project (Empowering universities' Learning and rEsearch caPacities in the one Health Approach for the maNagement of animals at the wildlife, livestock and human interface in SouTh Africa). This project involves universities, six from South Africa and two from Europe, and aims at embedding OH in research and learning to enable the control of diseases at the human, animal, and environmental interface, and to emphasize the interests of local African communities with wildlife conservation. The methodology adopts the NEOH method, developed in 2018 by the EU-COST Action, \u201cNetwork for the Evaluation of One Health.\u201d The approach is based on questionnaires delivered to participants, which focus on the six OH dimensions, and then translate answers into quantitative metrics through the OH Index (OHI) and the OH Ratio (OHR). The following two evaluation levels are foreseen: the whole project and the single partner institutions. The evaluations are carried on in parallel, with preliminary, mid-term, and final assessments, to monitor the efficacy of the project actions. The preliminary evaluation of the UNIBO Team resulted in the OHI of 0.23 and the OHR of 1.69 which indicate a low degree of OH-ness and an imbalance between OH operation and OH infrastructure. The UNIBO case study will be the baseline for the evaluation of the other partner institutions involved in the ELEPHANT project. This type of evaluation can support the implementation of OH practices inside a project and underpin the strategies that allow to achieving more effective results. Any improvement in the OH-ness of each single academic team can be also considered as a result of the ELEPHANT project, thus showing its multiplier effect in the context

Arcobacter spp. in raw milk from vending machines in Piedmont and occurrence of virulence genes in isolates

Arcobacter spp. has been recognized as an emerging foodborne pathogen and a hazard to human health. In the dairy chain, it has been isolated from different sources, nevertheless data on Arcobacter occurrence in raw milk provided by vending machines are few. This study aimed to identify potentially pathogenic Arcobacter spp. in raw milk intended for human consumption sold through vending machines located in Piedmont. In an 8-month period, 37 raw milk samples were collected from 24 dairy farms: 12 (32,4%) were collected directly in farm from bulk tank milk and 25 (67,6%) from vending machines. Eight (21,6%) out of the 37 milk samples and 7 (29,2%) out of the 24 dairy farms were positive for Arcobacter spp. by culture examination. Four (16%) out of the 25 samples from vending machines and 4 (33,3%) out of the 12 samples from bulk tank milk were positive. All 8 isolates were identified as A. butzleri both by MALDI-TOF MS and multiplex end-point PCR. According to the detection of virulence genes, a total of four Patho-types were highlighted: 5 isolates in P-type 1 and only one isolate for each of the P-types 2-3-4. A. butzleri isolates carrying encoding virulence factors genes were isolated from raw milk intended for human consumption: these findings strengthen the compulsory consumption after boiling as required by current legislation and suggest the need of enlarging the analytical investigations to other microorganisms not yet included in the food safety criteria

The Politicization of the European Union

What are the consequences of EU politicization for the EU and European societies? This book shifts the analytical focus from EU politicization processes to their empirical and normative research on the effects of politicization on public opinion, public discourses, policymaking and European integration