analisi del contributo delle poliproteine GAG e POL nello sviluppo della resistenza in pazienti HIV-1 positivi sottoposti a terapia antiretrovirale

The introduction in the mid 1990s of Antiretroviral Therapy in the cure of AIDS has dramatically decreased the morbidity and mortality rate and has significantly extended the lifespan and the quality of life of HIV-1 positive patients. Today there are more than 20 drugs licensed for clinical use, targeting different steps of viral life cycle including viral entry (coreceptor antagonists and fusion inhibitors), reverse transcription (nucleoside, NRTIs, and non-nucleoside inhibitors, NNRTIs, of the viral reverse transcriptase), integration (integrase inhibitors) and viral maturation (protease inhibitors, PIs). Current Guidelines recommend the use of combination therapies, including drugs of almost two classes, in particular one or two NRTIs and one NNRTIs or one PIs, those are considered the standard of care for the treatment of HIV infection. Despite the success of pharmacological combination strategy, the emergence of drug resistance is still a major factor contributing to the therapy failure. The mechanisms of resistance mainly involve mutations directly altering the interaction of viral enzymes and inhibitors (Menéndez-Arias, 2010). Nowadays, most of these mutations, defined drug resistance mutations, are well characterized (Johnson et al., 2010) and are used in standard genotypic tests as predictive clues of treatment failure, but in some cases such limited information is not sufficient to explain the virological failure. Emerging studies reveal that, besides the ones encoding Gag and Pol polyproteins, other regions might contribute to the development of resistance. In particular, some specific cleavage sites and non-cleavage site mutations (Ho et al., 2008; Parry et al., 2009; Dam et al., 2009; Nijhuis et al., 2007), as well as frameshift-regulating site mutations in Gag (Doyon et al., 1998), increase cleavage sites accessibility and polyprotein processing, thus compensating for the catalytic loss of function of the viral Protease (PR) induced by primary resistance mutations. Moreover, some residues in the HIV-1 Reverse Transcriptase (RT) are critical for proteolytic processing of Gag-Pol precursors (Nishitsuji et al., 2011; Chiang et al., 2012). Although the augmenting consciousness of the relevance of other viral protein domains, besides PR and RT enzymes, in the prediction and acquisition of resistance to the antiviral formulations, little is known about the function of such specific mutations in the viral life cycle. Moreover, the available data are not unanimous in defining the relative importance of each analyzed region, for example within the Gag protein (Dam et al., 2009; Parry et al., 2007). In this context, we are interested in studying the functional role of HIV-1 Pr55Gag protein as natural substrates of PR and its contribution in resistance mechanisms. We optimized PCR amplification experimental settings and sequencing conditions of the gag gene from clinical isolates of different subtypes. In collaboration with Professor Parisi, University of Padua, we analyzed the gag sequence derived from clinical samples of HIV-1 infected patients failing PR Inhibitors (PIs) and RT Inhibitors (RTIs), selected among a cohort of five infectious diseases units located in Veneto in Northeastern Italy. In order to determine the contribution of the Pr55Gag protein to the resistance mechanisms and its specific function in the viral life cycle, we designed a cloning strategy that allows to analyze the differential contribution of N-terminal and/or C-terminal regions of Gag in the presence or in the absence of the mutated PR-RT. Indeed, each patient derived PCR products could be inserted in an HIV-1 proviral genome. In particular, we used a modified version of the previously described env-complementation system, in which an env-deleted provirus expresses a reporter gene under the transcriptional control of the viral LTR and it is capable of one single round of replication. The HIV-1 envelope glycoprotein is given in trans along with the Rev protein. Among all the patient samples sequenced so far, we selected one characterized by resistant mutations only in the PR coding region to analyze the effect of the mutated N-terminal region of Pr55Gag protein. Our results indicated that: (i) the patient-derived mutations in the N-terminal region of Pr55Gag enhance the RT activity and the p24 content in the supernatant of producing cells in comparison to the wild type context; (ii) the patient-derived MA/CA cleavage site amino acid sequence doesn’t affect the processing ability of the wild type PR; (iii) the infectivity of virions carrying the patient-derived N-terminal region of Pr55Gag is reduced. Our results would contribute to better characterize the role of Gag and the relations with PR and RT in resistance development, their relevance in viral replication and evolution in the presence or in the absence of drug

I sistemi informativi geografici nella gestione del territorio aperto: il censimento delle strade vicinali nel Comune di Pontassieve (FI)

The research started from a need of the local authority to provide an updated data bank to create a dynamic mangement system for open terrority through the construction of a geographic relational database with web interface for various users

Mechanisms of HIV-1 Nucleocapsid Protein Inhibition by Lysyl-Peptidyl-Anthraquinone Conjugates

The Nucleocapsid protein NCp7 (NC) is a nucleic acid chaperone responsible for essential steps of the HIV-1 life cycle and an attractive candidate for drug development. NC destabilizes nucleic acid structures and promotes the formation of annealed substrates for HIV-1 reverse transcription elongation. Short helical nucleic acid segments bordered by bulges and loops, such as the Trans-Activation Response element (TAR) of HIV-1 and its complementary sequence (cTAR), are nucleation elements for helix destabilization by NC and also preferred recognition sites for threading intercalators. Inspired by these observations, we have recently demonstrated that 2,6-disubstituted peptidylanthraquinone-conjugates inhibit the chaperone activities of recombinant NC in vitro, and that inhibition correlates with the stabilization of TAR and cTAR stem-loop structures. We describe here enhanced NC inhibitory activity by novel conjugates that exhibit longer peptidyl chains ending with a conserved Nterminal lysine. Their efficient inhibition of TAR/cTAR annealing mediated by NC originates from the combination of at least three different mechanisms, namely, their stabilizing effects on nucleic acids dynamics by threading intercalation, their ability to target TAR RNA substrate leading to a direct competition with the protein for the same binding sites on TAR, and, finally, their effective binding to the NC protein. Our results suggest that these molecules may represent the stepping-stone for the future development of NC-inhibitors capable of targeting the protein itself and its recognition site in RNA

How Can I Trust You? The Role of Facial Trustworthiness in the Development of Epistemic and Interpersonal Trust

Recently, researchers from developmental and clinical psychology highlighted epistemic trust (ET) as a key factor for personality disorders. ET is intended as the mental openness to information coming from others during social exchanges. ET develops from signals called ostensive cues, delivered through facial expressions during interactions in a secure attachment context. Similarly, interpersonal trust (IT) refers to the perception of others as not harmful, which is also developed through secure attachment relationships. Our purpose was to suggest a conceptualization of ET as a specific facet of IT. We hypothesize that positive experiences of caregiving promote IT development that includes a specific sense of trust toward others’ knowledge. Moreover, we suggest that the early ability to infer a judgment of trustworthiness from facial cues is the starting point for developing both IT and ET. This conceptualization supports the role of considering both IT and ET in the development of borderline pathology. Our purpose is to suggest a conceptualization of ET as a specific facet of IT. We hypothesize that positive experiences of caregiving promote IT development that includes a specific sense of trust toward others’ knowledge. Moreover, we suggest that the early ability to infer a judgment of trustworthiness from facial cues is the starting point for developing both IT and ET. This conceptualization supports the role of considering both IT and ET in the development of borderline pathology

I.S.Mu.L.T. Achilles Tendon Ruptures Guidelines

This work provides easily accessible guidelines for the diagnosis, treatment and rehabilitation of Achilles tendon ruptures. These guidelines could be considered as recommendations for good clinical practice developed through a process of systematic review of the literature and expert opinion, to improve the quality of care for the individual patient and rationalize the use of resources. This work is divided into two sessions: 1) questions about hot topics; 2) answers to the questions following Evidence Based Medicine principles. Despite the frequency of the pathology andthe high level of satisfaction achieved in treatment of Achilles tendon ruptures, a global consensus is lacking. In fact, there is not a uniform treatment and rehabilitation protocol used for Achilles tendon ruptures

Evaluation of Coaguchek®Pro II coagulation testing device performance to assess direct oral anticoagulant action. The DOAC-CHECK study

Direct oral anticoagulants (DOAC) measurement is recommended in specific conditions. A point-of-care testing should be used in emergency to qualitatively rule out relevant DOAC concentrations. The DOAC-CHECK Study aims to evaluate whether the use of CoaguChek® Pro II (Roche Diagnostics International Ltd, Rotkreuz, Switzerland) coagulation testing device can provide reliable information in patients treated with DOAC. The study was carried out in two FCSA (Italian Federation of Thrombosis Centers) centers. We choose 3 different concentration thresholds for our analysis (30, 50 and 100 ng/mL) and by ROC curves the ideal cut-off point was selected to be the one that yielded a sensitivity of at least 95% associated with the highest possible specificity. 512 patients were enrolled. For Edoxaban and Rivaroxaban, both CoaguChek® Pro II prothrombin time (PT) and activated partial thromboplastin time (aPTT) tests showed a sensitivity >95% corresponding to satisfying specificity values; negative predictive values resulted in the range 90-100%. At variance, CoaguChek® Pro II PT and aPTT tests did not seem to be useful for identifying Apixaban and Dabigatran concentrations higher than the pre-defined thresholds. Our results suggest that CoaguChek® Pro II coagulation testing device can be used to qualitatively identify relevant concentrations of Edoxaban or Rivaroxaban, but not of Apixaban or Dabigatran

The importance of a taste. A comparative study on wild food plant consumption in twenty-one local communities in Italy

A comparative food ethnobotanical study was carried out in twenty-one local communities in Italy, fourteen of which were located in Northern Italy, one in Central Italy, one in Sardinia, and four in Southern Italy. 549 informants were asked to name and describe food uses of wild botanicals they currently gather and consume. Data showed that gathering, processing and consuming wild food plants are still important activities in all the selected areas. A few botanicals were quoted and cited in multiple areas, demonstrating that there are ethnobotanical contact points among the various Italian regions (Asparagus acutifolius, Reichardia picroides, Cichorium intybus, Foeniculum vulgare, Sambucus nigra, Silene vulgaris, Taraxacum officinale, Urtica dioica, Sonchus and Valerianella spp.). One taxon (Borago officinalis) in particular was found to be among the most quoted taxa in both the Southern and the Northern Italian sites

Shedding light on typical species : implications for habitat monitoring

Habitat monitoring in Europe is regulated by Article 17 of the Habitats Directive, which suggests the use of typical species to assess habitat conservation status. Yet, the Directive uses the term “typical” species but does not provide a definition, either for its use in reporting or for its use in impact assessments. To address the issue, an online workshop was organized by the Italian Society for Vegetation Science (SISV) to shed light on the diversity of perspectives regarding the different concepts of typical species, and to discuss the possible implications for habitat monitoring. To this aim, we inquired 73 people with a very different degree of expertise in the field of vegetation science by means of a tailored survey composed of six questions. We analysed the data using Pearson's Chi-squared test to verify that the answers diverged from a random distribution and checked the effect of the degree of experience of the surveyees on the results. We found that most of the surveyees agreed on the use of the phytosociological method for habitat monitoring and of the diagnostic and characteristic species to evaluate the structural and functional conservation status of habitats. With this contribution, we shed light on the meaning of “typical” species in the context of habitat monitoring

Predicting needlestick and sharps injuries in nursing students: Development of the SNNIP scale

© 2020 The Authors. Nursing Open published by John Wiley & Sons Ltd. Aim: To develop an instrument to investigate knowledge and predictive factors of needlestick and sharps injuries (NSIs) in nursing students during clinical placements. Design: Instrument development and cross-sectional study for psychometric testing. Methods: A self-administered instrument including demographic data, injury epidemiology and predictive factors of NSIs was developed between October 2018–January 2019. Content validity was assessed by a panel of experts. The instrument's factor structure and discriminant validity were explored using principal components analysis. The STROBE guidelines were followed. Results: Evidence of content validity was found (S-CVI 0.75; I-CVI 0.50–1.00). A three-factor structure was shown by exploratory factor analysis. Of the 238 participants, 39% had been injured at least once, of which 67.3% in the second year. Higher perceptions of “personal exposure” (4.06, SD 3.78) were reported by third-year students. Higher scores for “perceived benefits” of preventive behaviours (13.6, SD 1.46) were reported by second-year students