Quantitative Evaluation of Very Low Levels of HIV-1 Reverse Transcriptase by a Novel Highly Sensitive RT-qPCR Assay

Based on previous experience in our laboratory, we developed a real-time reverse transcriptase (RT) quantitative PCR (RT-qPCR) assay for the assessment of very low levels of HIV-1 RT activity. The RNA, acting as a template for reverse transcription into cDNA by HIV-1 RT, consisted of a synthetic RNA ad hoc generated by in vitro transcription and included a coding sequence for HSV-1 gD (gD-RNA-synt). Different conditions of variables involved in the RT-qPCR reaction, notably different amounts of gD-RNA-synt, different mixes of the reaction buffer, and different dNTP concentrations, were tested to optimize the assay. The results indicated that the gD-RNA-synt-based RT assay, in its optimized formulation, could detect a specific cDNA reverse transcription even in the presence of 1 x 10(-9) U of HIV RT. This achievement greatly improved the sensitivity of the assay over previous versions. In summary, this constructed RT-qPCR assay may be considered a promising tool for providing accurate information on very low HIV-1 RT activity

In-hospital and web-based intervention to counteract vaccine hesitancy in very preterm infants’ families: a NICU experience

Background Vaccine hesitancy is a global problem, carrying significant health risks for extremely vulnerable population as that of preterm infants. Social media are emerging as significant tools for public health promotion. Our aim was to evaluate both the coverage and the timeliness of routine immunizations in a cohort of preterm infants (< 33 weeks of gestational age) at 24 months of age whose families have been subjected to in-hospital and web-based interventions to counteract vaccine hesitancy. Methods For a period of 2 years parents of preterm infants were instructed during their follow up visits by a member of the NICU team to get correct informations about vaccines from a social network page. Vaccination rates of preterm infants were assessed at 24 months of chronological age with an electronic database and compared to both general population and historical cohort. Results Coverage and timeliness of vaccinations at 24 months of age of 170 preterm infants were analyzed in December 2019. Gestational age and birth weight median (IQR) were, respectively, 31.0 (5.0) weeks and 1475.0 (843.8) g. Coverage rates were similar to those of the regional population (p > 0.05), while timeliness of administration was significantly delayed compared to the recommended schedule (p < 0.001). Age of administration was not correlated with either body weight and gestational age at birth (Spearman rank, p > 0.05). DTaP-IPV-HBV-Hib 2nd and 3rd doses, MMR and Varicella vaccines coverage data were higher compared to historical cohort (p < 0.05). Conclusion Increasing vaccine confidence through web-based interventions could have a positive impact on vaccination acceptance of parents of preterm infants, although timeliness results still delayed. There is a strong need to develop different and effective vaccination strategies to protect this very vulnerable population

Resources for assessing parents’ vaccine hesitancy: a systematic review of the literature

Vaccine hesitancy (VH) is a complex and country-specific issue, responsible for the decreasing vaccination rate and subsequent spread of vaccine-preventable diseases. In literature, several questionnaires were developed to assess VH. The aim of this systematic review was to evaluate the published questionnaires assessing parental VH. The search was conducted in PubMed/Medline, Web of Science and The Cochrane Library, in December 2017, following the PRISMA guidelines. The search strategy included 4 types of keywords: parents, vaccine hesitancy/acceptance, immunization and survey. Only English and Italian original papers were included. 17 reviewers independently screened titles and abstracts. Only the included articles were downloaded in full and, after a second screening, data were extracted and recorded in an ad hoc spreadsheet. A total of 5,139 articles were retrieved, after duplicates elimination 3,508 papers were screened. After a screening selection, 334 studies were included in the analysis. Most studies were cross-sectional (92.8%), followed by case-control (4.8%) and cohort studies (2.4%). The population interviewed was mainly parents, without any further details (73.1%); mothers were the only parent surveyed in approximately 20% of the studies, while only 1 study involved selectively the fathers. The sample size ranged from 7 to 59,897. Only 38% of the included studies reported both the number and type of items used. Regarding the type, more than half consisted of closed questions, followed by Likert scales, while open-ended questions were used in 14.8% of the surveys. Frequently, the survey was conducted using a self-reported questionnaire or interview. The questionnaires were mostly administered on paper, while online forms were used in 20.1% of the cases. However, 80.2% of the questionnaires were not attached to the paper. HPV vaccine was the most frequently investigated (39.2%), followed by influenza (13.5%) and measles (10.8%). While 22.4% of the articles referred to paediatrics vaccinations in general. Data about the immunization behaviours were reported in 294 studies: the subjects involved showed a behaviour defined as “acceptance” in 129 studies (38.6%), as “hesitancy/scepticism/doubt” in 145 studies (43.1%) and as “refusal” in 22 studies (6.6%). This information was not reported in 12% of the studies. VH is still a public health challenge, as confirmed by the high number of studies and questionnaires retrieved. This study offers a deeper perspec- tive on the available questionnaires, helping to identify the best one in terms of aim and study setting.     &nbsp

Sex-Related Disparities in Cardiac Masses: Clinical Features and Outcomes

Background. Cardiac masses (CM) represent a heterogeneous clinical scenario, and sex-related differences of these patients remain to be established. Purpose: To evaluate sex-related disparities in CMs regarding clinical presentation and outcomes. Material and Methods. The study cohort included 321 consecutive patients with CM enrolled in our Centre between 2004 and 2022. A definitive diagnosis was achieved by histological examination or, in the case of cardiac thrombi, with radiological evidence of thrombus resolution after anticoagulant treatment. All-cause mortality at follow-up was evaluated. Multivariable regression analysis assessed the potential prognostic disparities between men and women. Results. Out of 321 patients with CM, 172 (54%) were female. Women were more frequently younger (p = 0.02) than men. Regarding CM histotypes, females were affected by benign masses more frequently (with cardiac myxoma above all), while metastatic tumours were more common in men (p < 0.001). At presentation, peripheral embolism occurred predominantly in women (p = 0.03). Echocardiographic features such as greater dimension, irregular margin, infiltration, sessile mass and immobility were far more common in men. Despite a better overall survival in women, no sex-related differences were observed in the prognosis of benign or malignant masses. In fact, in multivariate analyses, sex was not independently associated with all-cause death. Conversely, age, smoking habit, malignant tumours and peripheral embolism were independent predictors of mortality. Conclusions. In a large cohort of cardiac masses, a significant sex-related difference in histotype prevalence was found: Benign CMs affected female patients more frequently, while malignant tumours affected predominantly men. Despite better overall survival in women, sex did not influence prognosis in benign and malignant masses

SARS-CoV-2 Breakthrough Infections: Incidence and Risk Factors in a Large European Multicentric Cohort of Health Workers

The research aimed to investigate the incidence of SARS-CoV-2 breakthrough infections and their determinants in a large European cohort of more than 60,000 health workers

Non-coding RNAs as prognostic biomarkers of cardiac arrest

L'arrêt cardiaque extra-hospitalier (ACEH) est l'une des principales causes de décès, touchant environ 100 personnes sur 100 000 par an en Europe. Une réanimation a été tentée dans 50 à 60 % des cas, et en moyenne, 8 % des survivants sont sortis de l'hôpital. Un pronostic précis de l'évolution de ces patients permettrait de mieux adapter les soins de santé. Malgré les progrès réalisés, le pronostic post-ACEH reste encore trop peu fiable. De ce fait, la découverte de nouveaux biomarqueurs spécifiques pourrait améliorer les approches actuelles. Les ARN non-codants détectés dans le sang représentent un réservoir de nouveaux biomarqueurs. Parmi eux, certains microARN (miARN) ont déjà été associés au pronostic des patients après un ACEH.Sur cette base, une première étude a pour but de déterminer si des miARN circulants pourraient refléter l'étendue des lésions cérébrales post-ACEH. À cette fin, des miARN ont été identifiés par séquençage du plasma prélevé 48h post-ACEH chez 50 patients provenant de l'essai TTM (Targeted Temperature Management) et sélectionnés selon leur évolution neurologique favorable ou défavorable, 6 mois post-ACEH. Ces miARN ont été corrélés avec l'énolase spécifique des neurones (NSE), un marqueur de lésions cérébrales. Parmi ces miARN, les miR-9-3p, miR-124-3p et miR-129-5p, connus comme étant enrichis dans le cerveau, ont montré une corrélation positive significative avec la NSE. De plus, ces 3 miARN se sont avérés être des prédicteurs de l’évolution neurologique post-ACEH. Ces résultats suggèrent que les niveaux circulants de ces miARN peuvent refléter l'étendue des lésions cérébrales, renforçant ainsi leur potentiel pour aider au pronostic de l’évolution post-ACEH.Dans une deuxième étude, l'objectif était d'identifier des longs ARN non-codants (ARNlnc) et des ARN circulaires (ARNcirc) capables de prédire l'évolution des patients après un ACEH. À cette fin, les échantillons sanguins prélevés 48h post-ACEH chez 46 patients TTM sélectionnés en fonction de leur évolution neurologique à 6 mois ont été séquencés. Parmi les candidats identifiés, 5 ARNcirc (circAGO2, circDLG1, circDNM2, circFAM13b et circNFAT5) et 1 ARNlnc (lnc-IL1R1-1:2) ont été sélectionnés et mesurés par qPCR chez les 542 patients restants de l’étude TTM. La capacité de ces 6 candidats à prédire l’évolution neurologique a été évaluée par régression logistique, et la survie grâce aux courbes de Kaplan-Meier et aux modèles de Cox. Parmi les candidats étudiés, circNFAT5 fût le meilleur pour prédire l’évolution neurologique et la survie 6 mois post-ACEH. Par conséquent, cet ARNcirc fût sélectionné pour des études fonctionnelles in-vitro. Dans les modèles in-vitro imitant un syndrome post-arrêt cardiaque, circNFAT5 fût modulé lors de l'activation des cellules T et des monocytes, suggérant un lien possible entre cet ARNcirc et le processus inflammatoire post-ACEH. De plus, des expériences préliminaires ont suggéré une relation indépendante de l'expression de circNFAT5 de celle de son gène parental, renforçant un rôle fonctionnel indépendant de circNFAT5. Après avoir investigué plusieurs pistes, aucune d’entre elles n’a permis d’identifier formellement les fonctions de circNFAT5. Par conséquent, des explorations futures seront nécessaires pour comprendre les fonctions biologiques de circNFAT5. Enfin, les 3 ARNcirc les plus performants identifiés dans l'étude TTM ont également été mesurés par qPCR chez les 674 patients de l'essai TTM2. Les 3 ARNcirc ont confirmés leur capacité de prédiction de l’évolution neurologique et de la survie, mais uniquement dans le groupe de patients traités en normothermie. CircDNM2 fût le candidat le plus performant dans cette étude.En conclusion, ce projet visait à investiguer l'utilisation potentielle des ARNnc comme biomarqueurs après un ACEH et représente un point de départ pour de futures études sur l'utilité clinique et les mécanismes d'action des candidats identifiés pour le pronostic des patients après un ACEH.Cardiac arrest is one of the leading causes of death worldwide, affecting on average 100 out of 100,000 people per year in Europe. Of these, resuscitation is attempted in 50-60% of cases, and survivors to hospital discharge average 8%. Accurate prediction of the outcome of these patients would help adapt health care. However, despite progress in this field, the prognosis of these patients remains poor. Therefore, the discovery of new specific biomarkers could improve current multimodal prediction approaches. Non-coding RNAs (ncRNAs) detected in blood represent a reservoir of novel biomarkers. Among them, circulating levels of micro RNAs (miRNA) have already been shown to be associated with outcome prediction of patients after out-of-hospital cardiac arrest (OHCA).In light of this, an initial study was designed to determine whether the levels of miRNAs identified in patients after OHCA may indeed reflect the extent of brain damage. To this end, circulating levels of miRNAs were assessed by sequencing plasma samples collected 48h after ROSC of 50 patients from the Targeted Temperature Management (TTM) trial grouped according to their favourable or unfavourable neurological outcome, 6 months after OHCA. These miRNAs were correlated with neuron-specific enolase (NSE), a marker of brain damage. Among miRNAs, brain-enriched miR9-3p, miR124-3p and miR129-5p showed significant positive correlation with NSE. Furthermore, all 3 miRNAs showed to be predictors of neurological outcome. Thus, these results indicated that circulating levels of brain-enriched microRNAs may indeed reflect the extent of brain damage, strengthening their potential to aid in the outcome prognostication after OHCA.In a second study, the goal was to identify potential long ncRNAs (lncRNA) and circular RNAs (circRNA) able to predict the outcome of patients after OHCA. To this end, whole blood samples of 46 TTM patients grouped according to their neurological outcome were sequenced. Among the lncRNAs and circRNAs identified, 5 circRNAs (circAGO2, circDLG1, circDNM2, circFAM13b and circNFAT5) and 1 lncRNA (lnc-IL1R1-1:2) were selected and measured by qPCR in the remaining TTM samples (n=542). The ability of candidates to predict neurological outcome was assessed by logistic regression and survival with Kaplan-Meier and Cox proportional hazards curves. Among the selected candidates, circNFAT5 performed best in predicting neurological outcome and survival 6 months after OHCA. Therefore, this circRNA was selected for preliminary in-vitro functional studies. Among the in-vitro treatments mimicking a post-cardiac arrest syndrome, circNFAT5 was found to be significantly modulated upon activation of T-cells and monocytes, suggesting an association of this circRNA with the inflammatory process occurring after OHCA. Furthermore, preliminary experiments suggested an independent relationship of circNFAT5 expression with its parental gene, strengthening the belief of a functional regulatory role of circNFAT5. Although, different attempts were made to elucidate the regulatory mechanisms of action of circNFAT5, no potential functions could be delineated in this work. Therefore, future explorations are needed to shed light on the possible biological functions performed by circNFAT5. Finally, the 3 best performing circRNAs identified in the TTM-trial were also measured by qPCR in 674 patients of the TTM2-trial. Following the same analyses presented in TTM, the three circRNAs were confirmed as independent predictors of neurological outcome and survival, but only in the normothermia group of TTM2, with circDNM2 as best performing candidate.In conclusion, this project aimed to deepen the understanding of the potential use of ncRNAs as biomarkers after OHCA and represented a starting point for future studies focusing on the clinical utility and mechanisms of action of the identified candidates in the outcome prognostication of patients after OHCA

Les ARNs non-codants comme biomarqueurs pronostiques de l’arrêt cardiaque

Cardiac arrest is one of the leading causes of death worldwide, affecting on average 100 out of 100,000 people per year in Europe. Of these, resuscitation is attempted in 50-60% of cases, and survivors to hospital discharge average 8%. Accurate prediction of the outcome of these patients would help adapt health care. However, despite progress in this field, the prognosis of these patients remains poor. Therefore, the discovery of new specific biomarkers could improve current multimodal prediction approaches. Non-coding RNAs (ncRNAs) detected in blood represent a reservoir of novel biomarkers. Among them, circulating levels of micro RNAs (miRNA) have already been shown to be associated with outcome prediction of patients after out-of-hospital cardiac arrest (OHCA).In light of this, an initial study was designed to determine whether the levels of miRNAs identified in patients after OHCA may indeed reflect the extent of brain damage. To this end, circulating levels of miRNAs were assessed by sequencing plasma samples collected 48h after ROSC of 50 patients from the Targeted Temperature Management (TTM) trial grouped according to their favourable or unfavourable neurological outcome, 6 months after OHCA. These miRNAs were correlated with neuron-specific enolase (NSE), a marker of brain damage. Among miRNAs, brain-enriched miR9-3p, miR124-3p and miR129-5p showed significant positive correlation with NSE. Furthermore, all 3 miRNAs showed to be predictors of neurological outcome. Thus, these results indicated that circulating levels of brain-enriched microRNAs may indeed reflect the extent of brain damage, strengthening their potential to aid in the outcome prognostication after OHCA.In a second study, the goal was to identify potential long ncRNAs (lncRNA) and circular RNAs (circRNA) able to predict the outcome of patients after OHCA. To this end, whole blood samples of 46 TTM patients grouped according to their neurological outcome were sequenced. Among the lncRNAs and circRNAs identified, 5 circRNAs (circAGO2, circDLG1, circDNM2, circFAM13b and circNFAT5) and 1 lncRNA (lnc-IL1R1-1:2) were selected and measured by qPCR in the remaining TTM samples (n=542). The ability of candidates to predict neurological outcome was assessed by logistic regression and survival with Kaplan-Meier and Cox proportional hazards curves. Among the selected candidates, circNFAT5 performed best in predicting neurological outcome and survival 6 months after OHCA. Therefore, this circRNA was selected for preliminary in-vitro functional studies. Among the in-vitro treatments mimicking a post-cardiac arrest syndrome, circNFAT5 was found to be significantly modulated upon activation of T-cells and monocytes, suggesting an association of this circRNA with the inflammatory process occurring after OHCA. Furthermore, preliminary experiments suggested an independent relationship of circNFAT5 expression with its parental gene, strengthening the belief of a functional regulatory role of circNFAT5. Although, different attempts were made to elucidate the regulatory mechanisms of action of circNFAT5, no potential functions could be delineated in this work. Therefore, future explorations are needed to shed light on the possible biological functions performed by circNFAT5. Finally, the 3 best performing circRNAs identified in the TTM-trial were also measured by qPCR in 674 patients of the TTM2-trial. Following the same analyses presented in TTM, the three circRNAs were confirmed as independent predictors of neurological outcome and survival, but only in the normothermia group of TTM2, with circDNM2 as best performing candidate.In conclusion, this project aimed to deepen the understanding of the potential use of ncRNAs as biomarkers after OHCA and represented a starting point for future studies focusing on the clinical utility and mechanisms of action of the identified candidates in the outcome prognostication of patients after OHCA.L'arrêt cardiaque extra-hospitalier (ACEH) est l'une des principales causes de décès, touchant environ 100 personnes sur 100 000 par an en Europe. Une réanimation a été tentée dans 50 à 60 % des cas, et en moyenne, 8 % des survivants sont sortis de l'hôpital. Un pronostic précis de l'évolution de ces patients permettrait de mieux adapter les soins de santé. Malgré les progrès réalisés, le pronostic post-ACEH reste encore trop peu fiable. De ce fait, la découverte de nouveaux biomarqueurs spécifiques pourrait améliorer les approches actuelles. Les ARN non-codants détectés dans le sang représentent un réservoir de nouveaux biomarqueurs. Parmi eux, certains microARN (miARN) ont déjà été associés au pronostic des patients après un ACEH.Sur cette base, une première étude a pour but de déterminer si des miARN circulants pourraient refléter l'étendue des lésions cérébrales post-ACEH. À cette fin, des miARN ont été identifiés par séquençage du plasma prélevé 48h post-ACEH chez 50 patients provenant de l'essai TTM (Targeted Temperature Management) et sélectionnés selon leur évolution neurologique favorable ou défavorable, 6 mois post-ACEH. Ces miARN ont été corrélés avec l'énolase spécifique des neurones (NSE), un marqueur de lésions cérébrales. Parmi ces miARN, les miR-9-3p, miR-124-3p et miR-129-5p, connus comme étant enrichis dans le cerveau, ont montré une corrélation positive significative avec la NSE. De plus, ces 3 miARN se sont avérés être des prédicteurs de l’évolution neurologique post-ACEH. Ces résultats suggèrent que les niveaux circulants de ces miARN peuvent refléter l'étendue des lésions cérébrales, renforçant ainsi leur potentiel pour aider au pronostic de l’évolution post-ACEH.Dans une deuxième étude, l'objectif était d'identifier des longs ARN non-codants (ARNlnc) et des ARN circulaires (ARNcirc) capables de prédire l'évolution des patients après un ACEH. À cette fin, les échantillons sanguins prélevés 48h post-ACEH chez 46 patients TTM sélectionnés en fonction de leur évolution neurologique à 6 mois ont été séquencés. Parmi les candidats identifiés, 5 ARNcirc (circAGO2, circDLG1, circDNM2, circFAM13b et circNFAT5) et 1 ARNlnc (lnc-IL1R1-1:2) ont été sélectionnés et mesurés par qPCR chez les 542 patients restants de l’étude TTM. La capacité de ces 6 candidats à prédire l’évolution neurologique a été évaluée par régression logistique, et la survie grâce aux courbes de Kaplan-Meier et aux modèles de Cox. Parmi les candidats étudiés, circNFAT5 fût le meilleur pour prédire l’évolution neurologique et la survie 6 mois post-ACEH. Par conséquent, cet ARNcirc fût sélectionné pour des études fonctionnelles in-vitro. Dans les modèles in-vitro imitant un syndrome post-arrêt cardiaque, circNFAT5 fût modulé lors de l'activation des cellules T et des monocytes, suggérant un lien possible entre cet ARNcirc et le processus inflammatoire post-ACEH. De plus, des expériences préliminaires ont suggéré une relation indépendante de l'expression de circNFAT5 de celle de son gène parental, renforçant un rôle fonctionnel indépendant de circNFAT5. Après avoir investigué plusieurs pistes, aucune d’entre elles n’a permis d’identifier formellement les fonctions de circNFAT5. Par conséquent, des explorations futures seront nécessaires pour comprendre les fonctions biologiques de circNFAT5. Enfin, les 3 ARNcirc les plus performants identifiés dans l'étude TTM ont également été mesurés par qPCR chez les 674 patients de l'essai TTM2. Les 3 ARNcirc ont confirmés leur capacité de prédiction de l’évolution neurologique et de la survie, mais uniquement dans le groupe de patients traités en normothermie. CircDNM2 fût le candidat le plus performant dans cette étude.En conclusion, ce projet visait à investiguer l'utilisation potentielle des ARNnc comme biomarqueurs après un ACEH et représente un point de départ pour de futures études sur l'utilité clinique et les mécanismes d'action des candidats identifiés pour le pronostic des patients après un ACEH

The right light for paintings: an ambitious challenge

Light is fundamental in architectural design: spaces and objects perception is strictly influenced by luminous scenarios. This is especially true as regards museums applications. In these cases, lighting is essential to assure the complete perception of colours and shapes of artworks, guaranteeing visual comfort conditions. A reproduction of museum installation was set in the Laboratory of Photometry and Lighting at the Department of Industrial Engineering of University of Study of Naples “Federico II” and two different paintings were lit by four light scenes. Then tests on subjects were carried out, in order to investigate human preferences about artworks lighting; results are reported in this paper