MicroRNAs Expression in Response to rhNGF in Epithelial Corneal Cells: Focus on Neurotrophin Signaling Pathway.

PURPOSE: Nerve growth factor efficacy was demonstrated for corneal lesions treatment, and recombinant human NGF (rhNGF) was approved for neurotrophic keratitis therapy. However, NGF-induced molecular responses in cornea are still largely unknown. We analyzed microRNAs expression in human epithelial corneal cells after time-dependent rhNGF treatment. METHODS: Nearly 700 microRNAs were analyzed by qRT-PCR. MicroRNAs showing significant expression differences were examined by DIANA-miRpath v.3.0 to identify target genes and pathways. Immunoblots were performed to preliminarily assess the strength of the in silico results. RESULTS: Twenty-one microRNAs (miR-26a-1-3p, miR-30d-3p, miR-27b-5p, miR-146a-5p, miR-362-5p, mir-550a-5p, mir-34a-3p, mir-1227-3p, mir-27a-5p, mir-222-5p, mir-151a-5p, miR-449a, let7c-5p, miR-337-5p, mir-29b-3p, miR-200b-3p, miR-141-3p, miR-671-3p, miR-324-5p, mir-411-3p, and mir-425-3p) were significantly regulated in response to rhNGF. In silico analysis evidenced interesting target genes and pathways, including that of neurotrophin, when analyzed in depth. Almost 80 unique target genes (e.g., PI3K, AKT, MAPK, KRAS, BRAF, RhoA, Cdc42, Rac1, Bax, Bcl2, FasL) were identified as being among those most involved in neurotrophin signaling and in controlling cell proliferation, growth, and apoptosis. AKT and RhoA immunoblots demonstrated congruence with microRNA expression, providing preliminary validation of in silico data. CONCLUSIONS: MicroRNA levels in response to rhNGF were for the first time analyzed in corneal cells. Novel insights about microRNAs, target genes, pathways modulation, and possible biological responses were provided. Importantly, given the putative role of microRNAs as biomarkers or therapeutic targets, our results make available data which might be potentially exploitable for clinical applications.

Next-generation sequencing: recent applications to the analysis of colorectal cancer.

Since the establishment of the Sanger sequencing method, scientists around the world focused their efforts to progress in the field to produce the utmost technology. The introduction of next-generation sequencing (NGS) represents a revolutionary step and promises to lead to massive improvements in our understanding on the role of nucleic acids functions. Cancer research began to use this innovative and highly performing method, and interesting results started to appear in colorectal cancer (CRC) analysis. Several studies produced high-quality data in terms of mutation discovery, especially about actionable or less frequently mutated genes, epigenetics, transcriptomics. Analysis of results is unveiling relevant perspectives aiding to evaluate the response to therapies. Novel evidences have been presented also in other directions such as gut microbiota or CRC circulating tumor cells. However, despite its unquestioned potential, NGS poses some issues calling for additional studies. This review intends to offer a view of the state of the art of NGS applications to CRC through examination of the most important technologies and discussion of recent published results.

Bioinformatics approach to predict target genes for dysregulated microRNAs in hepatocellular carcinoma: study on a chemically-induced HCC mouse model.

BACKGROUND: Hepatocellular carcinoma (HCC) is an aggressive epithelial tumor which shows very poor prognosis and high rate of recurrence, representing an urgent problem for public healthcare. MicroRNAs (miRNAs/miRs) are a class of small, non-coding RNAs that attract great attention because of their role in regulation of processes such as cellular growth, proliferation, apoptosis. Because of the thousands of potential interactions between a single miR and target mRNAs, bioinformatics prediction tools are very useful to facilitate the task for individuating and selecting putative target genes. In this study, we present a chemically-induced HCC mouse model to identify differential expression of miRNAs during the progression of the hepatic injury up to HCC onset. In addition, we describe an established bioinformatics approach to highlight putative target genes and protein interaction networks where they are involved. RESULTS: We describe four miRs (miR-125a-5p, miR-27a, miR-182, miR-193b) which showed to be differentially expressed in the chemically-induced HCC mouse model. The miRs were subjected to four of the most used predictions tools and 15 predicted target genes were identified. The expression of one (ANK3) among the 15 predicted targets was further validated by immunoblotting. Then, enrichment annotation analysis was performed revealing significant clusters, including some playing a role in ion transporter activity, regulation of receptor protein serine/threonine kinase signaling pathway, protein import into nucleus, regulation of intracellular protein transport, regulation of cell adhesion, growth factor binding, and regulation of TGF-beta/SMAD signaling pathway. A network construction was created and links between the selected miRs, the predicted targets as well as the possible interactions among them and other proteins were built up. CONCLUSIONS: In this study, we combined miRNA expression analysis, obtained by an in vivo HCC mouse model, with a bioinformatics-based workflow. New genes, pathways and protein interactions, putatively involved in HCC initiation and progression, were identified and explored.

A method to comprehensively identify germline SNVs, INDELs and CNVs from whole exome sequencing data of BRCA1/2 negative breast cancer patients.

In the rapidly evolving field of genomics, understanding the genetic basis of complex diseases like breast cancer, particularly its familial/hereditary forms, is crucial. Current methods often examine genomic variants-such as Single Nucleotide Variants (SNVs), insertions/deletions (Indels), and Copy Number Variations (CNVs)-separately, lacking an integrated approach. Here, we introduced a robust, flexible methodology for a comprehensive variants' analysis using Whole Exome Sequencing (WES) data. Our approach uniquely combines meticulous validation with an effective variant filtering strategy. By reanalyzing two germline WES datasets from BRCA1/2 negative breast cancer patients, we demonstrated our tool's efficiency and adaptability, uncovering both known and novel variants. This contributed new insights for potential diagnostic, preventive, and therapeutic strategies. Our method stands out for its comprehensive inclusion of key genomic variants in a unified analysis, and its practical resolution of technical challenges, offering a pioneering solution in genomic research. This tool presents a breakthrough in providing detailed insights into the genetic alterations in genomes, with significant implications for understanding and managing hereditary breast cancer.

Trauma-spectrum symptoms among the Italian general population in the time of the COVID-19 outbreak.

Background: Recent evidence showed adverse mental health outcomes associated with the COVID-19 pandemic, including trauma-related symptoms. The Global Psychotrauma Screen (GPS) is a brief instrument designed to assess a broad range of trauma-related symptoms with no available validation in the Italian population. Aims: This study aimed to examine the factor structure of the Italian version of the GPS in a general population sample exposed to the COVID-19 pandemic and to evaluate trauma-related symptoms in the context of COVID-19 related risk factors associated with lockdown measures. Methods: We conducted a cross-sectional web-based observational study as part of a long-term monitoring programme of mental health outcomes in the general population. Eighteen thousand one hundred forty-seven participants completed a self-report online questionnaire to collect key demographic data and evaluate trauma-related symptoms using the GPS, PHQ-9, GAD-7, ISI, and PSS. Validation analyses included both exploratory and confirmatory factor analysis and correlation analyses. The relation with putative COVID-19 related risk factors was explored by multivariate regression analysis. Results: Exploratory factor analyses supported a two-factor model. Confirmatory factor analysis showed that the best fitting model was a three-factor solution, with core Post-traumatic Stress Symptoms (PTSS) (re-experiencing, avoidance, hyperarousal), Negative Affect (symptoms of depressed mood, anxiety, irritability), and Dissociative symptoms. GPS Risk factors and specific COVID-19 related stressful events were associated with GPS total and the three factor scores. Conclusions: Our data suggest that a wide range of trauma-spectrum symptoms were reported by a large Italian sample during the COVID-19 pandemic. The GPS symptoms clustered best in three factors: Negative Affect symptoms, Core PTSS, and Dissociative symptoms. In particular, high rates of core PTSS and negative affect symptoms were associated with the COVID-19 pandemic in Italy and should be routinely assessed in clinical practice.

Antecedentes: Evidencias recientes revelaron resultados adversos para la salud mental asociados con la pandemia de COVID-19, incluyendo síntomas relacionados con el trauma. El Mapeo Global de Psicotrauma (GPS, en sus siglas en inglés) es un breve instrumento diseñado para evaluar una amplia gama de síntomas relacionados con el trauma, sin validación disponible en la población italiana.Objetivos: El objetivo de este estudio fue examinar la estructura de factores de la versión italiana del GPS en una muestra de la población general expuesta a la pandemia de COVID-19 y evaluar los síntomas relacionados con el trauma en el contexto de los factores de riesgo relacionados con COVID-19 asociados con las medidas de confinamiento.Métodos: Realizamos un estudio observacional transversal basado en la web como parte de un programa de vigilancia a largo plazo de los resultados de salud mental en la población general. Dieciocho mil ciento cuarenta y siete participantes completaron un cuestionario en línea de autorreporte para recopilar datos demográficos claves y evaluar los síntomas relacionados con el trauma utilizando el GPS, PHQ-9, GAD-7, ISI y PSS. Los análisis de validación incluyeron análisis factoriales y de correlación tanto exploratorios como confirmatorios. La relación con los posibles factores de riesgo relacionados con COVID-19 se exploró mediante un análisis de regresión multivariante.Resultados: Los análisis factoriales exploratorios apoyaron un modelo de dos factores. El análisis factorial confirmatorio mostró que el modelo que mejor se ajustaba era una solución de tres factores, con los principales síntomas de estrés postraumático (PTSS, en sus siglas en inglés) (reexperimentación, evitación, hiperactivación), el efecto negativo (síntomas de depresión, ansiedad, irritabilidad) y los síntomas disociativos. Los factores de riesgo del GPS y los eventos estresantes específicos relacionados con COVID-19 se asociaron con el total del GPS y las tres puntuaciones de los factores.Conclusiones: Nuestros datos sugieren que una amplia gama de síntomas de espectro traumático fueron reportados por una gran muestra italiana durante la pandemia de COVID-19. Los síntomas del GPS se agruparon mejor en tres factores: Síntomas de Afecto Negativo, PTSS Central y Síntomas Disociativos. En particular, las altas tasas de PTSS central y los síntomas de afecto negativo se asociaron con la pandemia de COVID-19 en Italia y deben ser evaluados rutinariamente en la práctica clínica.

Carfilzomib Enhances the Suppressive Effect of Ruxolitinib in Myelofibrosis.

As the first FDA-approved tyrosine kinase inhibitor for treatment of patients with myelofibrosis (MF), ruxolitinib improves clinical symptoms but does not lead to eradication of the disease or significant reduction of the mutated allele burden. The resistance of MF clones against the suppressive action of ruxolitinib may be due to intrinsic or extrinsic mechanisms leading to activity of additional pro-survival genes or signalling pathways that function independently of JAK2/STAT5. To identify alternative therapeutic targets, we applied a pooled-shRNA library targeting ~5000 genes to a JAK2V617F-positive cell line under a variety of conditions, including absence or presence of ruxolitinib and in the presence of a bone marrow microenvironment-like culture medium. We identified several proteasomal gene family members as essential to HEL cell survival. The importance of these genes was validated in MF cells using the proteasomal inhibitor carfilzomib, which also enhanced lethality in combination with ruxolitinib. We also showed that proteasome gene expression is reduced by ruxolitinib in MF CD34+ cells and that additional targeting of proteasomal activity by carfilzomib enhances the inhibitory action of ruxolitinib in vitro. Hence, this study suggests a potential role for proteasome inhibitors in combination with ruxolitinib for management of MF patients.

Mental Health Outcomes Among Healthcare Workers and the General Population During the COVID-19 in Italy.

INTRODUCTION: During the COVID-19 pandemic, healthcare workers in Italy have been exposed to an unprecedented pressure and traumatic events. However, no direct comparison with the general population is available so far. The aim of this study is to detail mental health outcomes in healthcare workers compared to the general population. METHODS: 24050 respondents completed an on-line questionnaire during the contagion peak, 21342 general population, 1295 second-line healthcare workers, and 1411 front-line healthcare workers. Depressive, anxious, post-traumatic symptoms and insomnia were assessed. Specific COVID-19 related potential risk factors were also considered in healthcare workers. RESULTS: Depressive symptoms were more frequent in the general population (28.12%) and front-line healthcare workers (28.35%) compared to the second-line healthcare workers (19.98%) groups. Anxiety symptoms showed a prevalence of 21.25% in the general population, 18.05% for second-line healthcare workers, and 20.55% for front-line healthcare workers. Insomnia showed a prevalence of 7.82, 6.58, and 9.92% for the general population, second-line healthcare workers, and front-line healthcare workers, respectively. Compared to the general population, front-line healthcare workers had higher odds of endorsing total trauma-related symptoms. Both second-line healthcare workers and front-line healthcare workers had higher odds of endorsing core post-traumatic symptoms compared to the general population, while second-line healthcare workers had lower odds of endorsing negative affect and dissociative symptoms. Higher total traumatic symptom score was associated with being a front-line healthcare worker, having a colleague infected, hospitalized, or deceased, being a nurse, female gender, and younger age. CONCLUSION: This study suggests a significant psychological impact of the COVID-19 pandemic on the Italian general population and healthcare workers. Front-line healthcare workers represent a specific at-risk population for post-traumatic symptoms. These findings underline the importance of monitoring and intervention strategies.

COVID-19 Pandemic and Lockdown Measures Impact on Mental Health Among the General Population in Italy.

BACKGROUND: The psychological impact of the COronaVIrus Disease 2019 (COVID-19) outbreak and lockdown measures on the Italian population are unknown. The current study assesses rates of mental health outcomes in the Italian general population three to 4 weeks into lockdown measures and explores the impact of COVID-19 related potential risk factors. METHODS: A web-based survey spread throughout the internet between March 27th and April 6th 2020. Eighteen thousand one hundred forty-seven individuals completed the questionnaire, 79.6% women. Selected outcomes were post-traumatic stress symptoms (PTSS), depression, anxiety, insomnia, perceived stress, and adjustment disorder symptoms (ADS). Seemingly unrelated logistic regression analysis was performed to identify COVID-19 related risk factors. RESULTS: Endorsement rates for PTSS were 6,604 (37%), 3,084 (17.3%) for depression, 3,700 (20.8%) for anxiety, 1,301 (7.3%) for insomnia, 3,895 (21.8%) for high perceived stress and 4,092 (22.9%) for adjustment disorder. Being woman and younger age were associated with all of the selected outcomes. Quarantine was associated with PTSS, anxiety and ADS. Any recent COVID-related stressful life event was associated with all the selected outcomes. Discontinued working activity due to the COVID-19 was associated with all the selected outcomes, except for ADS; working more than usual was associated with PTSS, Perceived stress and ADS. Having a loved one deceased by COVID-19 was associated with PTSS, depression, perceived stress, and insomnia. CONCLUSION: We found high rates of negative mental health outcomes in the Italian general population 3 weeks into the COVID-19 lockdown measures and different COVID-19 related risk factors. These findings warrant further monitoring on the Italian population's mental health.

Mesenchymal stem cells of Systemic Sclerosis patients, derived from different sources, show a profibrotic microRNA profiling.

Systemic Sclerosis (SSc) is a disease with limited therapeutic possibilities. Mesenchymal stem cells (MSCs)-therapy could be a promising therapeutic option, however the ideal MSCs source has not yet been found. To address this problem, we perform comparison between bone marrow (BM)-MSCs and adipose (A)-MSCs, by the miRs expression profile, to identify the gene modulation in these two MSCs source. MicroRNAs (miRs) are RNAs sequences, regulating gene expression and MSCs, derived from different tissues, may differently respond to the SSc microenvironment. The miRs array was used for the miRs profiling and by DIANA-mirPath tool we identified the biological functions of the dysregulated miRs. In SSc-BM-MSCs, 6 miRs were significantly down-regulated and 4 miRs up-regulated. In SSc-A-MSCs, 11 miRs were significantly down-regulated and 3 miRs up-regulated. Interestingly, in both the sources, the involved pathways included the senescence mechanisms and the pro-fibrotic behaviour. Furthermore, both the MSCs sources showed potential compensatory ability. A deeper knowledge of this miRs signature might give more information about some pathogenic steps of the disease and in the same time clarify the possible therapeutic role of autologous MSCs in the regenerative therapy in SSc.