Gender differences in cognitive reserve: implication for subjective cognitive decline in women.

BACKGROUND: Subjective Cognitive Decline (SCD) is a self-experienced decline in cognitive capacity with normal performance on standardized cognitive tests, showing to increase risk of Alzheimer's Disease (AD). Cognitive reserve seems to influence the progression from SCD to Mild Cognitive Impairment (MCI) and to AD. The aim of our study was to investigate gender differences in cognitive reserve evaluating how sex might modulate the role of cognitive reserve on SCD. METHODS: We included 381 SCD patients who underwent clinical evaluation, neuropsychological assessment, evaluation of premorbid intelligence by the Test di Intelligenza Breve (TIB), cognitive complaints by the Memory Assessment Clinics Questionnaire (MAC-Q), and apolipoprotein E (APOE) genotyping. RESULTS: The proportion between women and men was significantly different (68.7% [95% CI 63.9-73.4 vs 31.4%, 95% CI 26.6-36.0]). Women were younger than men at onset of SCD and at the baseline visit (p = 0.021), had lower years of education (p = 0.007), lower TIB scores (p < 0.001), and higher MAC-Q scores (p = 0.012). TIB was directly associated with age at onset of SCD in both women and men, while years of education was inversely associated with age at onset only in women. Multivariate analysis showed that sex influences TIB independently from years of education. TIB was directly associated with MAC-Q in men. CONCLUSIONS: Sex interacts with premorbid intelligence and education level in influencing the age at onset and the severity of SCD. As the effect of education was different between men and women, we speculated that education might act as a minor contributor of cognitive reserve in women.

Hepatitis B protein HBx binds the DLEU2 lncRNA to sustain cccDNA and host cancer-related gene transcription.

OBJECTIVE: The HBV HBx regulatory protein is required for transcription from the covalently closed circular DNA (cccDNA) minichromosome and affects the epigenetic control of both viral and host cellular chromatin. DESIGN: We explored, in relevant cellular models of HBV replication, the functional consequences of HBx interaction with DLEU2, a long non-coding RNA (lncRNA) expressed in the liver and increased in human hepatocellular carcinoma (HCC), in the regulation of host target genes and the HBV cccDNA. RESULTS: We show that HBx binds the promoter region, enhances the transcription and induces the accumulation of DLEU2 in infected hepatocytes. We found that nuclear DLEU2 directly binds HBx and the histone methyltransferase enhancer of zeste homolog 2 (EZH2), the catalytic active subunit of the polycomb repressor complex 2 (PRC2) complex. Computational modelling and biochemical evidence suggest that HBx and EZH2 share two preferential binding sites in DLEU2 intron 1. HBx and DLEU2 co-recruitment on the cccDNA displaces EZH2 from the viral chromatin to boost transcription and viral replication. DLEU2-HBx association with target host promoters relieves EZH2 repression and leads to the transcriptional activation of a subset of EZH2/PRC2 target genes in HBV-infected cells and HBV-related HCCs. CONCLUSIONS: Our results highlight the ability of HBx to bind RNA to impact on the epigenetic control of both viral cccDNA and host genes and provide a new key to understand the role of DLEU2 and EZH2 overexpression in HBV-related HCCs and HBx contribution to hepatocytes transformation.

Genome-wide identification of direct HBx genomic targets.

BACKGROUND: The Hepatitis B Virus (HBV) HBx regulatory protein is required for HBV replication and involved in HBV-related carcinogenesis. HBx interacts with chromatin modifying enzymes and transcription factors to modulate histone post-translational modifications and to regulate viral cccDNA transcription and cellular gene expression. Aiming to identify genes and non-coding RNAs (ncRNAs) directly targeted by HBx, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) to analyse HBV recruitment on host cell chromatin in cells replicating HBV. RESULTS: ChIP-Seq high throughput sequencing of HBx-bound fragments was used to obtain a high-resolution, unbiased, mapping of HBx binding sites across the genome in HBV replicating cells. Protein-coding genes and ncRNAs involved in cell metabolism, chromatin dynamics and cancer were enriched among HBx targets together with genes/ncRNAs known to modulate HBV replication. The direct transcriptional activation of genes/miRNAs that potentiate endocytosis (Ras-related in brain (RAB) GTPase family) and autophagy (autophagy related (ATG) genes, beclin-1, miR-33a) and the transcriptional repression of microRNAs (miR-138, miR-224, miR-576, miR-596) that directly target the HBV pgRNA and would inhibit HBV replication, contribute to HBx-mediated increase of HBV replication. CONCLUSIONS: Our ChIP-Seq analysis of HBx genome wide chromatin recruitment defined the repertoire of genes and ncRNAs directly targeted by HBx and led to the identification of new mechanisms by which HBx positively regulates cccDNA transcription and HBV replication.

miRNA-27a-3p is involved in the plasticity of differentiated hepatocytes.

BACKGROUND: Epigenetic mechanisms, including DNA methylation, histone modifications, and chromatin remodeling, are highly involved in the regulation of hepatocyte viability, proliferation, and plasticity. We have previously demonstrated that repression of H3K27 methylation in differentiated hepatic HepaRG cells by treatment with GSK-J4, an inhibitor of JMJD3 and UTX H3K27 demethylase activity, changed their phenotype, inducing differentiated hepatocytes to proliferate. In addition to the epigenetic enzymatic role in the regulation of the retro-differentiation process, emerging evidence indicate that microRNAs (miRNAs) are involved in controlling hepatocyte proliferation during liver regeneration. Hence, the aim of this work is to investigate the impact of H3K27 methylation on miRNAs expression profile and its role in the regulation of the differentiation status of human hepatic progenitors HepaRG cells. METHODS: A miRNA-sequencing was carried out in differentiated HepaRG cells treated or not with GSK-J4. Target searching and Gene Ontology analysis were performed to identify the molecular processes modulated by differentially expressed miRNAs. The biological functions of selected miRNAs was further investigated by transfection of miRNAs inhibitors or mimics in differentiated HepaRG cells followed by qPCR analysis, albumin ELISA assay, CD49a FACS analysis and EdU staining. RESULTS: We identified 12 miRNAs modulated by GSK-J4; among these, miR-27a-3p and miR- 423-5p influenced the expression of several proliferation genes in differentiated HepaRG cells. MiR-27a-3p overexpression increased the number of hepatic cells reentering proliferation. Interestingly, both miR-27a-3p and miR-423-5p did not affect the expression levels of genes involved in the differentiation of progenitors HepaRG cells. CONCLUSIONS: Modulation of H3K27me3 methylation in differentiated HepaRG cells, by GSK-J4 treatment, influenced miRNA' s expression profile pushing liver cells towards a proliferating phenotype. We demonstrated the involvement of miR-27a-3p in reinducing proliferation of differentiated hepatocytes suggesting a potential role in liver plasticity.

Facilitators and barriers in HIV testing and continuum of care among migrant transgender women who are sex workers residing in Florence, Italy.

Background: An increased risk of contracting HIV infection, suboptimal adherence, and a loss to follow-up have been observed in migrants, particularly if those individuals are transgender or sex workers. A clear picture of the HIV epidemic among migrants is complex due to the lack of specific national data. Aims: We developed a qualitative study that describes the barriers and facilitators (cultural, social, and personal) in HIV testing and the continuum of care for a group of migrant transgender women who are sex workers. Methods: A semi-structured interview was conducted with a group of migrant transgender women who are sex workers living with HIV or with unknown HIV serostatus residing in the Florentine metropolitan area. Results: We included 12 participants: 3 had unknown HIV serostatus and 9 were living with HIV in follow-up at the Clinic of Infectious and Tropical Diseases, Careggi University hospiral, Florence, Italy. Among barriers, the perceived stigma due to their identity as migrants and transgender people, the language lack of ability and the legal position in the host country played a significant role. Moreover, the interviewees claimed having no alternative to sex work: for those individuals, changing their lifestyle condition is perceived as difficult or impossible due to social prejudices. Conversely, the interviewees considered support services, such as cultural mediators/interpreters and street units, as facilitators to HIV testing, access to care, and continuum of care. Having regular and accessible ART and the availability of a more consistent health care system, represent reasons for HIV-positive migrants living with HIV to move to Italy. Conclusions: Knowledge of this population's personal experience regarding the barriers and factors that facilitate access to the HIV care system is essential for planning public health interventions capable of responding to the real needs of patients.

Molecular mechanisms of HBV-associated hepatocarcinogenesis.

Hepatitis B virus (HBV) contributes to hepatocellular carcinoma (HCC) development through direct and indirect mechanisms. HBV-DNA integration into the host genome occurs at early steps of clonal tumor expansion and induces both genomic instability and direct insertional mutagenesis of diverse cancer-related genes. Prolonged expression of the viral regulatory protein HBx and the large envelope protein deregulate the cellular transcription program and proliferation control and sensitize liver cells to carcinogenic factors. Epigenetic changes targeting the expression of tumor suppressor genes occur early in the development of HCC. A major role is played by HBx that is recruited on cellular chromatin and modulates chromatin dynamics at specific gene loci. Compared with tumors associated with other risk factors, HBV-related tumors have a higher rate of chromosomal alterations and p53 inactivation by mutations, overexpress fetal liver/hepatic progenitor cells genes, and show a specific activation of the AKT pathway. The wnt/ß-catenin pathway is also often activated, but HBV-related tumors display a low rate of activating ß-catenin mutations. All available evidence strongly supports the notion that chronic HBV infection triggers both common and etiology-specific oncogenic pathways, thus playing a direct role beyond stimulation of host immune responses and chronic necroinflammatory liver disease.

The Individual- and Organization-Related Stressors in Pandemic Scale for Healthcare Workers (IOSPS-HW): Development and Psychometric Properties of a New Instrument to Assess Individual and Organizational Stress Factors in Periods of Pandemics.

The validation and psychometric properties of the Individual and Organization related Stressors in Pandemic Scale for Healthcare Workers (IOSPS-HW) were presented. This is a new measure to assess individual factors related to the health and well-being of individuals, such as family and personal relationships, as well as organizational factors related to the management of the pandemic, including workplace relationships, job management and communication. Across two studies conducted at different time points of the pandemic, psychometric evidence of the IOSPS-HW is presented. In Study 1, through a cross-sectional design, we conducted exploratory and confirmatory factor analysis through which the originally developed 43 items scale was reduced to a 20-item bidimensional scale with two correlated dimensions: Organization-related Stressors (O-S; 12 items) and Individual- and Health-related Stressors (IH-S; 8 items). Internal consistency and criterion validity were also provided by investigating the relationship with post-traumatic stress. In Study 2, we provided evidence for the temporal invariance of the measure and for temporal stability through a Multigroup-CFA through a longitudinal design. We also supported the criterion and predictive validity. The results suggest that IOSPS-HW is a good instrument to simultaneously investigating individual and organizational factors related to sanitary emergencies in healthcare workers.

Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function.

HBV cccDNA, the template for transcription of all viral mRNAs, accumulates in the nucleus of infected cells as a stable episome organized into minichromosomes by histones and non-histone viral and cellular proteins. Using a cccDNA-specific chromatin immunoprecipitation (ChIP)-based quantitative assay, we have previously shown that transcription of the HBV minichromosome is regulated by epigenetic changes of cccDNA-bound histones and that modulation of the acetylation status of cccDNA-bound H3/H4 histones impacts on HBV replication. We now show that the cellular histone acetyltransferases CBP, p300, and PCAF/GCN5, and the histone deacetylases HDAC1 and hSirt1 are all recruited in vivo onto the cccDNA. We also found that the HBx regulatory protein produced in HBV replicating cells is recruited onto the cccDNA minichromosome, and the kinetics of HBx recruitment on the cccDNA parallels the HBV replication. As expected, an HBV mutant that does not express HBx is impaired in its replication, and exogenously expressed HBx transcomplements the replication defects. p300 recruitment is severely impaired, and cccDNA-bound histones are rapidly hypoacetylated in cells replicating the HBx mutant, whereas the recruitment of the histone deacetylases hSirt1 and HDAC1 is increased and occurs at earlier times. Finally, HBx mutant cccDNA transcribes significantly less pgRNA. Altogether our results further support the existence of a complex network of epigenetic events that influence cccDNA function and HBV replication and identify an epigenetic mechanism (i.e., to prevent cccDNA deacetylation) by which HBx controls HBV replication.

Perception of the COVID-19 pandemic among people with spinal cord injury: an Italian survey.

STUDY DESIGN: An observational study based on an online survey to explore if the participant had experienced (1) cancellation or delay of scheduled health services (2) reduction of assistance provided by a caregiver (3) barriers to social participation and recreational activities. Three validated questionnaires to investigate well-being and symptoms of anxiety and depression were also administered. OBJECTIVES: Our main aim was to quantify the obstacles experienced by adults living with SCI in Italy during COVID-19 pandemic, to explore the presence of depression and anxiety symptoms and to quantify subjective well-being. SETTING: Outpatient clinic of a Spinal Unit in Italy. METHODS: Online survey via direct contact and by e-mail lists. RESULTS: In total, 101 individuals completed the survey. Of, 82.2% participants reported a history of deferment or cancellation of non-COVID-19 health services. The majority (56.4%) revealed that, at least seldom, they have chosen to reduce their usual everyday activities and more than one third (37.6%) affirmed that they had been forced to renounce to one or more of their occupations. Discontinuation of assistance by caregivers was uncommon. The median score of questionnaires measuring depression and anxiety symptoms do not differ significatively when compared with prior studies. The variable that explored the limitations experienced in everyday activities showed a significant correlation with the results of the questionnaires measuring well-being and symptoms of anxiety. CONCLUSIONS: We believe that our results could contribute to the discussion ongoing inside our community on how to answer to the new challenges of this pandemic period and of the post-pandemic future.

CAG Repeats Within the Non-pathological Range in the HTT Gene Influence Personality Traits in Patients With Subjective Cognitive Decline: A 13-Year Follow-Up Study.

Objective: HTT is a gene containing a key region of CAG repeats. When expanded beyond 39 repeats, Huntington disease (HD) develops. HTT genes with <35 repeats are not associated with HD. The biological function of CAG repeat expansion below the non-pathological threshold is not well understood. In fact higher number of repeats in HTT confer advantageous changes in brain structure and general intelligence, but several studies focused on establishing the association between CAG expansions and susceptibility to psychiatric disturbances and to other neurodegenerative disease than HD. We hypothesized that HTT CAG repeat length below the pathological threshold might influence mood and personality traits in a longitudinal sample of individuals with Subjective Cognitive Decline. Methods: We included 54 patients with SCD. All patients underwent an extensive neuropsychological battery at baseline, APOE genotyping and analysis of HTT alleles. We used the Big Five Factors Questionnaire (BFFQ) and Hamilton Depression Rating Scale (HDRS), respectively, to assess personality traits of patients and depression at baseline. Patients who did not progress to Mild Cognitive Impairment (MCI) had at least 5-year follow-up time. Results: In the whole sample, CAG repeat number in the shorter HTT allele was inversely correlated with conscientiousness (Pearson = -0.364, p = 0.007). There was no correlation between HDRS and CAG repeats. During the follow-up, 14 patients [25.93% (95% C.I. = 14.24-37.61)] progressed to MCI (MCI+) and 40 [74.07% (95% C.I. = 62.39-85.76)] did not (MCI-). When we performed the same analysis in the MCI+ group we found that: CAG repeat length on the shorter allele was inversely correlated with energy (Pearson = 0.639, p = 0.014) and conscientiousness (Pearson = -0.695, p = 0.006). CAG repeat length on the longer allele was inversely correlated with conscientiousness (Pearson = -0.901, p < 0.001) and directly correlated with emotional stability (Pearson = 0.639, p = 0.014). These associations were confirmed also by multivariate analysis. We found no correlations between BFFQ parameters and CAG repeats in the MCI- group. Discussion: Personality traits and CAG repeat length in the intermediate range have been associated with progression of cognitive decline and neuropathological findings consistent with AD. We showed that CAG repeat lengths in the HTT gene within the non-pathological range influence personality traits.

Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection.

BACKGROUND & AIMS: The molecular biology of hepatitis B virus (HBV) has been extensively studied but the exact role of the hepatitis B X protein (HBx) in the context of natural HBV infections remains unknown. METHODS: Primary human hepatocytes and differentiated HepaRG cells allowing conditional trans complementation of HBx were infected with wild type (HBV(wt)) or HBx deficient (HBV(x-)) HBV particles and establishment of HBV replication was followed. RESULTS: We observed that cells inoculated with HBx-deficient HBV particles (HBV(x-)) did not lead to productive HBV infection contrary to cells inoculated with wild type HBV particles (HBV(wt)). Although equal amounts of nuclear covalently closed circular HBV-DNA (cccDNA) demonstrated comparable uptake and nuclear import, active transcription was only observed from HBV(wt) genomes. Trans-complementation of HBx was able to rescue transcription from the HBV(x-) genome and led to antigen and virion secretion, even weeks after infection. Constant expression of HBx was necessary to maintain HBV antigen expression and replication. Finally, we demonstrated that HBx is not packaged into virions during assembly but is expressed after infection within the new host cell to allow epigenetic control of HBV transcription from cccDNA. CONCLUSIONS: Our results demonstrate that HBx is required to initiate and maintain HBV replication and highlight HBx as the key regulator during the natural infection process.

Differential regulation of E2F1 apoptotic target genes in response to DNA damage.

E2F1, a member of the E2F family of transcription factors, in addition to its established proliferative effect, has also been implicated in the induction of apoptosis through p53-dependent and p53-independent pathways. Several genes involved in the activation or execution of the apoptotic programme have recently been shown to be upregulated at the transcriptional level by E2F1 overexpression, including the genes encoding INK4a/ARF, Apaf-1, caspase 7 and p73 (refs 3-5). E2F1 is stabilized in response to DNA damage but it has not been established how this translates into the activation of specific subsets of E2F target genes. Here, we applied a chromatin immunoprecipitation approach to show that, in response to DNA damage, E2F1 is directed from cell cycle progression to apoptotic E2F target genes. We identify p73 as an important E2F1 apoptotic target gene in DNA damage response and we show that acetylation is required for E2F1 recruitment on the P1p73 promoter and for its transcriptional activation.

Early intra-intensive care unit psychological intervention promotes recovery from post traumatic stress disorders, anxiety and depression symptoms in critically ill patients.

INTRODUCTION: Critically ill patients who require intensive care unit (ICU) treatment may experience psychological distress with increasing development of psychological disorders and related morbidity. Our aim was to determine whether intra-ICU clinical psychologist interventions decrease the prevalence of anxiety, depression and posttraumatic stress disorder (PTSD) after 12 months from ICU discharge. METHODS: Our observational study included critical patients admitted before clinical psychologist intervention (control group) and patients who were involved in a clinical psychologist program (intervention group). The Hospital Anxiety and Depression Scale (HADS) and Impact of Event Scale-Revised questionnaires were used to assess the level of posttraumatic stress, anxiety and depression symptoms. RESULTS: The control and intervention groups showed similar demographic and clinical characteristics. Patients in the intervention group showed lower rates of anxiety (8.9% vs. 17.4%) and depression (6.5% vs. 12.8%) than the control group on the basis of HADS scores, even if the differences were not statistically significant. High risk for PTSD was significantly lower in patients receiving early clinical psychologist support than in the control group (21.1% vs. 57%; P < 0.0001). The percentage of patients who needed psychiatric medications at 12 months was significantly higher in the control group than in the patient group (41.7% vs. 8.1%; P < 0.0001). CONCLUSIONS: Our results suggest that that early intra-ICU clinical psychologist intervention may help critically ill trauma patients recover from this stressful experience.

From Development to Aging: The Path to Cellular Senescence.

Significance: Senescence is a cellular state induced by internal or external stimuli, which result in cell cycle arrest, morphological changes, and dysfunctions in mitochondrial and lysosomal functionality as well as the senescence-associated secretory phenotype. Senescent cells accumulate in tissues in physiological and pathological conditions such as development, tissue repair, aging, and cancer. Recent Advances: Growing evidences indicate that senescent cells in vivo are a heterogeneous cell population due to different cell-autonomous activated pathways and distinct microenvironmental contexts. Critical Issues: In this review, we discuss the different contexts where senescence assumes a key role with beneficial or harmful outcomes. The heterogeneous nature of senescence pushes toward resolution of the specific molecular profile and secretome to typify senescent cells in physiological and pathological contexts. Future Directions: Future research will enable exploring the heterogeneity of the senescent population to precisely map the progression of cells through senescent trajectories and study the impact of the therapeutic advantage of senolytic drugs for translational strategies toward supporting the health span. Antioxid. Redox Signal. 34, 294-307.

Comparing prognostic utility between the 8th edition of TNM staging system and the lymph node ratio for oral squamous cell carcinoma.

BACKGROUND: Lymph node metastasis in oral squamous cell carcinoma (OSCC) is associated with poor prognosis. The 8th edition of TNM has implemented new nodal staging criteria. We assess the prognostic utility of the lymph node ratio (LNR) and compare it to that of pN in the TNM 8th edition. METHODS: One hundred and forty-two patients with OSCC were retrospectively studied. Nodal staging was performed using the TMN 8th edition and the prognostic value of the LNR in terms of overall survival (OS) and disease-free survival (DFS) was evaluated. RESULTS: Fifty-seven patients were eligible for inclusion. The LNR was independently prognostic of OS (p = 0.02). Instead N classification was not significantly predictive of OS (p = 0.10). High LNRs resulted in decreases in OS of approximately 40% within 6 months after surgery. CONCLUSIONS: The LNR identifies patients with poor outcomes better than N classification. The lack of reliable LNR cutoffs compromises its utility in clinical practice.

SARS-CoV-2 Entry Genes Expression in Relation with Interferon Response in Cystic Fibrosis Patients.

The expression rate of SARS-CoV-2 entry genes, angiotensin-converting enzyme 2 (ACE2), the main viral receptor and the proteases, furin and transmembrane serine protease 2 (TMPRSS2) in cystic fibrosis (CF) individuals is poorly known. Hence, we examined their levels in upper respiratory samples of CF patients (n = 46) and healthy controls (n = 45). Moreover, we sought to understand the interplay of type I interferon (IFN-I) with ACE2, furin and TMPRSS2 by evaluating their gene expression with respect to ISG15, a well-known marker of IFN activation, in upper respiratory samples and after ex vivo IFNß exposure. Lower ACE2 levels and trends toward the reduction of furin and TMPRSS2 were found in CF patients compared with the healthy controls; decreased ACE2 amounts were also detected in CF individuals with pancreatic insufficiency and in those receiving inhaled antibiotics. Moreover, there was a strong positive correlation between ISG15 and ACE2 levels. However, after ex vivo IFNß stimulation of nasopharyngeal cells, the truncated isoform (dACE2), recently demonstrated as the IFN stimulated one with respect to the full-length isoform (flACE2), slightly augmented in cells from CF patients whereas in those from healthy donors, dACE2 levels showed variable levels of upregulation. An altered expression of SARS-COV-2 entry genes and a poor responsiveness of dACE2 to IFN-I stimulation might be crucial in the diffusion of SARS-CoV-2 infection in CF.

Specific Protein 1 and p53 Interplay Modulates the Expression of the KCTD-Containing Cullin3 Adaptor Suppressor of Hedgehog 2.

The Hedgehog (Hh) signaling pathway plays a crucial role in normal embryonic development and adult tissue homeostasis. On the other end, dysregulated Hh signaling triggers a prolonged mitogenic response that may prompt abnormal cell proliferation, favoring tumorigenesis. Indeed, about 30% of medulloblastomas (MBs), the most common malignant childhood cerebellar tumors, exhibit improper activation of the Hh signaling. The oncosuppressor KCASH2 has been described as a suppressor of the Hh signaling pathway, and low KCASH2 expression was observed in Hh-dependent MB tumor. Therefore, the study of the modulation of KCASH2 expression may provide fundamental information for the development of new therapeutic approaches, aimed to restore physiological KCASH2 levels and Hh inhibition. To this end, we have analyzed the TATA-less KCASH2 proximal promoter and identified key transcriptional regulators of this gene: Sp1, a TF frequently overexpressed in tumors, and the tumor suppressor p53. Here, we show that in WT cells, Sp1 binds KCASH2 promoter on several putative binding sites, leading to increase in KCASH2 expression. On the other hand, p53 is involved in negative regulation of KCASH2. In this context, the balance between p53 and Sp1 expression, and the interplay between these two proteins determine whether Sp1 acts as an activator or a repressor of KCASH2 transcription. Indeed, in p53-/- MEF and p53 mutated tumor cells, we hypothesize that Sp1 drives promoter methylation through increased expression of the DNA methyltransferase 1 (DNMT1) and reduces KCASH2 transcription, which can be reversed by Sp1 inhibition or use of demethylating agents. We suggest therefore that downregulation of KCASH2 expression in tumors could be mediated by gain of Sp1 activity and epigenetic silencing events in cells where p53 functionality is lost. This work may open new venues for novel therapeutic multidrug approaches in the treatment of Hh-dependent tumors carrying p53 deficiency.

Reducing the risk of patient suicide in Tuscany.

Patient suicide is one of the most frequent incidents in healthcare facilities to be reported to the National Observatory of Sentinel Events in Italy. Despite national initiatives, in Tuscany potentially preventable patient suicides still occur in both acute and community care settings. We describe here an aggregated qualitative analysis of 14 patient suicides that took place in public health services between 2017 and 2018. We outline the methodology and results of an improvement action we enacted in the healthcare system that involved reviewing and reinforcing relevant managerial strategies and clinical activities, with the aim of reducing potentially preventable patient suicides.

A Hyper-Glycosylation of HBV Surface Antigen Correlates with HBsAg-Negativity at Immunosuppression-Driven HBV Reactivation in Vivo and Hinders HBsAg Recognition in Vitro.

Immune-suppression driven Hepatitis B Virus (HBV)-reactivation poses serious concerns since it occurs in several clinical settings and can result in severe forms of hepatitis. Previous studies showed that HBV strains, circulating in patients with HBV-reactivation, are characterized by an enrichment of immune-escape mutations in HBV surface antigen (HBsAg). Here, we focused on specific immune-escape mutations associated with the acquisition of N-linked glycosylation sites in HBsAg (NLGSs). In particular, we investigated profiles of NLGSs in 47 patients with immunosuppression-driven HBV-reactivation and we evaluated their impact on HBsAg-antigenicity and HBV-replication in vitro. At HBV-reactivation, despite a median serum HBV-DNA of 6.7 [5.3-8.0] logIU/mL, 23.4% of patients remained HBsAg-negative. HBsAg-negativity at HBV-reactivation correlated with the presence of >1 additional NLGSs (p < 0.001). These NLGSs are located in the major hydrophilic region of HBsAg (known to be the target of antibodies) and resulted from the single mutation T115N, T117N, T123N, N114ins, and from the triple mutant S113N+T131N+M133T. In vitro, NLGSs strongly alter HBsAg antigenic properties and recognition by antibodies used in assays for HBsAg-quantification without affecting HBsAg-secretion and other parameters of HBV-replication. In conclusion, additional NLGSs correlate with HBsAg-negativity despite HBV-reactivation, and hamper HBsAg-antigenicity in vitro, supporting the role of NGSs in immune-escape and the importance of HBV-DNA for a proper diagnosis of HBV-reactivation.

Control of cccDNA function in hepatitis B virus infection.

The template of hepatitis B virus (HBV) transcription, the covalently closed circular DNA (cccDNA), plays a key role in the life cycle of the virus and permits the persistence of infection. Novel molecular techniques have opened new possibilities to investigate the organization and the activity of the cccDNA minichromosome in vivo, and recent advances have started to shed light on the complexity of the mechanisms controlling cccDNA function. Nuclear cccDNA accumulates in hepatocyte nuclei as a stable minichromosome organized by histone and non-histone viral and cellular proteins. Identification of the molecular mechanisms regulating cccDNA stability and its transcriptional activity at the RNA, DNA and epigenetic levels in the course of chronic hepatitis B (CH-B) infection may reveal new potential therapeutic targets for anti-HBV drugs and hence assist in the design of strategies aimed at silencing and eventually depleting the cccDNA reservoir.