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Background: the protein phosphatase 3 catalytic subunit alpha (PPP3CA) gene encodes for the alpha isoform of the calcineurin catalytic subunit, which controls the phosphorylation status of many targets. Currently, 23 pathogenic variants of PPP3CA are known, with clinical manifestations varying by mutation type and domain., Results: through whole exome sequencing, we found two de novo variants in PPP3CA: a frameshift variant predicted leading to a truncated protein in Pt.1 and a splicing variant in Pt.2 associated with mild phenotype. PPP3CA is ubiquitously expressed with tissue-specificity of; namely, splicing isoform 1 prevailing over isoform 2 in the central nervous system. By analyzing isoform distribution in patient-derived cell lines, we highlight a skewed expression of both isoforms in Pt.1, whereas only isoform 2 shows a moderate reduction in Pt.2. In contrast, we did not observe significant abundance changes at the protein level. Cell lines derived from Pt.1 showed a reduced proliferation, associated with an increase in cell death and the upregulation of the unfolded protein response (UPR) pathway., Conclusion: data suggest that an aberrant PPP3CA protein in Pt.1 could lead to UPR activation resulting in increased cell death. In Pt.2 an imbalance between the two main isoforms possibly explains the peculiar pathological manifestations, such as a moderate developmental delay., Competing Interests: Declarations. Ethics approval and consent to participate: This study was performed in line with the principles of the Declaration of Helsinky and its later amendments. Approval of use of cell line was granted by the Ethics Committee of University of Milan (number 99/20, 17 November 2020). Written informed consent was obtained from the parents. Consent for publication: Not applicable. Competing interests: The authors have no relevant financial or non-financial interests to disclose., (© 2024. The Author(s).)

Background and Aims: The best adjuvant chemotherapy for resected biliary tract cancer (BTC) is under debate, with capecitabine supported by weak evidence. Aim of this network meta-analysis is to estimate the efficacy of different phase II/III regimens, comparing monotherapies (gemcitabine or fluoropyrimidines) head-to-head, against observation and combination regimens., Methods: A comprehensive literature search was conducted on PubMed and EMBASE for phase II/III randomized clinical trials (RCTs) available as of December 2023, reporting hazard ratios (HRs) of overall survival (OS) and event-free survival (EFS). A frequentist framework employing a random-effects model was applied; treatment rankings were outlined according to P-score, based on direct and indirect evidence. Exploratory subgroup analyses for OS were also performed (primary site, resected margin status and nodal involvement)., Results: Six RCTs (1979 total patients) were identified. Fluoropyrimidine monotherapy showed significantly better OS (HR .84 [.72-.97]) and EFS (HR .79 [.69-.91]) than observation, as any monotherapy did (HR .84 [.74-.96]; HR .79 [.70-.89]). In the head-to-head comparison for OS, only S1 confirmed to be superior to observation alone (HR .69 [.49-.98]) while fluoropyrimidines achieved the best P score (.81), similarly to any monotherapy (0.92). Combinations failed to prove superior to monotherapies with respect both to OS and EFS. Subgroup analyses were inconclusive due to results' inconsistency and limited sample size., Conclusions: Our work confirmed that adjuvant chemotherapy grants OS and EFS benefit for resected BTC patients. Fluoropyrimidines appeared the most effective option, confirming capecitabine as the preferred choice for the Western population., (© 2024 The Author(s). Liver International published by John Wiley & Sons Ltd.)

Background: We previously demonstrated that the human amniotic fluid (hAF) from II trimester of gestation is a feasible source of stromal progenitors (human amniotic fluid stem cells, hAFSC), with significant paracrine potential for regenerative medicine. Extracellular vesicles (EVs) separated and concentrated from hAFSC secretome can deliver pro-survival, proliferative, anti-fibrotic and cardioprotective effects in preclinical models of skeletal and cardiac muscle injury. While hAFSC-EVs isolation can be significantly influenced by in vitro cell culture, here we profiled EVs directly concentrated from hAF as an alternative option and investigated their paracrine potential against oxidative stress., Methods: II trimester hAF samples were obtained as leftover material from prenatal diagnostic amniocentesis following written informed consent. EVs were separated by size exclusion chromatography and concentrated by ultracentrifugation. hAF-EVs were assessed by nanoparticle tracking analysis, transmission electron microscopy, Western Blot, and flow cytometry; their metabolic activity was evaluated by oximetric and luminometric analyses and their cargo profiled by proteomics and RNA sequencing. hAF-EV paracrine potential was tested in preclinical in vitro models of oxidative stress and dysfunction on murine C2C12 cells and on 3D human cardiac microtissue., Results: Our protocol resulted in a yield of 6.31 ± 0.98 × 10 9 EVs particles per hAF milliliter showing round cup-shaped morphology and 209.63 ± 6.10 nm average size, with relevant expression of CD81, CD63 and CD9 tetraspanin markers. hAF-EVs were enriched in CD133/1, CD326, CD24, CD29, and SSEA4 and able to produce ATP by oxygen consumption. While oxidative stress significantly reduced C2C12 survival, hAF-EV priming resulted in significant rescue of cell viability, with notable recovery of ATP synthesis and concomitant reduction of cell damage and lipid peroxidation activity. 3D human cardiac microtissues treated with hAF-EVs and experiencing H 2 O 2 stress and TGFβ stimulation showed improved survival with a remarkable decrease in the onset of fibrosis., Conclusions: Our results suggest that leftover samples of II trimester human amniotic fluid can represent a feasible source of EVs to counteract oxidative damage on target cells, thus offering a novel candidate therapeutic option to counteract skeletal and cardiac muscle injury., Competing Interests: Declaration of competing interest The authors have nothing to disclose nor competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

SMC1A epilepsy syndrome or developmental and epileptic encephalopathy-85 with or without midline brain defects (DEE85, OMIM #301044) is an X-linked neurologic disorder associated with mutations of the SMC1A gene, which is also responsible for about 5% of patients affected by Cornelia de Lange syndrome spectrum (CdLS). Only described in female patients, SMC1A epilepsy syndrome is characterized by the onset of severe refractory epileptic seizures in the first year of life, global developmental delay, a variable degree of intellectual disability, and dysmorphic facial features not typical of CdLS. This was a descriptive observational study for the largest international cohort with this specific disorder. The main goal of this study was to improve the knowledge of the natural history of this phenotype with particular attention to the psychomotor development and the epilepsy data. The analyzed cohort shows normal prenatal growth with the subsequent development of postnatal microcephaly. The incidence of neonatal problems (seizures and respiratory compromise) is considerable (51.4%). There is a significant prevalence of central nervous system (20%) and cardiovascular malformations (20%). Motor skills are generally delayed. The presence of drug-resistant epilepsy is confirmed; the therapeutic role of a ketogenic diet is still uncertain. The significant regression of previously acquired skills following the onset of seizures has been observed. Facial dysmorphisms are variable and no patient shows a classic CdLS phenotype. To sum up, SMC1A variants caused drug-resistant epilepsy in these patients, more than two-thirds of whom were shown to progress to developmental and epileptic encephalopathy. The SMC1A gene variants are all different from each other (apart from a couple of monozygotic twins), demonstrating the absence of a mutational hotspot in the SMC1A gene. Owing to the absence of phenotypic specificity, whole-exome sequencing is currently the diagnostic gold standard., (© 2024 Wiley Periodicals LLC.)

Background: Chromatinopathies are a heterogeneous group of genetic disorders caused by pathogenic variants in genes coding for chromatin state balance proteins. Remarkably, many of these syndromes present unbalanced postnatal growth, both under- and over-, although little has been described in the literature. Fetal growth measurements are common practice in pregnancy management and values within normal ranges indicate proper intrauterine growth progression; on the contrary, abnormalities in intrauterine fetal growth open the discussion of possible pathogenesis affecting growth even in the postnatal period., Methods: Among the numerous chromatinopathies, we have selected six of the most documented in the literature offering evidence about two fetal overgrowth (Sotos and Weaver syndrome) and four fetal undergrowth syndromes (Bohring Opitz, Cornelia de Lange, Floating-Harbor, and Meier Gorlin syndrome), describing their molecular characteristics, maternal biochemical results and early pregnancy findings, prenatal ultrasound findings, and postnatal characteristics., Results/conclusion: To date, the scarce data in the literature on prenatal findings are few and inconclusive, even though these parameters may contribute to a more rapid and accurate diagnosis, calling for a better and more detailed description of pregnancy findings., (© 2024 Wiley Periodicals LLC.)

Histone deacetylases (HDACs) are enzymes pivotal for histone modification (i.e. acetylation marks removal), chromatin accessibility and gene expression regulation. Class I HDACs (including HDAC1, 2, 3, 8) are ubiquitously expressed and they often participate in multi-molecular protein complexes. To date, three neurodevelopmental disorders caused by mutations in genes encoding for HDACs (HDAC4, HDAC6 and HDAC8) and thus belonging to the group of chromatinopathies, have been described. We performed whole exome sequencing (WES) for a patient (#249) clinically diagnosed with the chromatinopathy Rubinstein-Taybi syndrome (RSTS) but negative for mutations in RSTS genes, identifying a de novo frameshift variant in HDAC2 gene. We then investigated its molecular effects in lymphoblastoid cell lines (LCLs) derived from the patient compared to LCLs from healthy donors (HD). As the variant was predicted to be likely pathogenetic and to affect the sequence of nuclear localization signal, we performed immunocytochemistry and lysates fractionation, observing a nuclear mis-localization of HDAC2 compared to HD LCLs. In addition, HDAC2 total protein abundance resulted altered in patient, and we found that newly identified variant in HDAC2 affects also acetylation levels, with significant difference in acetylation pattern among patient #249, HD and RSTS cells and in expression of a known molecular target. Remarkably, RNA-seq performed on #249, HD and RSTS cells shows differentially expressed genes (DEGs) common to #249 and RSTS. Interestingly, our reported patient was clinically diagnosed with RSTS, a chromatinopathy which known causative genes encode for enzymes antagonizing HDACs. These results support the role of HDAC2 as causative gene for chromatinopathies, strengthening the genotype-phenotype correlations in this relevant group of disorders., (© 2024. The Author(s).)

Background: Limited evidence exists regarding the association between COVID-19 and Long COVID manifestations in children, particularly concerning variants of concern (VOCs). We aimed to characterize a cohort of pediatric patients hospitalized with confirmed acute SARS-CoV-2 and monitor them for Long COVID symptoms. Additionally, it seeks to explore any potential correlations between VOCs and clinical symptoms., Methods: We conducted a prospective study involving children hospitalized from November 2021 to March 2023, with confirmed acute SARS-CoV-2 infection. A telephone survey was conducted at 3-6-12 months after discharge., Results: We included 167 patients (77 F/90 M). Upon hospital admission, 95.5% of patients presented as symptomatic. Regarding patients for whom it was feasible to determine the SARS-CoV-2 variant (n = 51), the Delta variant was identified in 11 children (21.6%) and Omicron variant in the remaining 40 patients (78.4%: 27.5% BA.1 variant; 15% BA.2 variant; 57.5% BA.5 variant). 19 patients (16.5%) reported experiencing at least one symptom indicative of Long COVID (weight loss 31.6%, inappetence 26.3%, chronic cough 21.1%, fatigue 21.1%, and sleep disturbances, wheezing, abdominal pain and mood disorders 15.8%). In only 4 patients with Long COVID we could identified a specific SARS-CoV-2 variant (3 Omicron: 2 BA.1 and 1 BA.2; 1 Delta)., Conclusions: this study underscores that long COVID is a significant concern in the pediatric population. Our data reinforce the importance of continuously monitoring the impact of long-COVID in infants, children, and adolescents. A follow-up following SARS-CoV-2 infection is therefore advisable, with symptom investigation tailored to the patient's age., (© 2024. The Author(s).)

Aim: We retrospectively evaluated the effect of dabrafenib/trametinib combination in patients with BRAF-mutated non-small-cell lung cancer (NSCLC) treated in a single center from 2017 to 2022. Patients: The response and safety data of 42 patients (27 treated in first-line and 15 as second/subsequent lines) were analyzed. Results: The objective response was 73.8%, with no differences between patients undergoing first- or second-line. A longer, statistically significant median progression-free survival (PFS) was observed in patients receiving the combination in first-line vs those in the second/subsequent lines (19.9 months [95% CI: 19.7-20] vs 13.1 months [95% CI: 8.6-17.6], respectively; p = 0.012). The median overall survival (OS) was 29.9 months (95% CI: 14.1-45.7) for patients treated with the combination in first-line and 22.4 months (95% CI: 14.6-30.2) for those treated in subsequent lines. The combination was well tolerated Conclusion: We confirm the efficacy of dabrafenib/trametinib in BRAF-V600-mutated NSCLC.

Introduction: Mismatch repair (MMR) deficiency represents a biomarker and therapeutic target in various neoplasms, but its role in biliary tract cancers (BTCs) remains misunderstood., Methods: MMR status was retrospectively assessed using immunohistochemistry in 163-BTCs patients. We identified MMR proficiency (pMMR)/deficiency (dMMR) according to the loss of MMR proteins (MLH1, PMS2, MSH2, MSH6). The primary objective of the study was to assess the incidence of dMMR in BTCs; the secondary purpose was to explore its association with prognosis and clinical features., Results: dMMR was recorded in 9 patients, and it was strongly associated with mucinous histology (p < 0.01). Regarding the prognostic effect, in 122-radically resected patients, disease-free survival (DFS) resulted significantly shorter in dMMR patients compared to pMMR patients (10.7 vs. 31.3 months, p = 0.025) and so did nodal status (48.2 vs. 15.3 months in N0 vs. N+) (p < 0.01). Moreover, dMMR confirmed its prognostic role in terms of DFS at multivariate analysis (p = 0.03), together with nodal status (p = 0.01), and resection margin (p = 0.03). In 103 M+ patients (encompassing 41 metastatic de novo and 62 recurred after surgery patients) there were not differences between dMMR and pMMR regarding survival analyses., Conclusions: dMMR status is strongly correlated with mucinous histology and represents an independent prognostic factor in terms of disease relapse in patients with resected BTC., Implications for Practice: MMR may play an independent role in promoting an aggressive behaviour in patients with radically resected BTC. These results could be useful in improving the selection of patients after resection and, above all, should justify the evaluation of MMR status as a therapeutic target in BTC, especially in patients with atypical histology., (© 2023 S. Karger AG, Basel.)

Toll-like receptor-driven and interleukin-1 (IL-1) receptor-driven inflammation mediated by IL-1 receptor-associated kinase 4 (IRAK4) is involved in the pathophysiology of hidradenitis suppurativa (HS) and atopic dermatitis (AD). KT-474 (SAR444656), an IRAK4 degrader, was studied in a randomized, double-blind, placebo-controlled phase 1 trial where the primary objective was safety and tolerability. Secondary objectives included pharmacokinetics, pharmacodynamics and clinical activity in patients with moderate to severe HS and in patients with moderate to severe AD. KT-474 was administered as a single dose and then daily for 14 d in 105 healthy volunteers (HVs), followed by dosing for 28 d in an open-label cohort of 21 patients. Degradation of IRAK4 was observed in HV blood, with mean reductions after a single dose of ≥93% at 600-1,600 mg and after 14 daily doses of ≥95% at 50-200 mg. In patients, similar IRAK4 degradation was achieved in blood, and IRAK4 was normalized in skin lesions where it was overexpressed relative to HVs. Reduction of disease-relevant inflammatory biomarkers was demonstrated in the blood and skin of patients with HS and patients with AD and was associated with improvement in skin lesions and symptoms. There were no drug-related infections. These results, from what, to our knowledge, is the first published clinical trial using a heterobifunctional degrader, provide initial proof of concept for KT-474 in HS and AD to be further confirmed in larger trials. ClinicalTrials.gov identifier: NCT04772885 ., (© 2023. The Author(s).)

Gonadotropin-releasing hormone (GnRH) deficiency (GD) is a disorder characterized by absent or delayed puberty, with largely unknown genetic causes. The purpose of this study was to obtain and exploit gene expression profiles of GnRH neurons during development to unveil novel biological mechanisms and genetic determinants underlying GD. Here, we combined bioinformatic analyses of immortalized and primary embryonic GnRH neuron transcriptomes with exome sequencing from GD patients to identify candidate genes implicated in the pathogenesis of GD. Among differentially expressed and filtered transcripts, we found loss-of-function (LoF) variants of the autism-linked neuroligin 3 (NLGN3) gene in two unrelated patients co-presenting with GD and neurodevelopmental traits. We demonstrated that NLGN3 is upregulated in maturing GnRH neurons and that NLGN3 wild-type, but not mutant, protein promotes neuritogenesis when overexpressed in developing GnRH cells. Our data represent proof of principle that this complementary approach can identify new candidate GD genes and demonstrate that LoF NLGN3 variants can contribute to GD. This novel genotype-phenotype correlation implies common genetic mechanisms underlying neurodevelopmental disorders, such as GD and autistic spectrum disorder., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Diaphanous related formins are regulatory cytoskeletal protein involved in actin elongation and microtubule stabilization. In humans, defects in two of the three diaphanous genes (DIAPH1 and DIAPH3) have been associated with different types of hearing loss. Here, we investigate the role of the third member of the family, DIAPH2, in nonsyndromic hearing loss, prompted by the identification, by exome sequencing, of a predicted pathogenic missense variant in DIAPH2. This variant occurs at a conserved site and segregated with nonsyndromic X-linked hearing loss in an Italian family. Our immunohistochemical studies indicated that the mouse ortholog protein Diaph2 is expressed during development in the cochlea, specifically in the actin-rich stereocilia of the sensory outer hair cells. In-vitro studies showed a functional impairment of the mutant DIAPH2 protein upon RhoA-dependent activation. Finally, Diaph2 knock-out and knock-in mice were generated by CRISPR/Cas9 technology and auditory brainstem response measurements performed at 4, 8 and 14 weeks. However, no hearing impairment was detected. Our findings indicate that DIAPH2 may play a role in the inner ear; further studies are however needed to clarify the contribution of DIAPH2 to deafness., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chiereghin et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Introduction: In locally advanced gastric cancer (GC), FLOT represents the standard perioperative regimen and combination with immunotherapy is under investigation. However, the role of immune tumor microenvironment (TME) is poorly recognized in this setting. We aimed to study TME characteristics and dynamics during FLOT., Methods: Paired biopsy (PRE) and surgical (POST) samples of 25 patients treated with FLOT were prospectively analyzed. After collection of clinic-pathological data, NanoString analyses were performed. The primary objective of the study was to assess the changes induced by chemotherapy in POST compared to PRE samples., Results: The unsupervised hierarchical method analysis clearly distinguished PRE and POST samples, even though some cases showed high immune gene expression at baseline. When POST samples were compared with PRE, a differential expression in hyper-expressed gene sets related to cytotoxicity, T-cell functions, complement system, tumor necrosis factor superfamily, cell cycle, and regulation was recognized. Downstaging of the primary tumor (T-regression, measured by pathologic compared to clinical T stage) was the covariate most frequently associated with these changes. Using the immune cell profiling, cases with T-regression reported a significant increase of T, CD8+ T and B cells and a decrease in mast cells, while nonresponders demonstrated an increase of T, B, cytotoxic, and mast cells., Conclusion: Our analysis shows that FLOT significantly influences immune TME of GC. While relevant modifications preferentially occur in tumors showing primary tumor regression, response to treatment seems to be associated with a specific immune profile., (© 2023 S. Karger AG, Basel.)

The aim of the present study was to investigate the effect of the substitution, in dairy cow rations, of traditional protein and starch sources with more sustainable "circular" feeds to increase the sustainability of dairy production. For this purpose, eight multiparous mid-lactating cows were blocked and assigned to one of four treatments and were used in a replicated 4 × 4 Latin squares design with 21-days periods (14 days of adaptation and 7 of data collection). Two different circular feedstuffs were tested: a bakery's former foodstuff (FF) and a wheat distiller's grain with solubles (WDGS). These ingredients were used, alone and in combination, in three experimental diets (FF, WDGS; FF + WDGS) and compared to a standard ration (CTR). Dry matter intake and rumination time were not influenced by these diets. Conversely, dietary treatments partially influenced the milk yield, rumen pH, Volatile Fatty Acids (VFA) production, and fibre digestibility. In particular, the combined inclusion of FF and WDGS increased milk production (37.39 vs. 36.92, 35.48, 35.71 kg/day, for FF, WDGS and CTR diets, respectively) and reduced milk urea content (13.14 vs. 16.19, 15.58, 16.95 mg/dL for FF, WDGS, and CTR diets, respectively). No effects of this association were found in the milk composition, acetic and propionic production, and fibre digestibility. These results suggest that the association of former foodstuff and wheat distillers' grains could be safely included in dairy cow rations to increase the sustainability of cow nutrition and improve milk production without impairing animal health, dry matter intake, and fibre digestibility.

Objective: In recent decades, the trend for women is to delay childbearing. However, worldwide, advanced maternal age is an independent risk factor for stillbirth, as well as advanced gestational age. National data are not available about stillbirths in the Italian population. We explored whether, at term of pregnancy, advanced maternal age is associated with an increased risk of stillbirth in Italy. We speculate that a policy of induction of labor at term of pregnancy in older mothers may significantly reduce the stillbirth., Methods: Data provided by Italian Ministry of Health and National Statistical Institute were used to identify all singleton deliveries ≥22 weeks of gestation during a four years study period. We evaluated the outcome of pregnancy (livebirths or stillbirths) and we stratified data by gestational age and by maternal age at delivery. The hazard risk and the relative risk of stillbirth were calculated., Results: The overall stillbirth rate was 3.4 per 1000, with a total of 6451 cases of stillbirths in the four years study period. Overall, the risk of stillbirth increases at term of pregnancy in all maternal age groups, especially in older mothers. A total of 674 stillbirths occurred in women aged 40 years or older and 24.2% of them ( n  = 163) occurred at term of pregnancy. Among women aged 40 years and above, 7.3% of stillbirths (49/674) occurred beyond 39 weeks of gestation. The hazard risk doubles from 39 to 40 weeks, from 0.60 per 1000 ongoing pregnancies to 1.16 per 1000 ongoing pregnancies; the relative risk at 40 weeks of gestation was the highest in the older mothers and was 5.17 (95% CI 3.16-8.46)., Conclusions: The effect of maternal age on birth outcomes is a relevant aspect in Italy. If the association between maternal age and stillbirth is supposed to be part of the pathophysiology of fetal death, our data indicate that induction of labor before 40 weeks of gestation in women aged 40 years old or older might prevent overall 7.3% of stillbirths for induction at 39 weeks, 13% of stillbirths for induction at 38 weeks. To reduce potentially preventable stillbirths, caregivers should perform a specific risk assessment for each pregnant woman. The impact of maternal age should be seriously considered, and an individualized approach should be planned at term of pregnancy in older mothers, including the possibility of a slightly anticipation of induction of labor if spontaneously undelivered.

Using the timely re-activation of WNT signalling in neuralizing human induced pluripotent stem cells (hiPSCs), we have produced neural progenitor cells with a gene expression profile typical of human embryonic dentate gyrus (DG) cells. Notably, in addition to continuous WNT signalling, a specific laminin isoform is crucial to prolonging the neural stem state and to extending progenitor cell proliferation for over 200 days in vitro. Laminin 511 is indeed specifically required to support proliferation and to inhibit differentiation of hippocampal progenitor cells for extended time periods when compared with a number of different laminin isoforms assayed. Global gene expression profiles of these cells suggest that a niche of laminin 511 and WNT signalling is sufficient to maintain their capability to undergo typical hippocampal neurogenesis. Moreover, laminin 511 signalling sustains the expression of a set of genes responsible for the maintenance of a hippocampal neurogenic niche. Finally, xenograft of human DG progenitors into the DG of adult immunosuppressed host mice produces efficient integration of neurons that innervate CA3 layer cells spanning the same area of endogenous hippocampal neuron synapses., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Immune checkpoint inhibitors (ICIs) are a key component of different stages of hepatocellular carcinoma (HCC) treatment, particularly in the first line of treatment. A lesson on the primary resistance which hampers their efficacy and activity was learned from the failure of the trials which tested them as first-line mono-therapies. Despite the combination of anti-PD(L)1 agents with anti-VEGF, anti CTLA4, or TKIs demonstrating relevant improvements in efficacy, the "doublets strategy" still shows room for improvement, due to a limited overall survival benefit and a high rate of progressive disease as best response. In this review, we discuss the results from the currently tested doublet strategies (i.e., atezolizumab+bevacizumab, durvalumab+tremelimumab with a mention to the newly presented ICIs/TKIs combinations), which highlight the need for therapeutic improvement. Furthermore, we examine the rationale and provide an overview of the ongoing trials testing the treatment intensification strategy with triplet drugs: anti-PD1+anti-CTLA4+anti-VEGF/TKIs and anti-PD1+anti-VEGF+alternative immunity targets. Lastly, we report on the alternative strategy to integrate ICIs into the new paradigm of immune therapeutics constituted by CAR-T and anti-cancer vaccines. This review provides up-to-date knowledge of ongoing clinical trials of the aforementioned strategies and critical insight into their mechanistic premises.

Purpose: The worldwide pandemic caused by SARS-CoV-2 virus posed many challenges to the scientific and medical communities, including the protection and management of fragile populations. People with epilepsy (PWE) are a heterogenous group of subjects, with different treatment regimens and severity of symptoms. During the National lockdown, in Italy many patients with chronic conditions lost their regular follow-up program. The aim of this study was to investigate the impact of COVID-19 on their health status, from the start of the pandemic (March 2020) to July 2021 and one year later., Methods: We proposed an online questionnaire to subjects followed up at different epilepsy centers located in Milano, Monza & Lodi, three of Lombardy, Northern Italy, the most affected areas by the pandemic. Survey evaluated age, sex, characteristics of patients, type of epilepsy and therapies, COVID-19 diagnosis, vaccines, sleep quality, and anxiety status., Results: Among 178 analyzed surveys, 37 individuals reported symptoms of COVID-19 in closed contacts, including 9 with molecular diagnosis and 16 PWE performing the nasopharyngeal swab with 3 positive cases. One year later, 35 individuals reported at least one symptom overlapping with those typical of COVID-19, 8 received COVID-19 diagnosis, among which 6 were positive for SARS-CoV-2 infection. According to the sleep quality scale assessment, most PWE (52.3%) had poor sleep quality. Assessing anxiety status, 32 (38.1%) had a pathological score., Conclusion: In this multicenter study, we observed that PWE do not appear to be at a higher risk of severe COVID-19. It will be fundamental monitoring this group to assess possible differences in long-COVID-19 and/or neuro-COVID-19 prevalence. On the other hand, our survey confirmed the impact of the pandemic on anxiety and quality of sleep in PWE. Thus, it is important to promptly recognize and treat psychological distress in PWE, because it could be a risk factor in seizure aggravation and quality-of-life deterioration. Telemedicine appears to be a useful tool to support patients with chronic diseases, such as epilepsy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Chromatinopathies are defined as genetic disorders caused by mutations in genes coding for protein involved in the chromatin state balance. So far 82 human conditions have been described belonging to this group of congenital disorders, sharing some molecular features and clinical signs. For almost all of these conditions, no specific treatment is available. For better understanding the molecular cascade caused by chromatin imbalance and for envisaging possible therapeutic strategies it is fundamental to combine clinical and basic research studies. To this end, animal modelling systems represent an invaluable tool to study chromatinopathies. In this review, we focused on available data in the literature of animal models mimicking the human genetic conditions. Importantly, affected organs and abnormalities are shared in the different animal models and most of these abnormalities are reported as clinical manifestation, underlying the parallelism between clinics and translational research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Di Fede, Grazioli, Lettieri, Parodi, Castiglioni, Taci, Colombo, Ancona, Priori, Gervasini and Massa.)

Background and Aim: The need to estimate prognosis of advanced BTC (aBTC) patients treated with first-line chemotherapy is compelling. The aim of the study is to evaluate the ECSIPOT (psECogSIiPnigOT) index, influenced by PECS (PsECogSii) index, prognostic nutritional index (PNI), and GOT., Methods: This international study was conducted on a training cohort of 126 patients and in three validation cohorts, both European and Korean. ECSIPOT index formula: (PECS:0 = 1 point; PECS:1 = 1.4 points; PECS:2 = 3.2 points) + (PNI > 36.7 = 1 point; PNI < 36.7 = 2 points) + (GOT < 100 = 1 point; GOT > 100 = 2 points). Event-time distributions were estimated using the Kaplan-Meier method, and survival curves were compared using the log-rank test., Results: In the training cohort, mOS was 12.9, 6.3, and 2.8 months for patients with ECSIPOT-0, ECSIPOT-1, and ECSIPOT-2, respectively (ECSIPOT-0: HR 1; ECSIPOT-1: HR 2.11; ECSIPOT-2: HR 4.93; p < 0.0001). In the first validation cohort, mOS was 11.5, 7.3, and 3.3 months for ECSIPOT-0, ECSIPOT-1, and ECSIPOT-2, respectively (ECSIPOT-0: HR 1; ECSIPOT-1: HR 1.74; ECSIPOT-2: HR 3.41; p < 0.0001). In the second validation cohort, mOS was 25.2, 12.5, and 3.0 months for ECSIPOT-0, ECSIPOT-1, and ECSIPOT-2, respectively (ECSIPOT-0: HR = 1; ECSIPOT-1: HR 2.33; ECSIPOT-2: HR 8.46; p < 0.0001). In the third validation cohort, mOS was 11.8, 8.1, and 4.6 months for ECSIPOT-0, ECSIPOT-1, and ECSIPOT-2, respectively (ECSIPOT-0: HR = 1; ECSIPOT-1: HR 1.47; ECSIPOT-2: HR 3.17; p < 0.0001). Multivariate analysis in all cohorts confirmed the ECSIPOT index as an independent prognostic factor for OS., Conclusion: The easy assessment and good risk-stratification performance make the ECSIPOT index a promising tool to comprehensively estimate the prognosis of aBTC patients., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Saliva molecular tests have shown a similar sensitivity and specificity compared to nasopharyngeal test for SARS-CoV-2 diagnosis in both symptomatic and asymptomatic individuals. The SARS-CoV-2 pandemic affected Lombardy prisons, generating the need for extensive contact tracing activities and for detecting asymptomatic carriers. The availability of a less invasive test in a setting that hosts a high-risk and often hard-to-reach population, suggests its possible use in prisons., Methods: The study was carried out on a population of new incomers in Milan San Vittore pre-trial prison. All the new incomers were submitted to quarantine and to saliva test and nasopharyngeal swab (NPS) for SARS-CoV-2 detection at the entry and at the end of quarantine before their admission in community (Protocol 1-February 2 nd to March 5 th , 2021). Starting from March 8 th to July 30 th , 2021, the screening protocol was adjusted to avoid biases in sample collection (Protocol II), and saliva testing was performed at entrance., Results: 12/1,120 enrolled subjects were excluded from the study. Among the 1,080 processed samples, 1 tested positive, 5 weakly positive, 1,069 negative, 3 were invalid, and 2 samples tested positive for the viral gene N2 only, with Ct value above 38. During Protocol I, 6/156 coupled saliva/NPS tests were discordant due to food ingestion prior saliva collection, prompting us to establishing Protocol II., Conclusions: Saliva molecular testing is feasible in prison setting, being less invasive and easier to use, and reliable. Acceptability was very high even in a complex context as that of newly incarcerated persons., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer VC declared a past co-authorship/collaboration with the authors RG and RR to the handling Editor., (Copyright © 2022 Parodi, Ottaviano, Cocco, Ancona, Bianchi, Massa, Bartolotti, Pezzoni, Giuliani, Borghi and Ranieri.)

Ramucirumab plus paclitaxel is considered the standard of care in the second-line treatment of gastric carcinoma (GC). The aim of this study was to evaluate plasma vascular endothelial growth factor-A (VEGF-A), VEGF-D, and circulating soluble VEGF receptor-2 (sVEGFR-2) as possible markers of resistance or response to ramucirumab administered with paclitaxel in pretreated metastatic GC patients. Plasma samples were collected at different time points (on days 1 and 15 of the first 3 cycles, at best radiologic response and at disease progression). VEGF-A, VEGF-D and sVEGFR-2 were analysed by ELISA. Correlations of biomarker baseline levels or dynamic changes with outcome measures were assessed. Progression-free survival (PFS) was the primary endpoint of the study. Forty-one patients were enrolled. VEGF-A and VEGF-D, but not sVEGFR-2, values significantly increased during treatment compared to baseline (P < 0.001). A positive correlation between VEGF-A and sVEGFR-2 at cycle 2 was found (P=0.045). At univariate analysis, higher baseline levels of VEGF-A were associated with worse OS (P=0.015). Early increase of sVEGFR-2 levels after the first treatment cycle was the only factor associated with longer PFS (6.6 vs. 3.6 months, P=0.049) and OS (18.6 vs. 5.2 months, P=0.008). Significance of sVEGFR-2 early increase was retained at multivariate analysis for OS (HR 0.32; 95% CI 0.12-0.91; P=0.032). The reported results confirmed the prognostic role of baseline VEGF-A and, with the limitations of the limited sample size and the lack of a control arm, suggested that the early increase of sVEGFR-2 after 1 cycle of treatment could be a potential predictive biomarker of benefit from second-line ramucirumab plus paclitaxel in GC., Competing Interests: None., (AJCR Copyright © 2022.)

The Rubinstein-Taybi syndrome (RSTS) is a rare developmental disorder characterized by craniofacial dysmorphisms, broad thumbs and toes, intellectual disability, growth deficiency, and recurrent infections. Mutations in the cyclic adenosine monophosphate response element-binding protein (CREB)-binding protein (CREBBP) or in the E1A-associated protein p300 (EP300) genes have been demonstrated in 55% (RSTS1) and up to 8% of the patients (RSTS2), respectively. Dysfunction of immune response has been reported in a subgroup of individuals with RSTS. Here we characterize two patients carrying the same EP300 variant and distinctive RSTS features (including congenital heart abnormalities, short stature, feeding problems, and gastroesophageal reflux). Whole exome sequencing did not support a dual molecular diagnosis hypothesis. Nonetheless, patients showed distinct clinical manifestations and immunological features. The most severe phenotype was associated with reduced T-cell production and diversity. This latter feature was confirmed in a control group of four RSTS patients., (© 2022 Wiley Periodicals LLC.)

Senescence is a stress-response process characterized by the irreversible inhibition of cell proliferation, associated to the acquisition of a senescence-associated secretory phenotype (SASP), that may drive pathological conditions. Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells, featuring the hyperactivation of the mammalian Target of Rapamycin Complex 1 (mTORC1) for the absence of tuberin expression, cause the disruption of the lung parenchyma. Considering that LAM cells secrete SASP factors and that mTOR is also a driver of senescence, we deepened the contribution of senescence in LAM cell phenotype. We firstly demonstrated that human primary tuberin-deficient LAM cells (LAM/TSC cells) have senescent features depending on mTOR hyperactivation, since their high positivity to SA-β galactosidase and to phospho-histone H2A.X are reduced by inducing tuberin expression and by inhibiting mTOR with rapamycin. Then, we demonstrated the capability of LAM/TSC cells to induce senescence. Indeed, primary lung fibroblasts (PLFs) grown in LAM/TSC conditioned medium increased the positivity to SA-β galactosidase and to phospho-histone H2A.X, as well as p21 WAF1/CIP1 expression, and enhanced the mRNA expression and the secretion of the SASP component IL-8. Taken together, these data make senescence a novel field of study to understand LAM development and progression.

Hearing relies on the proper functioning of auditory hair cells and on actin-based cytoskeletal structures. Diaphanous-related formins (DRFs) are evolutionarily conserved cytoskeletal proteins that regulate the nucleation of linear unbranched actin filaments. They play key roles during metazoan development, and they seem particularly pivotal for the correct physiology of the reproductive and auditory systems. Indeed, in Drosophila melanogaster , a single diaphanous (dia) gene is present, and mutants show sterility and impaired response to sound. Vertebrates, instead, have three orthologs of the diaphanous gene: DIAPH1 , DIAPH2 , and DIAPH3 . In humans, defects in DIAPH1 and DIAPH3 have been associated with different types of hearing loss. In particular, heterozygous mutations in DIAPH1 are responsible for autosomal dominant deafness with or without thrombocytopenia ( DFNA1 , MIM #124900), whereas regulatory mutations inducing the overexpression of DIAPH3 cause autosomal dominant auditory neuropathy 1 ( AUNA1 , MIM #609129). Here, we provide an overview of the expression and function of DRFs in normal hearing and deafness.

Background: Predictive biomarkers for advanced hepatocellular carcinoma are lacking. EZH2 drives sorafenib resistance through H3K27me3 and is counteracted by SETD2, which catalyzes H3K36me3. The authors tested the predictive power of circulating H3K27me3 and H3K36me3 in advanced hepatocellular carcinoma patients treated with sorafenib. Methods: A total of 80 plasma samples were tested for histone variants by ELISA. Changes from baseline to best response or progressive disease were correlated with patient survival. Results: A higher EZH2 / SETD2 ratio predicted worse prognosis in this setting. H3K27me3 and H3K36me3 decreased from baseline to best response. The H3K27me3/H3K36me3 ratio increased from baseline to progressive disease. Higher ratios at best response were associated with shorter progression-free survival. Conclusion: The authors suggest that circulating H3K27me3/H3K36me3 ratio level acts as a predictive biomarker for sorafenib treatment outcomes in patients with advanced hepatocellular carcinoma.

Individuals with Chronic Kidney Disease (CKD) are at high risk for cardiovascular morbidity and mortality. The aim of this study was to examine the relationship between renal dysfunction and all-cause mortality in a sample of subjects undergoing coronary angiography (CA). We evaluated 1017 subjects who consecutively underwent CA. Glomerular filtration rate (eGFR) was estimated by CKD-EPI and urinary albumin excretion reported as urinary albumin-to-creatinine ratio. Vital status was ascertained by interrogating the Italian Health Card Database. One-thousand-seventeen subjects (759 M/258F) were enrolled into the study from 2016 to 2018. One-hundred-fourteen deaths occurred during a median follow-up of 44 months. The whole population was divided in two subgroups according to the presence/absence of low eGFR (i.e. < 60 ml/min/1.73 m 2 ). Subjects with low baseline eGFR had a worse clinical profile than subjects with preserved kidney function. The risk of death in subjects with eGFR < 60 ml/min/1.73 m 2 was almost three times higher than in subjects with preserved kidney function: fully adjusted HR 2.70 (95% CI 1.56-4.67). The presence of albuminuria also predicted a high risk of death: fully adjusted HR 2.09 (95% CI 1.17-3.73) and HR 4.26 (95% CI 2.18-8.33), microalbuminuria or macroalbuminuria, respectively, being normoalbuminuria the reference group. Again, the increased risk remained significant after adjusting for several potential confounders. In conclusion, kidney disease measures (i.e. low eGFR or albuminuria) independently predict increased risk for all-cause death in a large sample of subjects undergoing CA. These results have a relevant clinical impact., (© 2021. Società Italiana di Medicina Interna (SIMI).)

KMT2A (Lysine methyltransferase 2A) is a member of the epigenetic machinery, encoding a lysine methyltransferase responsible for the transcriptional activation through lysine 4 of histone 3 (H3K4) methylation. KMT2A has a crucial role in gene expression, thus it is associated to pathological conditions when found mutated. KMT2A germinal mutations are associated to Wiedemann-Steiner syndrome and also in patients with initial clinical diagnosis of several other chromatinopathies (i.e., Coffin-Siris syndromes, Kabuki syndrome, Cornelia De Lange syndrome, Rubinstein-Taybi syndrome), sharing an overlapping phenotype. On the other hand, KMT2A somatic mutations have been reported in several tumors, mainly blood malignancies. Due to its evolutionary conservation, the role of KMT2A in embryonic development, hematopoiesis and neurodevelopment has been explored in different animal models, and in recent decades, epigenetic treatments for disorders linked to KMT2A dysfunction have been extensively investigated. To note, pharmaceutical compounds acting on tumors characterized by KMT2A mutations have been formulated, and even nutritional interventions for chromatinopathies have become the object of study due to the role of microbiota in epigenetic regulation.

Background: The clinical course of COVID-19 is more severe in elderly patients with cardio-metabolic co-morbidities. Chronic kidney disease is considered an independent cardiovascular risk factor. We aimed to evaluate the impact of reduced eGFR on the composite outcome of admission to ICU and death in a sample of consecutive COVID-19 hospitalized patients., Methods: We retrospectively evaluated clinical records of a consecutive sample of hospitalized COVID-19 patients. A total of 231 patients were considered for statistical analysis. The whole sample was divided in two groups on the basis of eGFR value, e.g., ≥ or <60 mL/min/1.73 m 2 . Patients with low eGFR were further divided among those with a history of chronic kidney disease (CKD) and those without (AKI, acute kidney injury). The primary outcome was a composite of admission to ICU or death, whichever occurred first. The single components were secondary outcomes., Results: Seventy-nine (34.2%) patients reached the composite outcome. A total of 64 patients (27.7%) died during hospitalization, and 41 (17.7%) were admitted to the ICU. A significantly higher number of events was present among patients with low eGFR ( p < 0.0001). Age ( p < 0.001), SpO2 ( p < 0.001), previous anti-platelet treatment ( p = 0.006), Charlson's Comorbidities Index ( p < 0.001), serum creatinine ( p < 0.001), eGFR ( p = 0.003), low eGFR ( p < 0.001), blood glucose levels ( p < 0.001), and LDH ( p = 0.003) were significantly associated with the main outcome in univariate analysis. Low eGFR (HR 1.64, 95% CI 1.02-2.63, p = 0.040) and age (HR per 5 years 1.22, 95% CI 1.10-1.36, p < 0.001) were significantly and independently associated with the main outcome in the multivariate model. Patients with AKI showed an increased hazard ratio to reach the combined outcome ( p = 0.059), while those patients with both CKD had a significantly higher probability of developing the combined outcome ( p < 0.001)., Conclusions: Patients with reduced eGFR at admission should be considered at high risk for clinical deterioration and death, requiring the best supportive treatment in order to prevent the worst outcome.

Ischemic damage in brain tissue triggers a cascade of molecular and structural plastic changes, thus influencing a wide range of cell-to-cell interactions. Understanding and manipulating this scenario of intercellular connections is the Holy Grail for post-stroke neurorehabilitation. Here, we discuss the main findings in the literature related to post-stroke alterations in cell-to-cell interactions, which may be either detrimental or supportive for functional recovery. We consider both neural and non-neural cells, starting from astrocytes and reactive astrogliosis and moving to the roles of the oligodendrocytes in the support of vulnerable neurons and sprouting inhibition. We discuss the controversial role of microglia in neural inflammation after injury and we conclude with the description of post-stroke alterations in pyramidal and GABAergic cells interactions. For all of these sections, we review not only the spontaneous evolution in cellular interactions after ischemic injury, but also the experimental strategies which have targeted these interactions and that are inspiring novel therapeutic strategies for clinical application.

Objective: Stillbirth is one of the most devastating adverse pregnancy outcome, but it is often associated with a missing post-mortem histological examination. We aimed at evaluating whether the staining of amniotic fluid reflects the fetal conditions surrounding the death and if it correlates with any histologic sign of fetal distress., Study Design: Terminal gasping (represented by the massive presence of intra-alveolar squamous cells), thymic and adrenal cortex modifications were evaluated as histologic signs of fetal distress in stillbirths, and stratified according to the degree of staining of the amniotic fluid., Results: The presence of meconium-stained amniotic fluid did not correlate with the presence of gasping and/or thymic and/or adrenal cortex changes. Clear amniotic fluid was not associated with the absence of histologic signs of distress., Conclusions: The evaluation of the staining of the amniotic fluid fails to identify distressed fetuses. A histologic evaluation of fetal organs provides detailed information, irrespective of the presence/absence of meconium-stained amniotic fluid., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mouse models provide opportunities to investigate genetic interactions that cause or modify the frequency of neural tube defects (NTDs). Mutation of the PAX3 transcription factor prevents neural tube closure, leading to cranial and spinal NTDs whose frequency is responsive to folate status. Canonical Wnt signalling is implicated both in regulation of Pax3 expression and as a target of PAX3. This study investigated potential interactions of Pax3 mutation and canonical Wnt signalling using conditional gain- and loss-of-function models of β-catenin. We found an additive effect of β-catenin gain of function and Pax3 loss of function on NTDs and neural crest defects. β-catenin gain of function in the Pax3 expression domain led to significantly increased frequency of cranial but not spinal NTDs in embryos that are heterozygous for Pax3 mutation, while both cranial and spinal neural tube closure were exacerbated in Pax3 homozygotes. Similarly, deficits of migrating neural crest cells were exacerbated by β-catenin gain of function, with almost complete ablation of spinal neural crest cells and derivatives in Pax3 homozygous mutants. Pax3 expression was not affected by β-catenin gain of function, while we confirmed that loss of function led to reduced Pax3 transcription. In contrast to gain of function, β-catenin knockout in the Pax3 expression domain lowered the frequency of cranial NTDs in Pax3 null embryos. However, loss of function of β-catenin and Pax3 resulted in spinal NTDs, suggesting differential regulation of cranial and spinal neural tube closure. In summary, β-catenin function modulates the frequency of PAX3-related NTDs in the mouse., (© 2021 The Authors. genesis published by Wiley Periodicals LLC.)

The benefits of schools' closure, used as a containment strategy by many European countries, must be carefully considered against the adverse effects of child wellbeing. In this study, we assessed SARS-CoV-2 seroprevalence, which better estimates the real extent of the infection unraveling asymptomatic cases, among schoolchildren aged 3 to 18 in Milan, using dried blood spot, a safe and extremely viable methods for children, and then compared it between September 2020 and January 2021. Secondly, we evaluated the seroconversion rate and compared it between students attending schools in presence and those switched to distance-learning, using a logistic regression model, both as univariate and multivariate, adjusting for age and biological-sex. Among 1109 pupils, we found a seroprevalence of 2.8% in September before school reopening, while in January 2021, the seropositive rate was 12.5%, reflecting the general growth rate of infections during the second pandemic wave. The overall seroconversion rate was 10%, with no differences based on biological-sex and age groups; we observed no seroreversion. When considered age groups, the seroconversion rate was 10.5% (95%Confidence Interval, 2.9-24.8) among children attending preschools, 10.6% (95%Confidence Interval, 8.2-13.4) for primary schools, 9.9% (95%Confidence Interval, 6.8-13.8) for secondary schools, and 7.8% (95%Confidence Interval, 4-13.2) among high-school students. Interestingly, no differences in seroconversion rate were found between students who attended school compared to those who started remote learning in the first days of November. Furthermore, most patients (61%) reported that the contact occurred within the household. We reported a low seroconversion rate among school children in Milan, with no differences between those who attended from September 2020 to January 2021 compared to those who switched to remote learning in the first days of November. Our data suggest that schools do not amplify SARS-CoV-2 transmission, but rather reflect the level of the transmission in the community., Competing Interests: The authors have declared that no competing interests exist.

Gonadotropin releasing hormone (GnRH) neurons are hypothalamic neuroendocrine cells that control sexual reproduction. During embryonic development, GnRH neurons migrate from the nose to the hypothalamus, where they receive inputs from several afferent neurons, following the axonal scaffold patterned by nasal nerves. Each step of GnRH neuron development depends on the orchestrated action of several molecules exerting specific biological functions. Mutations in genes encoding for these essential molecules may cause Congenital Hypogonadotropic Hypogonadism (CHH), a rare disorder characterized by GnRH deficiency, delayed puberty and infertility. Depending on their action in the GnRH neuronal system, CHH causative genes can be divided into neurodevelopmental and neuroendocrine genes. The CHH genetic complexity, combined with multiple inheritance patterns, results in an extreme phenotypic variability of CHH patients. In this review, we aim at providing a comprehensive and updated description of the genes thus far associated with CHH, by dissecting their biological relevance in the GnRH system and their functional relevance underlying CHH pathogenesis.

Monitoring the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) community-wide transmission with a suitable and effective sampling method would be of great support for public health response to the spreading due to asymptomatic subjects in the community.Here, we describe how using saliva samples for SARS-CoV-2 detection has allowed for a weekly surveillance of a small business company and the early detection of coronavirus disease 2019 cases.As on 23rd March, two cases were detected and investigated, and control measures were rapidly applied.

Pancreatic ductal adenocarcinoma (PDAC) is a non-immunogenic tumor poorly responsive to immune checkpoint inhibitors. This study investigates the effect of 5-fluorouracil (5-FU), irinotecan, and oxaliplatin (FOLFIRINOX), and gemcitabine plus nab-paclitaxel (GEMnPAC) regimens on PD-L1 mRNA expression in plasma-derived microvesicles (MVs) in 50 PDAC patients. Plasma was collected before starting chemotherapy and after 3 months of treatment. mRNA was extracted from MVs, and PD-L1 expression was measured by digital droplet PCR. Twenty-eight patients were PD-L1 positive in MVs at baseline, of which 18 were in the GEMnPAC cohort and 10 in the FOLFIRINOX one. The amount of PD-L1 expression in MVs increased from baseline to 3 months of treatment in patients receiving GEMnPAC (median value 0.002 vs. 0.005; p = 0.01) compared to those treated with FOLFIRINOX (median 0.003 vs. 0.004; p = 0.97). The increase in PD-L1 mRNA expression in MVs was not related to tumor response (PR + SD: p = 0.08; PD: p = 0.28). Our findings demonstrate that GEMnPAC can increase PD-L1 mRNA expression in patient-derived circulating MVs, providing a rationale for testing the efficacy of this regimen in sequential or simultaneous combinations with immunotherapy in PDAC patients.

Background: In the current coronavirus disease-2019 (COVID-19) pandemic, lung ultrasound (LUS) has been extensively employed to evaluate lung involvement and proposed as a useful screening tool for early diagnosis in the emergency department (ED), prehospitalization triage, and treatment monitoring of COVID-19 pneumonia. However, the actual effectiveness of LUS in characterizing lung involvement in COVID-19 is still unclear. Our aim was to evaluate LUS diagnostic performance in assessing or ruling out COVID-19 pneumonia when compared with chest CT (gold standard) in a population of SARS-CoV-2-infected patients. Methods: A total of 260 consecutive RT-PCR confirmed SARS-CoV-2-infected patients were included in the study. All the patients underwent both chest CT scan and concurrent LUS at admission, within the first 6-12 h of hospital stay. Results: Chest CT scan was considered positive when showing a "typical" or "indeterminate" pattern for COVID-19, according to the RSNA classification system. Disease prevalence for COVID-19 pneumonia was 90.77%. LUS demonstrated a sensitivity of 56.78% in detecting lung alteration. The concordance rate for the assessment of abnormalities by both methods increased in the case of peripheral distribution and middle-lower lung location of lesions and in cases of more severe lung involvement. A total of nine patients had a "false-positive" LUS examination. Alternative diagnosis included chronic heart disease (six cases), bronchiectasis (two cases), and subpleural emphysema (one case). LUS specificity was 62.50%. Collateral findings indicative of overlapping conditions at chest CT were recorded also in patients with COVID-19 pneumonia and appeared distributed with increasing frequency passing from the group with mild disease (17 cases) to that with severe disease (40 cases). Conclusions: LUS does not seem to be an adequate tool for screening purposes in the ED, due to the risk of missing some lesions and/or to underestimate the actual extent of the disease. Furthermore, the not specificity of LUS implies the possibility to erroneously classify pre-existing or overlapping conditions as COVID-19 pneumonia. It seems more safe to integrate a positive LUS examination with clinical, epidemiological, laboratory, and radiologic findings to suggest a "virosis." Viral testing confirmation is always required., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Quarato, Mirijello, Maggi, Borelli, Russo, Lacedonia, Foschino Barbaro, Scioscia, Tondo, Rea, Simeone, Feragalli, Massa, Greco, De Cosmo and Sperandeo.)

Cornelia de Lange syndrome (CdLS) is a genetic disease that exemplifies the evolution of knowledge in the field of rare genetic disorders. Originally described as a unique pattern of major and minor anomalies, over time this syndrome has been shown to be characterized by a significant variability of clinical expression. By increasing the number of patients described, knowledge of the natural history of the condition has been enriched with the demonstration of the relative frequency of various potential comorbidities. Since 2006, the discovery of CdLS's molecular basis has shown an equally vast genetic heterogeneity linked to the presence of variants in genes encoding for the cohesin complex pathway. The most recent clinical-genetic data led to the classification of the "original syndrome" into a "clinical spectrum" that foresees the presence of classic patients, of non-classic forms, and of conditions that show a modest phenotypic overlapping with the original disease. Finally, the knowledge of the molecular basis of the disease has allowed the development of basic research projects that could lay the foundations for the development of possible innovative pharmacological treatments.

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

FOLFIRINOX (FFX) and gemcitabine + nab-paclitaxel (GN) are the most common chemotherapy regimens in first-line treatment of metastatic pancreatic cancer (PC). They have not been compared each other in a prospective trial, but only in retrospective studies, which can thus be affected by several biases. In order to overcome these biases, we took advantage of matching-adjusted indirect comparison (MAIC), that allows an indirect comparison by reducing cross-trial differences, and compared data from 268 patients treated with GN in a real-world setting with data from the 171 patients included in the FFX arm of the PRODIGE trial. Survival outcomes did not differ between the two populations. Overall survival was 11.1 months for both treatments (hazard ratio (HR) of FFX 1.10, 95% confidence interval (CI) 0.81-1.49; p = 0.527). Progression-free survival was 6.0 months with GN and 6.4 months with FFX (HR of FFX 1.11, 95% CI 0.82-1.50; p = 0.520). On the other hand, we observed a difference in the toxicity profiles: grade 3/4 anemia was more frequent with GN, whereas a higher occurrence of grade 3/4 vomiting and diarrhea was reported with FFX. FFX and GN show an equivalent efficacy but different safety profiles in the first-line therapy of metastatic pancreatic cancer. Searching for reliable predictive biomarkers is advised in order to improve therapeutic strategy in metastatic PC.

Background: Regorafenib has been shown to improve clinical outcomes compared to placebo, becoming a standard second-line therapy for sorafenib-progressed and -tolerated hepatocellular carcinoma (HCC) patients., Objective: We performed a multicentre, retrospective study in Italy and Korea to evaluate the effectiveness of the treatment sequence sorafenib-regorafenib compared with sorafenib and physician's choice in a real-life setting., Patients and Methods: A propensity score model was developed to control the results for baseline variable imbalances between the arm treated with sorafenib and regorafenib (S-R) and the arm treated with sorafenib and physician's choice (S-P). Survival analysis was conducted on the matched population., Results: After the application of propensity score matching, we analysed 99 patients in the arm treated with S-R and 99 patients in the arm treated with S-P. For the S-R group, the median overall survival was 22.2 months (95% CI 17.1-27.4), compared to 17.9 months (95% CI 15.1-50.0) for the S-P group. The results of the univariate analysis showed a 31% reduction of death risk for patients treated with S-R (p = 0.0382) compared to patients treated with S-P. Interaction tests highlighted the predictive role of alpha-fetoprotein (AFP), neutrophil-to-lymphocyte ratio (NLR), and extrahepatic spread., Conclusion: This study provides additional proof of the superiority of the S-R treatment over the S-P treatment approach in advanced HCC patients from a real-life setting.

Prenatal exposure to valproate (VPA), an antiepileptic drug, has been associated with fetal valproate spectrum disorders (FVSD), a clinical condition including congenital malformations, developmental delay, intellectual disability as well as autism spectrum disorder, together with a distinctive facial appearance. VPA is a known inhibitor of histone deacetylase which regulates the chromatin state. Interestingly, perturbations of this epigenetic balance are associated with chromatinopathies, a heterogeneous group of Mendelian disorders arising from mutations in components of the epigenetic machinery. Patients affected from these disorders display a plethora of clinical signs, mainly neurological deficits and intellectual disability, together with distinctive craniofacial dysmorphisms. Remarkably, critically examining the phenotype of FVSD and chromatinopathies, they shared several overlapping features that can be observed despite the different etiologies of these disorders, suggesting the possible existence of a common perturbed mechanism(s) during embryonic development., Competing Interests: RF formerly held a leadership position in TeratOmic Consulting LLC. This now dissolved organization provided expert consulting support in birth defects litigation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Parodi, Di Fede, Peron, Viganò, Grazioli, Castiglioni, Finnell, Gervasini, Vignoli and Massa.)

CHD7 is a chromatin remodeler protein that controls gene expression via the formation of multi-protein complexes with specific transcription factors. During development, CHD7 controls several differentiation programs, mainly by acting on neural progenitors and neural crest (NC) cells. Thus, its roles range from the central nervous system to the peripheral nervous system and the organs colonized by NC cells, including the heart. Accordingly, mutated CHD7 is linked to CHARGE syndrome, which is characterized by several neuronal dysfunctions and by malformations of NC-derived/populated organs. Altered CHD7 has also been associated with different neoplastic transformations. Interestingly, recent evidence revealed that semaphorins, a class of molecules involved in developmental and pathological processes similar to those controlled by CHD7, are regulated by CHD7 in a context-specific manner. In this article, we will review the recent insights that support the existence of genetic interactions between these pathways, both during developmental processes and cancer progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lettieri, Oleari, Paganoni, Gervasini, Massa, Fantin and Cariboni.)

Modified FOLFIRINOX (mFOLFIRINOX) and gemcitabine + nab-paclitaxel (GemNab) regimens represent a standard treatment in advanced pancreatic cancer (aPC). DPYD and UGT1A1 variants are relevant predictors of fluoropyrimidine and irinotecan-associated adverse events (AEs). Furthermore, data about the associations between polymorphisms in ABCB and CDA genes and GemNab-related toxicities are still controversial. The present study analyzes the association between DPYD, UGT, ABCB1, CDA variants, and AEs in aPC patients (pts) treated with mFOLFIRINOX or GemNab. Blood samples collected from 104 aPC pts treated with mFOLFIRINOX and 63 with GemNab were tested for DPYD c.1679T>G, IVS14+1G>A, c.2194G>A, c.2846A>T, UGT1A1*28, CDA c.79A>C, and ABCB1 c.1236C>T, c.2677G>T/A, c.3435C>T by real-time PCR and automatic sequencing. In mFOLFIRINOX cohort, DPYD IVS14+1GA genotype was associated with G4 hematological AEs, while the UGT1A1*28 significantly correlated with the risk of thrombocytopenia (p = 0.006). In the GemNab cohort, a significant association between CDA c.79CC and high-grade nausea was observed (p = 0.002). Moreover, the presence of at least a mutant allele in ABCB1 increased the risk of overall hematological AEs (p = 0.01), both further strengthened by the presence of CDA c.79CC (p = 0.0002). DPYD IVS14+1A allele is confirmed to be associated with fluoropyrimidine life-threatening toxicities, and UGT1A1*28 is related with a higher risk of hematologic AEs following irinotecan treatment. CDA c.79C and ABCB1 c.1236T, c.2677T/A, and c.3435T mutant alleles are predictive biomarkers of GemNab-related AEs. All these variants should be considered in aPC pts candidate to mFOLFIRINOX or GemNab treatments.

The short-chain fatty acid butyrate, produced by the gut microbiota, acts as a potent histone deacetylase (HDAC) inhibitor. We assessed possible ameliorative effects of butyrate, relative to other HDAC inhibitors, in in vitro and in vivo models of Rubinstein-Taybi syndrome (RSTS), a severe neurodevelopmental disorder caused by variants in the genes encoding the histone acetyltransferases CBP and p300. In RSTS cell lines, butyrate led to the patient-specific rescue of acetylation defects at subtoxic concentrations. Remarkably, we observed that the commensal gut microbiota composition in a cohort of RSTS patients is significantly depleted in butyrate-producing bacteria compared to healthy siblings. We demonstrate that the effects of butyrate and the differences in microbiota composition are conserved in a Drosophila melanogaster mutant for CBP, enabling future dissection of the gut-host interactions in an in vivo RSTS model. This study sheds light on microbiota composition in a chromatinopathy, paving the way for novel therapeutic interventions.