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Background: SARS-CoV-2 is the coronavirus responsible for the COVID-19 pandemic. Even though we are no longer in a pandemic situation, people are still getting infected, some of them need hospitalization and a few of them die. Methods: We conducted a retrospective study including 445 patients who accessed the Emergency Section of Policlinico Umberto I, Rome, Italy, where they had routine blood exams. In this study, we focused on the complete blood count, serum creatinine and azotemia. The data were analyzed using ANOVA, Spearman correlation and ROC analyses. They were divided into four groups based on their clinical outcomes: (1) the emergency group (patients who had mild forms and were quickly discharged); (2) the hospital ward group (patients who were admitted to the emergency section and were then hospitalized in a COVID-19 ward); (3) the intensive care unit (ICU) group (patients who required intensive assistance after the admission in the emergency section); (4) the deceased group (patients who had a fatal outcome after admission to the emergency section). Results: We found significant changes for creatinine, azotemia, hematocrit, mean corpuscular hemoglobin concentration, basophils, monocytes, red blood cell distribution width, hemoglobin, hematocrit and red blood cell numbers using ANOVA according to their clinical outcomes, particularly for the deceased group. Also, we found linear correlations of clinical outcomes with eosinophils, hemoglobin, hematocrit, mean corpuscular hemoglobin concentration, lymphocyte, neutrophil, platelet and red blood cell number and red blood cell distribution width. Conclusions: This study discloses an early association between “classical” routine blood biomarkers and the severity of clinical outcomes in Omicron patients.

Nerve growth factor (NGF) plays a dual role both in inflammatory states and cancer, acting both as a pro-inflammatory and oncogenic factor and as an anti-inflammatory and pro-apoptotic mediator in a context-dependent way based on the signaling networks and its interaction with diverse cellular components within the microenvironment. This report aims to provide a summary and subsequent review of the literature on the role of NGF in regulating the inflammatory microenvironment and tumor cell growth, survival, and death. The role of NGF in inflammation and tumorigenesis as a component of the inflammatory system, its interaction with the various components of the respective microenvironments, its ability to cause epigenetic changes, and its role in the treatment of cancer have been highlighted in this paper.

NGF plays a crucial immunomodulatory role and increased levels are found in numerous tissues during autoimmune states. NGF directly modulates innate and adaptive immune responses of B and T cells and causes the release of neuropeptides and neurotransmitters controlling the immune system activation in inflamed tissues. Evidence suggests that NGF is involved in the pathogenesis of numerous immune diseases including autoimmune thyroiditis, chronic arthritis, multiple sclerosis, systemic lupus erythematosus, mastocytosis, and chronic granulomatous disease. Furthermore, as NGF levels have been linked to disease severity, it could be considered an optimal early biomarker to identify therapeutic approach efficacy. In conclusion, by gaining insights into how these molecules function and which cells they interact with, future studies can devise targeted therapies to address various neurological, immunological, and other disorders more effectively. This knowledge may pave the way for innovative treatments based on NGF manipulation aimed at improving the quality of life for individuals affected by diseases involving neurotrophins.

Genetic features of alcohol dependence have been extensively investigated in recent years. A large body of studies has underlined the important role of genetic variants not only in metabolic pathways but also in the neurobiology of alcohol dependence, mediated by the neuronal circuits regulating reward and craving. Serotonin transporter (5-HTT), encoded by the SLC6A4 gene (Solute carrier family 6-neurotransmitter transporter-member 4), is targeted by antidepressant drugs such as selective serotonin reuptake inhibitors (SSRIs) and plays a pivotal role in serotoninergic transmission; it has been associated with psychiatric diseases and alcohol dependence. Transcriptional regulation and expression of 5-HTT depend not only on epigenetic modifications, among which DNA methylation (CpG and non-CpG) is primarily involved, but also on sequence variations occurring in intron/exon regions and in untranslated regions in 5′ and 3′, being the first sequences important for the splicing machinery and the last for the binding of transcription factors and micro RNAs. This work intends to shed light on the role of sequence variations known to affect the expression or function of 5-HTT in alcohol-dependent individuals. We found a statistically significant difference in the allelic (p = 0.0083) and genotypic (p = 0.0151) frequencies of the tri-allelic polymorphism, with higher function alleles and genotypes more represented in the control population. Furthermore, we identified three haplotypes more frequent in subjects with AUD (p < 0.0001) and one more frequent in the control population (p < 0.0001). The results obtained for the tri-allelic polymorphism in alcohol dependence confirm what is already present in part of the literature. The role of haplotypes requires further studies to be clarified.

Abstract Cystic fibrosis (CF) is caused by defects of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR-modulating drugs may overcome specific defects, such as the case of Trikafta, which is a clinically approved triple combination of Elexacaftor, Tezacaftor and Ivacaftor (ETI) that exhibited a strong ability to rescue the function of the most frequent F508del pathogenic variant even in genotypes with the mutated allele in single copy. Nevertheless, most rare genotypes lacking the F508del allele are still not eligible for targeted therapies. Via the innovative approach of using nasal conditionally reprogrammed cell (CRC) cell-based models that mimic patient disease in vitro, which are obtainable from each patient due to the 100% efficiency of the cell culture establishment, we theratyped orphan CFTR mutation L1077P. Protein studies, Forskolin-induced organoid swelling, and Ussing chamber assays congruently proved the L1077P variant function rescue by ETI. Notably, this rescue takes place even in the context of a single-copy L1077P allele, which appears to enhance its expression. Thus, the possibility of single-allele treatment also arises for rare genotypes, with an allele-specific modulation as part of the mechanism. Of note, besides providing indication of drug efficacy with respect to specific CFTR pathogenic variants or genotypes, this approach allows the evaluation of the response of single-patient cells within their genetic background. In this view, our studies support in vitro guided personalized CF therapies also for rare patients who are nearly excluded from clinical trials.

Background: We assessed the long-term (24 months) efficacy and safety of monthly calcifediol (0.266 mg) in the correction and maintenance of total 25(OH)D levels in postmenopausal women with basal values p for trend < 0.001). However, mean values at month 24 (36.7 ± 15.9) were not significantly different in respect to values at month 12 (41.2 ± 11.18). At 24 months, only 1 out 19 patients had a value p < 0.0216), with no significant changes between 12 and 24 months. No significant changes were observed as far as ionised calcium or creatinine were concerned. Conclusions: The long-term administration of calcifediol maintains stable and sustained 25(OH)D concentrations, with no safety concerns.

Fetal alcohol spectrum disorders (FASD) represent a continuum of lifelong impairments resulting from prenatal exposure to alcohol, with significant global impact. The “spectrum” of disorders includes a continuum of physical, cognitive, behavioral, and developmental impairments which can have profound and lasting effects on individuals throughout their lives, impacting their health, social interactions, psychological well-being, and every aspect of their lives. This narrative paper explores the intricate relationship between oxidative stress and epigenetics in FASD pathogenesis and its therapeutic implications. Oxidative stress, induced by alcohol metabolism, disrupts cellular components, particularly in the vulnerable fetal brain, leading to aberrant development. Furthermore, oxidative stress is implicated in epigenetic changes, including alterations in DNA methylation, histone modifications, and microRNA expression, which influence gene regulation in FASD patients. Moreover, mitochondrial dysfunction and neuroinflammation contribute to epigenetic changes associated with FASD. Understanding these mechanisms holds promise for targeted therapeutic interventions. This includes antioxidant supplementation and lifestyle modifications to mitigate FASD-related impairments. While preclinical studies show promise, further clinical trials are needed to validate these interventions’ efficacy in improving clinical outcomes for individuals affected by FASD. This comprehensive understanding of the role of oxidative stress in epigenetics in FASD underscores the importance of multidisciplinary approaches for diagnosis, management, and prevention strategies. Continued research in this field is crucial for advancing our knowledge and developing effective interventions to address this significant public health concern.

Klinefelter syndrome (KS) is a male genetic disease caused by the presence of an extra X chromosome, causing endocrine disorders mainly responsible for a high rate of infertility and metabolic disorders in adulthood. Scientific research is interested in identifying new biomarkers that can be predictive or prognostic of alterations strictly connected to KS. Lipocalin-2 (LCN-2, also known as NGAL) is a small protein initially identified within neutrophils as a protein related to innate immunity. Serum LCN-2 estimation seems to be a useful tool in predicting the metabolic complications caused by several pathological conditions. However, little is known about its potential role in infertility conditions. The present pilot study aims to investigate the presence of LCN-2 in the serum of a group of pre-pubertal and post-pubertal children affected by KS, compared to healthy controls. We demonstrated for the first time the presence of elevated levels of LCN-2 in the serum of KS patients, compared to controls. This increase was accompanied, in pre-pubertal KS patients, by the loss of correlation with LH and HDL, which instead was present in the healthy individuals. Moreover, in all KS individuals, a positive correlation between LCN-2 and inhibin B serum concentration was found. Despite the limited size of the sample analyzed, our preliminary data encourage further studies to confirm the findings and to extend the study to KS adult patients, to verify the predictive/prognostic value of LCN-2 as new biomarker for metabolic diseases and infertility associated with the pathology.

Besides its role in coagulation, vitamin K seems to be involved in various other mechanisms, including inflammation and age-related diseases, also at the level of gene expression. This work examined the roles of two vitamin K2 (menaquinones) vitamers, namely, menaquinone-4 (MK4) and reduced menaquinone-7 (MK7R), as gene modulator compounds, as well as their potential role in the epigenetic regulation of genes involved in amyloidogenesis and neuroinflammation. The SK-N-BE human neuroblastoma cells provided a “first-line” model for screening the neuroinflammatory and neurodegenerative molecular pathways. MK7R, being a new vitamin K form, was first tested in terms of solubilization, uptake and cell viability, together with MK4 as an endogenous control. We assessed the expression of key factors in amyloidogenesis and neuroinflammation, observing that the MK7R treatment was associated with the downregulation of neurodegeneration- (PSEN1 and BACE1) and neuroinflammation- (IL-1β and IL-6) associated genes, whereas genes retaining protective roles toward amiloidogenesis were upregulated (ADAM10 and ADAM17). By profiling the DNA methylation patterns of genes known to be epigenetically regulated, we observed a correlation between hypermethylation and the downregulation of PSEN1, IL-1β and IL-6. These results suggest a possible role of MK7R in the treatment of cognitive impairment, giving a possible base for further preclinical experiments in animal models of neurodegenerative disease.

Background: Despite the feasibility and promising activity data on intensity-modulated RT and simultaneous integrated boost (IMRT-SIB) dose escalation in preoperative chemoradiation (CRT) for locally advanced rectal cancer (LARC), few data are currently available on long-term outcomes. Patients and Methods: A cohort of 288 LARC patients with cT3-T4, cN0-2, cM0 treated with IMRT-SIB and capecitabine from March 2013 to December 2019, followed by a total mesorectal excision (TME) or an organ-preserving strategy, was collected from a prospective database of 10 Italian institutions. A dose of 45 Gy in 25 fractions was prescribed to the tumor and elective nodes, while the SIB dose was prescribed according to the clinical practice of each institution on the gross tumor volume (GTV). Concurrent capecitabine was administered at a dose of 825 mg/m2 twice daily, 7 days a week. The primary objective of the study was to evaluate long-term outcomes in terms of local control (LC), progression-free survival (PFS) and overall survival (OS). The secondary objective was to confirm the previously reported feasibility, safety and efficacy (pCR, TRG1-2 and downstaging rates) of the treatment in a larger patient population. Results: All patients received a dose of 45 Gy to the tumor and elective nodes, while the SIB dose ranged from 52.5 Gy to 57.5 Gy (median 55 Gy). Acute gastrointestinal and hematologic toxicity rates of grade 3–4 were 5.7% and 1.8%, respectively. At preoperative restaging, 36 patients (12.5%) with complete or major clinical responses (cCR or mCR) were offered an organ-preserving approach with local excision (29 patients) or a watch and wait strategy (7 patients). The complete pathologic response rate (pCR) in radically operated patients was 25.8%. In addition, 4 TME patients had pT0N1 and 19 LE patients had pT0Nx, corresponding to an overall pT0 rate of 31.3%. Of the 36 patients selected for organ preservation, 7 (19.5%) required the completion of TME due to unfavorable pathologic features after LE or tumor regrowth during W-W resulting in long-term rectal preservation in 29 of 288 (10.1%) of the total patient population. Major postoperative complications occurred in 14.2% of all operated patients. At a median follow-up of 50 months, the 5-year PFS and OS rates were 72.3% (95% CI: 66.3–77.4) and 85.9% (95% CI: 80.2–90.1), respectively. The 5-year local recurrence (LR) rate was 9.2% (95% CI: 6.0–13.2), while the distant metastasis (DM) rate was 21.3% (95% CI: 16.5–26.5). The DM rate was 24.5% in the high-risk subset compared to 16.2% in the low-intermediate risk group (p = 0.062) with similar LR rates (10% and 8%, respectively). On multivariable analysis, cT4 and TRG3–5 were significantly associated with worse PFS, OS and metastasis-free survival. Conclusions: Preoperative IMRT-SIB with the moderate dose intensification of 52.5–57.5 Gy (median 55 Gy) and the full dose of concurrent capecitabine confirmed to be feasible and effective in our real-life clinical practice. Organ preservation was shown to be feasible in carefully selected, responsive patients. The favorable long-term survival rates highlight the efficacy of this intensified treatment program. The incorporation of IMRT-SIB with a more effective systemic therapy component in high-risk patients could represent a new area of investigational interest.

In the precision medicine era of cystic fibrosis (CF), therapeutic interventions, by the so-called modulators, target the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The levels of targetable CFTR proteins are a main variable in the success of patient-specific therapy. In turn, the CFTR protein level depends, at least in part, on the level of CFTR mRNA. Many mechanisms can modulate the CFTR mRNA level, for example, transcriptional rate, stability of the mRNA, epigenetics, and pathogenic variants that can affect mRNA production and degradation. Independently from the causes of variable CFTR mRNA levels, their exact quantitative assessment is of great importance in CF. Methods with high analytical sensitivity, precision, and accuracy are mandatory for the quantitative evaluation aimed at the amelioration of the diagnostic, prognostic, and therapeutic aspects. This paper compares, for the first time, two CFTR gene expression quantification methods: a well-established method for the relative quantification of CFTR mRNA using a real-time PCR and an innovative method for its absolute quantification using a droplet digital PCR. No comprehensive methods for absolute CFTR quantification via droplet digital PCR have been published so far. The accurate quantification of CFTR expression at the mRNA level is a critical step for the personalized therapeutic approaches of CF.

DNA methylation, the main epigenetic modification regulating gene expression, plays a role in the pathophysiology of neurodegeneration. Previous evidence indicates that 5′-flanking hypomethylation of PSEN1, a gene involved in the amyloidogenic pathway in Alzheimer’s disease (AD), boosts the AD-like phenotype in transgenic TgCRND8 mice. Supplementation with S-adenosylmethionine (SAM), the methyl donor in the DNA methylation reactions, reverts the pathological phenotype. Several studies indicate that epigenetic signatures, driving the shift between normal and diseased aging, can be acquired during the first stages of life, even in utero, and manifest phenotypically later on in life. Therefore, we decided to test whether SAM supplementation during the perinatal period (i.e., supplementing the mothers from mating to weaning) could exert a protective role towards AD-like symptom manifestation. We therefore compared the effect of post-weaning vs. perinatal SAM treatment in TgCRND8 mice by assessing PSEN1 methylation and expression and the development of amyloid plaques. We found that short-term perinatal supplementation was as effective as the longer post-weaning supplementation in repressing PSEN1 expression and amyloid deposition in adult mice. These results highlight the importance of epigenetic memory and methyl donor availability during early life to promote healthy aging and stress the functional role of non-CpG methylation.

Alcohol consumption is associated with oxidative stress and an increased risk of carcinoma of the upper aero-digestive tract (UADT). Recently, it has been found that some microorganisms in the human oral cavity may locally metabolize ethanol, forming acetaldehyde, a carcinogenic metabolite of alcohol. In a cohort of patients first visited for UADT cancers, we estimated their alcohol consumption by measuring Ethyl Glucuronide/EtG (a long-lasting metabolite of ethanol) in the hair and carbohydrate-deficient transferrin/CDT (short-term index of alcohol intake) in the serum. Moreover, we analyzed, by culture-based methods, the presence of Neisseria subflava, Streptococcus mitis, Candida albicans, and glabrata (microorganisms generating acetaldehyde) in the oral cavity. According to the EtG values, we correlated drinking alcohol with endogenous oxidative stress and the investigated microorganism’s presence. We found that 55% of heavy drinkers presented microorganisms generating acetaldehyde locally. Moreover, we found that the presence of oral acetaldehyde-producing bacteria correlates with increased oxidative stress compared to patients without such bacteria. As for the study of alcohol dehydrogenase gene polymorphisms (the enzyme that transforms alcohol to acetaldehyde), we found that only the “CGTCGTCCC” haplotype was more frequent in the general population than in carcinoma patients. This pilot study suggests the importance of estimating alcohol consumption (EtG), the presence of bacteria producing acetaldehyde, and oxidative stress as risk factors for the onset of oral carcinomas.

Urinalysis is commonly used as a screening tool for kidney disease. In many cases, the dipstick urine assay includes the assessment of albumin/protein and creatinine; consequently, the value of their ratio is available on the urine section report. Identification of albuminuria/proteinuria at early stages is an important issue to prevent or at least delay the onset of chronic kidney disease (CKD), kidney failure, and the progression of cardiovascular damage linked to the kidney’s loss of function. Sensitive and specific diagnostic methods are required for the assessment of such an important biomarker: urine albumin, creatinine, and their ratio (ACR) measured with quantitative assays are considered the gold standard. Routine dipstick methods (more rapid and at a lower cost) are intended for wide population screening. The aim of our study was to verify the reliability of an automated urinalysis dipstick method by comparing the results with the quantitative test of creatinine and albumin performed on a clinical chemistry platform. The first-morning voids of 249 patients who arrived from different departments were analyzed in the Central Laboratory of the University Hospital Policlinico Umberto I in Rome. We found a good correlation between the two assays, even though we observed that the dipstick assessment tends to overestimate the ACR’s value, disclosing a higher number of false positives if compared to the reference method. As an important novelty in this study, we analyzed our data considering age (starting from pediatric to geriatric patients) and sex as variables for a sub-stratification of the participants. Our results show that positive values need to be confirmed with quantitative methods, especially in women and younger people, and that from samples that resulted as diluted at the dipstick assay, the ACR’s values can be obtained if they are reanalyzed with quantitative assays. Moreover, patients with microalbuminuria (ACR 30–300 mg/g) or severe albumin urinary excretion (ACR > 300 mg/g) should be reanalyzed using quantitative methods to obtain a more reliable calculation of the ACR.

Background: Since its outbreak, Coronavirus disease 2019 (COVID-19), a life-threatening respiratory illness, has rapidly become a public health emergency with a devastating social impact. Lately, the Omicron strain is considered the main variant of concern. Routine blood biomarkers are, indeed, essential for stratifying patients at risk of severe outcomes, and a huge amount of data is available in the literature, mainly for the previous variants. However, only a few studies are available on early routine biochemical blood biomarkers for Omicron-afflicted patients. Thus, the aim and novelty of this study were to identify routine blood biomarkers detected at the emergency room for the early prediction of severe morbidity and/or mortality. Methods: 449 COVID-19 patients from Sapienza University Hospital of Rome were divided into four groups: (1) the emergency group (patients with mild forms who were quickly discharged); (2) the hospital ward group (patients that after the admission in the emergency department were hospitalized in a COVID-19 ward); (3) the intensive care unit (ICU) group (patients that after the admission in the emergency department required intensive assistance); (4) the deceased group (patients that after the admission in the emergency department had a fatal outcome). Results: ANOVA and ROC data showed that high-sensitivity troponin-T (TnT), fibrinogen, glycemia, C-reactive protein, lactate dehydrogenase, albumin, D-dimer myoglobin, and ferritin for both men and women may predict lethal outcomes already at the level of the emergency department. Conclusions: Compared to previous Delta COVID-19 parallel emergency patterns of prediction, Omicron-induced changes in TnT may be considered other early predictors of severe outcomes.

The evaluation of morpho-functional sperm characteristics alone is not enough to explain infertility or to predict the outcome of Assisted Reproductive Technologies (ART): more sensitive diagnostic tools are needed in clinical practice. The aim of the present study was to analyze Sperm DNA Fragmentation (SDF) and sperm-borne miR-34c-5p and miR-449b-5p levels in men of couples undergoing ART, in order to investigate any correlations with fertilization rate, embryo quality and development. Male partners (n = 106) were recruited. Semen analysis, SDF evaluation and molecular profiling analysis of miR-34c-5p and miR-449b-5p (in 38 subjects) were performed. Sperm DNA Fragmentation evaluation- a positive correlation between SDF post sperm selection and the percentage of low-quality embryos and a negative correlation with viable embryo were found. SDF > 2.9% increased the risk of obtaining a non-viable embryo by almost 4-fold. Sperm miRNAs profile—we found an association with both miRNAs and sperm concentration, while miR-449b-5p is positively associated with SDF. Moreover, the two miRNAs are positively correlated. Higher levels of miR-34c-5p compared to miR-449b-5p increases by 14-fold the probability of obtaining viable embryos. This study shows that SDF, sperm miR-34c-5p, and miR-449b-5p have a promising role as biomarkers of semen quality and ART outcome.

The Presenilin1 (PSEN1) gene encodes the catalytic peptide of the γ-secretase complex, a key enzyme that cleaves the amyloid-β protein precursor (AβPP), to generate the amyloid-β (Aβ) peptides, involved in Alzheimer’s Disease (AD). Other substrates of the γ-secretase, such as E-cadherin and Notch1, are involved in neurodevelopment and haematopoiesis. Gene-specific DNA methylation influences PSEN1 expression in AD animal models. Here we evaluated canonical and non-canonical cytosine methylation patterns of the PSEN1 5ʹ-flanking during brain development and AD progression, in DNA extracted from the frontal cortex of AD transgenic mice (TgCRND8) and post-mortem human brain. Mapping CpG and non-CpG methylation revealed different methylation profiles in mice and humans. PSEN1 expression only correlated with DNA methylation in adult female mice. However, in post-mortem human brain, lower methylation, both at CpG and non-CpG sites, correlated closely with higher PSEN1 expression during brain development and in disease progression. PSEN1 methylation in blood DNA was significantly lower in AD patients than in controls. The present study is the first to demonstrate a temporal correlation between dynamic changes in PSEN1 CpG and non-CpG methylation patterns and mRNA expression during neurodevelopment and AD neurodegeneration. These observations were made possible by the use of an improved bisulphite methylation assay employing primers that are not biased towards non-CpG methylation. Our findings deepen the understanding of γ-secretase regulation and support the hypothesis that epigenetic changes can promote the pathophysiology of AD. Moreover, they suggest that PSEN1 DNA methylation in peripheral blood may provide a biomarker for AD.

Both physiological and pathological aging processes induce brain alterations especially affecting the speed of processing, working memory, conceptual reasoning and executive functions. Many therapeutic approaches to reduce the impact of brain aging on cognitive functioning have been tested; unfortunately, there are no satisfactory results as a single therapy. As aging is partly contributed by free radical reactions, it has been proposed that exogenous antioxidants could have a positive impact on both aging and its associated manifestations. The aim of this report is to provide a summary and a subsequent review of the literature evidence on the role of antioxidants in preventing and improving cognition in the aging brain. Manipulation of endogenous cellular defense mechanisms through nutritional antioxidants or pharmacological compounds represents an innovative approach to therapeutic intervention in diseases causing brain tissue damage, such as neurodegeneration. Coherently with this notion, antioxidants, especially those derived from the Mediterranean diet such as hydroxytyrosol and resveratrol, seem to be able to delay and modulate the cognitive brain aging processes and decrease the occurrence of its effects on the brain. The potential preventive activity of antioxidants should be evaluated in long-term exposure clinical trials, using preparations with high bioavailability, able to bypass the blood-brain barrier limitation, and that are well standardized.

Background: We report a mono-institutional experience regarding patient-perceived quality regarding the Chieti Radiotherapy Department, through RAMSI (Radiotherapy Amica Mia—SmileINTM(SI)—My Friend RadiotherapySI) project, in critical scenarios of limited equipment and COVID-19. Material and methods: Patient-reported experience measures (PREMs) were assessed as follows: Patient-centric welcome perception (PCWP), Comfort, Professional skills and Punctuality. Patients could give anonymous feedback using HappyOrNot technology through four totems located in strategic areas within the center. An internal benchmark was obtained using the feedback received after a preliminary observation period. The SI Experience Index was collected, analyzed and compared. Weekly and monthly reports were generated. Results: From February 2019 to February 2022, 8924 patients accessed the department; 17,464 daily treatments were recorded and 5830 points of feedback were collected: 896, 1267, 1125 and 2542 for PCWP, Comfort, Professional skills and Punctuality, respectively. A LINAC decommissioning period was analyzed, with decreases in the SI-Index score and Smile-IN approved percentage and an improvement after this period. Additionally, the COVID-19 pandemic was analyzed with a mild evaluations decrease for PREM’s Welcome, Comfort and Punctuality (Δ-value: −9%, −3% and −4%, respectively), while Professional skills were always optimal. Conclusion: The RAMSI project was effective for assessing treatment quality perception, allowing for improving clinical procedures with corrective actions. The RAMSI project is ongoing.

Abstract Background A clinical heterogeneity was reported in patients with Cystic Fibrosis (CF) with the same CFTR genotype and between siblings with CF. Methods We investigated all clinical aspects in a cohort of 101 pairs of siblings with CF (including 6 triplets) followed since diagnosis. Results Severe lung disease had a 22.2% concordance in sib-pairs, occurred early and the FEV1% at 12 years was predictive of the severity of lung disease in the adulthood. Similarly, CF liver disease occurred early (median: 15 years) and showed a concordance of 27.8% in sib-pairs suggesting a scarce contribution of genetic factors; in fact, only 2/15 patients with liver disease in discordant sib-pairs had a deficiency of alpha-1-antitrypsin (a known modifier gene of CF liver phenotype). CF related diabetes was found in 22 pairs (in 6 in both the siblings). It occurred later (median: 32.5 years) and is strongly associated with liver disease. Colonization by P. aeruginosa and nasal polyposis that required surgery had a concordance > 50% in sib-pairs and were poorly correlated to other clinical parameters. The pancreatic status was highly concordant in pairs of siblings (i.e., 95.1%) but a different pancreatic status was observed in patients with the same CFTR mutations. This suggests a close relationship of the pancreatic status with the “whole” CFTR genotype, including mutations in regulatory regions that may modulate the levels of CFTR expression. Finally, a severe course of CF was evident in a number of patients with pancreatic sufficiency. Conclusions Physicians involved in care of patients with CF and in genetic counseling must be aware of the clinical heterogeneity of CF even in sib-pairs that, at the state of the art, is difficult to explain.

Head and neck cancer (HNC) concerns more than 890,000 patients worldwide annually and is associated with the advanced stage at presentation and heavy outcomes. Alcohol drinking, together with tobacco smoking, and human papillomavirus infection are the main recognized risk factors. The tumorigenesis of HNC represents an intricate sequential process that implicates a gradual acquisition of genetic and epigenetics alterations targeting crucial pathways regulating cell growth, motility, and stromal interactions. Tumor microenvironment and growth factors also play a major role in HNC. Alcohol toxicity is caused both directly by ethanol and indirectly by its metabolic products, with the involvement of the oral microbiota and oxidative stress; alcohol might enhance the exposure of epithelial cells to carcinogens, causing epigenetic modifications, DNA damage, and inaccurate DNA repair with the formation of DNA adducts. Long-term markers of alcohol consumption, especially those detected in the hair, may provide crucial information on the real alcohol drinking of HNC patients. Strategies for prevention could include food supplements as polyphenols, and alkylating drugs as therapy that play a key role in HNC management. Indeed, polyphenols throughout their antioxidant and anti-inflammatory actions may counteract or limit the toxic effect of alcohol whereas alkylating agents inhibiting cancer cells’ growth could reduce the carcinogenic damage induced by alcohol. Despite the established association between alcohol and HNC, a concerning pattern of alcohol consumption in survivors of HNC has been shown. It is of primary importance to increase the awareness of cancer risks associated with alcohol consumption, both in oncologic patients and the general population, to provide advice for reducing HNC prevalence and complications.

Background. COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a virus belonging to the Coronaviridae family. This disease has spread rapidly around the world and soon became an international public health emergency leading to an unpredicted pressure on the hospital emergency units. Early routine blood biomarkers could be key predicting factors of COVID-19 morbidity and mortality as suggested for C-reactive protein (CRP), IL-6, prothrombin and D-dimer. This study aims to identify other early routine blood biomarkers for COVID-19 severity prediction disclosed directly into the emergency section. Methods. Our research was conducted on 156 COVID-19 patients hospitalized at the Sapienza University Hospital “Policlinico Umberto I” of Rome, Italy, between March 2020 and April 2020 during the paroxysm’s initial phase of the pandemic. In this retrospective study, patients were divided into three groups according to their outcome: (1) emergency group (patients who entered the emergency room and were discharged shortly after because they did not show severe symptoms); (2) intensive care unit (ICU) group (patients who attended the ICU after admission to the emergency unit); (3) the deceased group (patients with a fatal outcome who attended the emergency and, afterward, the ICU units). Routine laboratory tests from medical records were collected when patients were admitted to the emergency unit. We focused on Aspartate transaminase (AST), Alanine transaminase (ALT), Lactate dehydrogenase (LDH), Creatine kinase (CK), Myoglobin (MGB), Ferritin, CRP, and D-dimer. Results. As expected, ANOVA data show an age morbidity increase in both ICU and deceased groups compared with the emergency group. A main effect of morbidity was revealed by ANOVA for all the analyzed parameters with an elevation between the emergency group and the deceased group. Furthermore, a significant increase in LDH, Ferritin, CRP, and D-dimer was also observed between the ICU group and the emergency group and between the deceased group and ICU group. Receiver operating characteristic (ROC) analyses confirmed and extended these findings. Conclusions. This study suggests that the contemporaneous presence of high levels of LDH, Ferritin, and as expected, CRP, and D-dimer could be considered as potential predictors of COVID-19 severity and death.

We previously examined the safety and immunogenicity of multiple vaccines administered to a military cohort, divided into two groups, the first composed of students at military schools, thus operating inside the national borders for at least 3 years, and the other formed of soldiers periodically engaged in a 9-month-long mission abroad (Lebanon). In the current study, we analyzed 112 individuals of this cohort, 50 pertaining to the first group and 62 to the second group, in order to examine the possible late appearance of side effects and to calculate the half-life of the induced antibodies. Moreover, the possible involvement of B-cell polyclonal activation as a pathogenetic mechanism for long term antibody persistence has even been explored. No late side effects, as far as autoimmunity and/or lymphoproliferation appearance, have been noticed. The long duration of the vaccine induced anti-HAV antibodies has been confirmed, whereas the antibodies induced by tetravalent meningococcal polysaccharide vaccine have been found to persist above the threshold for putative protection for a longer time, and anti-tetanus, diphtheria, and polio 1 and 3 for a shorter time than previously estimated. No signs of polyclonal B-cell activation have been found, as a possible mechanism to understand the long antibody persistence.

The interplay between the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial sodium channel (ENaC) in respiratory epithelia has a crucial role in the pathogenesis of cystic fibrosis (CF). The comprehension of the mechanisms of transcriptional regulation of ENaC genes is pivotal to better detail the pathogenic mechanism and the genotype–phenotype relationship in CF, as well as to realize therapeutic approaches based on the transcriptional downregulation of ENaC genes. Since we aimed to study the epigenetic transcriptional control of ENaC genes, an assessment of their expression and DNA methylation patterns in different human cell lines, nasal brushing samples, and leucocytes was performed. The mRNA expression of CFTR and ENaC subunits α, β and γ (respectively SCNN1A, SCNN1B, and SCNN1G genes) was studied by real time PCR. DNA methylation of 5′-flanking region of SCNN1A, SCNN1B, and SCNN1G genes was studied by HpaII/PCR. The levels of expression and DNA methylation of ENaC genes in the different cell lines, brushing samples, and leukocytes were very variable. The DNA regions studied of each ENaC gene showed different methylation patterns. A general inverse correlation between expression and DNA methylation was evidenced. Leukocytes showed very low expression of all the 3 ENaC genes corresponding to a DNA methylated pattern. The SCNN1A gene resulted to be the most expressed in some cell lines that, accordingly, showed a completely demethylated pattern. Coherently, a heavy and moderate methylated pattern of, respectively, SCNN1B and SCNN1G genes corresponded to low levels of expression. As exceptions, we found that dexamethasone treatment appeared to stimulate the expression of all the 3 ENaC genes, without an evident modulation of the DNA methylation pattern, and that in nasal brushing a considerable expression of all the 3 ENaC genes were found despite an apparent methylated pattern. At least part of the expression modulation of ENaC genes seems to depend on the DNA methylation patterns of specific DNA regions. This points to epigenetics as a controlling mechanism of ENaC function and as a possible therapeutic approach for CF.

Two novel and related pathogenic variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene were structurally and functionally characterized. These alterations have not been previously described in literature. Two patients with diagnosis of Cystic Fibrosis (CF) based on the presence of one mutated allele, p.Phe508del, pathological sweat test and clinical symptoms were studied. To complete the genotypes of both patients, an extensive genetic and functional analysis of the CFTR gene was performed. Extensive genetic characterization confirmed the presence of p.Phe508del pathogenic variant and revealed, in both patients, the presence of an insertion of part of intron 10 in intron 9 of the CFTR gene, within the (TG)m repeat, with a variable poly-T stretch. The molecular lesions resulted to be very similar in both patients, with only a difference in the number of T in the poly-T stretch. The functional characterization at RNA level revealed a complete anomalous splicing, without exon 10, from the allele with the insertion of both patients. Consequently, the alleles with the insertions are expected not to contribute to the formation of a functional CFTR protein. Molecular and functional features of these alterations are compatible with the definition of novel CF-causing variants of the CFTR gene. This also allowed the completion of the genetic characterization of both patients.

Genetic analysis in cystic fibrosis (CF) is a difficult task. Within the many causes of variability and uncertainty, a major determinant is poor knowledge of the functional effect of most DNA variants of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. In turn, knowledge of the effect of a CFTR variant has dramatic diagnostic, prognostic and, in the era of CF precision medicine, also therapeutic consequences. One of the most challenging CFTR variants is the (TG)mTn haplotype, which has variable functional effect and controversial clinical consequences. The exact quantification of the anomalous splicing of CFTR exon 10 (in the HGVS name; exon 9 in the legacy name) and, consequently, of the residual wild-type functional CFTR mRNA, should be mandatory in clinical assessment of patients with potentially pathological haplotype of this tract. Here, we present a real time-based assay for the quantification of the proportion of exon 10+/exon 10− CFTR mRNA, starting from nasal brushing. Our assay proved rapid, economic and easy to perform. Specific primers used for this assay are either disclosed or commercially available, allowing any laboratory to easily perform it. A simplified analysis of the data is provided, facilitating the interpretation of the results. This method helps to enhance the comprehension of the genotype–phenotype relationship in CF and CFTR-related disorders (CFTR-RD), crucial for the diagnosis, prognosis and personalized therapy of CF.

Background: There are no predictive factors of evolution of cystic fibrosis (CF) screen positive inconclusive diagnosis subjects (CFSPIDs). Aim: to define the role of the second CFTR variant as a predictive factor of disease evolution in CFSPIDs carrying the D1152H variant. Methods: We retrospectively evaluated clinical characteristics and outcome of CFSPIDs carrying the D1152H variant followed at five Italian CF centers. CFSPIDs were divided in two groups: Group A: compound heterozygous for D1152H and a CF-causing variant; Group B: compound heterozygous for D1152H and a: (i) non CF-causing variant, (ii) variant with varying clinical consequences, or (iii) variant with unknown significance. The variants were classified according to CFTR2 mutation database. Results: We enrolled 43 CFSPIDs with at least one D1152H variant: 28 (65.1%) were classified in the group A, and 15 (34.9%) in the Group B. CFSPIDs of group A had the first IRT significantly higher compared to those of group B (p < 0.05) and had a more severe clinical outcome during the follow-up. At the end of the study period, after a mean follow-up of 40.6 months (range 6–91.6), 4 (9.3%) out of 43 CFSPIDs progressed to CFTR-RD or CF. All these subjects were in the group A. Conclusions: The genetic profile could help predict the risk of disease evolution in CFSPIDs carrying D1152H, revealing the subjects that need a more frequent follow-up.

Urinary tract infections (UTIs) are mainly caused by uropathogenic Escherichia coli (UPEC). Acute and recurrent UTIs are commonly treated with antibiotics, the efficacy of which is limited by the emergence of antibiotic resistant strains. The natural sugar d-mannose is considered as an alternative to antibiotics due to its ability to mask the bacterial adhesin FimH, thereby preventing its binding to urothelial cells. Despite its extensive use, the possibility that d-mannose exerts “antibiotic-like” activity by altering bacterial growth and metabolism or selecting FimH variants has not been investigated yet. To this aim, main bacterial features of the prototype UPEC strain CFT073 treated with d-mannose were analyzed by standard microbiological methods. FimH functionality was analyzed by yeast agglutination and human bladder cell adhesion assays. Our results indicate that high d-mannose concentrations have no effect on bacterial growth and do not interfere with the activity of different antibiotics. d-mannose ranked as the least preferred carbon source to support bacterial metabolism and growth, in comparison with d-glucose, d-fructose, and l-arabinose. Since small glucose amounts are physiologically detectable in urine, we can conclude that the presence of d-mannose is irrelevant for bacterial metabolism. Moreover, d-mannose removal after long-term exposure did not alter FimH’s capacity to bind to mannosylated proteins. Overall, our data indicate that d-mannose is a good alternative in the prevention and treatment of UPEC-related UTIs.

Tangier disease is an autosomal recessive disorder characterized by severe reduction in HDL-cholesterol and peripheral lipid storage. We describe a family with c.5094C>A p.Tyr16980* mutation in the ABCA1 gene, clinically characterized by syringomyelic-like anesthesia, demyelinating multineuropathy and reduction in intraepidermal small fibers innervation. In the proband patient, cardiac involvement determined a myocardial infarction; lipid storage was demonstrated in gut, cornea and aortic wall. The reported ABCA1 mutation has never been described before in a Tangier family.

Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR.

[This corrects the article DOI: 10.1371/journal.pone.0120099.].

Discordant results obtained in bisulfite assays using MethPrimers (PCR primers designed using MethPrimer software or assuming that non-CpGs cytosines are non methylated) versus primers insensitive to cytosine methylation lead us to hypothesize a technical bias. We therefore used the two kinds of primers to study different experimental models and methylation statuses. We demonstrated that MethPrimers negatively select hypermethylated DNA sequences in the PCR step of the bisulfite assay, resulting in CpG methylation underestimation and non-CpG methylation masking, failing to evidence differential methylation statuses. We also describe the characteristics of "Methylation-Insensitive Primers" (MIPs), having degenerated bases (G/A) to cope with the uncertain C/U conversion. As CpG and non-CpG DNA methylation patterns are largely variable depending on the species, developmental stage, tissue and cell type, a variable extent of the bias is expected. The more the methylome is methylated, the greater is the extent of the bias, with a prevalent effect of non-CpG methylation. These findings suggest a revision of several DNA methylation patterns so far documented and also point out the necessity of applying unbiased analyses to the increasing number of epigenomic studies.

The precursors of atherogenic dyslipidemia (AD) are not well defined. Therefore, we investigated 62 non-obese, non-diabetic AD and 221 normolipemic children. Anthropometric parameters, blood pressure and biochemical measures were obtained in index children, their parents and all available siblings. The heritability (h(2)) of anthropometric and biochemical traits was estimated by SOLAR. Rare and common variants in APOA1 and LPL genes were screened by re-sequencing. Compared to normolipemic, AD children showed increased body mass index, waist circumference, plasma glucose, insulin, ApoB, HOMA-IR, hs-CRP and lower adiponectin (p

We report data on the spatial structure and seasonal variation of the community of Valmarino cave, a medium sized sandstone cave, located a few kilometres from the coast line, in Central Italy. Due to both its habitat features and its relatively recent geological history, Valmarino cave is only inhabited by terrestrial, troglophilic elements, i.e facultative cave dwellers. By means of monthly censuses and density plot estimates we have investigated species abundance, diversity and their spatial organization, by considering separately samples from different cave sectors. Homogeneous sampling design allowed to compare series of samplings performed in 1974 and 1994. On the whole 21 arthropods and one snail species constitute the cave community. Ordination plots resulting from correspondence analyses of monthly samples outline a distinct spatial and temporal structure. Two main sub-communities can be identified: a inner subcommunity, mainly represented by eu-troglophilic species, showing a remarkable stability throughout the year and an outer sub-community, mainly represented by sub-troglophilic species, showing strong seasonal variation. Both spatial and temporal vectors show similar importance in shaping the community structure. An interesting result of this study is the long term stability of both spatial and seasonal components of the community structure which remained almost identical after 20 years, as shown by the comparison of ordination plots obtained from 1974 and 1994 sampling series. Therefore this study provides empirical evidence of a frequently hypothesised, albeit never demonstrated feature of the cave ecosystem.

IntroductionIn this study we investigated the effects of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene variants on the composition of faecal microbiota, in patients affected by Cystic Fibrosis (CF). CFTR mutations (F508del is the most common) lead to a decreased secretion of chloride/water, and to mucus sticky secretions, in pancreas, respiratory and gastrointestinal tracts. Intestinal manifestations are underestimated in CF, leading to ileum meconium at birth, or small bowel bacterial overgrowth in adult age.MethodsThirty-six CF patients, fasting and under no-antibiotic treatment, were CFTR genotyped on both alleles. Faecal samples were subjected to molecular microbial profiling through Temporal Temperature Gradient Electrophoresis and species-specific PCR. Ecological parameters and multivariate algorithms were employed to find out if CFTR variants could be related to the microbiota structure.ResultsPatients were classified by two different criteria: 1) presence/absence of F508del mutation; 2) disease severity in heterozygous and homozygous F508del patients. We found that homozygous-F508del and severe CF patients exhibited an enhanced dysbiotic faecal microbiota composition, even within the CF cohort itself, with higher biodiversity and evenness. We also found, by species-specific PCR, that potentially harmful species (Escherichia coli and Eubacterium biforme) were abundant in homozygous-F508del and severe CF patients, while beneficial species (Faecalibacterium prausnitzii, Bifidobacterium spp., and Eubacterium limosum) were reduced.ConclusionsThis is the first report that establishes a link among CFTR variants and shifts in faecal microbiota, opening the way to studies that perceive CF as a 'systemic disease', linking the lung and the gut in a joined axis.

Homologous Replacement is used to modify specific gene sequences of chromosomal DNA in a process referred to as "Small Fragment Homologous Replacement", where DNA fragments replace genomic target resulting in specific sequence changes. To optimize the efficiency of this process, we developed a reporter based assay system where the replacement frequency is quantified by cytofluorimetric analysis following restoration of a stably integrated mutated eGFP gene in the genome of SV-40 immortalized mouse embryonic fibroblasts (MEF-SV-40). To obtain the highest correction frequency with this system, several parameters were considered: fragment synthesis and concentration, cell cycle phase and methylation status of both fragment and recipient genome. In addition, different drugs were employed to test their ability to improve technique efficiency. SFHR-mediated genomic modification resulted to be stably transmitted for several cell generations and confirmed at transcript and genomic levels. Modification efficiency was estimated in a range of 0.01-0.5%, further increasing when PARP-1 repair pathway was inhibited. In this study, for the first time SFHR efficiency issue was systematically approached and in part addressed, therefore opening new potential therapeutic ex-vivo applications.