Development of a rapid LC-MS/MS method for simultaneous quantification of ten commonly used antibiotic drugs in human serum.

The use of TDM in clinical practice to monitor the plasma levels of antibiotics administered to critically ill patients is a well-established approach that allows for optimization of the patient's response to drug therapy, considering the characteristics of the drug, the clinical and physiological status of the patient and any peculiar of the pathogen that caused the clinical picture. In our laboratory, we have developed a single LC-MS/MS analysis for dosing the serum concentration of an antibacterial panel composed of eight antibacterial and two selective inhibitors. The method presented used a certified material furnished by a commercial company and was internally validated using the EMA guidelines. The results have shown high sensitivity, precision, and accuracy, a lower matrix effect combined with simple sample preparation and a time-saving procedure. We have evaluated the recovery rate and matrix effect by testing serum samples without pathological index and serum pools obtained from haemolysed, icteric, or lipemic samples. The assay has shown a recovery range between 94% and 101%.

Identification of a set of genes potentially responsible for resistance to ferroptosis in lung adenocarcinoma cancer stem cells.

Scientific literature supports the evidence that cancer stem cells (CSCs) retain inside low reactive oxygen species (ROS) levels and are, therefore, less susceptible to cell death, including ferroptosis, a type of cell death dependent on iron-driven lipid peroxidation. A collection of lung adenocarcinoma (LUAD) primary cell lines derived from malignant pleural effusions (MPEs) of patients was used to obtain 3D spheroids enriched for stem-like properties. We observed that the ferroptosis inducer RSL3 triggered lipid peroxidation and cell death in LUAD cells when grown in 2D conditions; however, when grown in 3D conditions, all cell lines underwent a phenotypic switch, exhibiting substantial resistance to RSL3 and, therefore, protection against ferroptotic cell death. Interestingly, this phenomenon was reversed by disrupting 3D cells and growing them back in adherence, supporting the idea of CSCs plasticity, which holds that cancer cells have the dynamic ability to transition between a CSC state and a non-CSC state. Molecular analyses showed that ferroptosis resistance in 3D spheroids correlated with an increased expression of antioxidant genes and high levels of proteins involved in iron storage and export, indicating protection against oxidative stress and low availability of iron for the initiation of ferroptosis. Moreover, transcriptomic analyses highlighted a novel subset of genes commonly modulated in 3D spheroids and potentially capable of driving ferroptosis protection in LUAD-CSCs, thus allowing to better understand the mechanisms of CSC-mediated drug resistance in tumors.

The MITF/mir-579-3p regulatory axis dictates BRAF-mutated melanoma cell fate in response to MAPK inhibitors.

Therapy of melanoma has improved dramatically over the last years thanks to the development of targeted therapies (MAPKi) and immunotherapies. However, drug resistance continues to limit the efficacy of these therapies. Our research group has provided robust evidence as to the involvement of a set of microRNAs in the development of resistance to target therapy in BRAF-mutated melanomas. Among them, a pivotal role is played by the oncosuppressor miR-579-3p. Here we show that miR-579-3p and the microphthalmia-associated transcription factor (MITF) influence reciprocally their expression through positive feedback regulatory loops. In particular we show that miR-579-3p is specifically deregulated in BRAF-mutant melanomas and that its expression levels mirror those of MITF. Luciferase and ChIP studies show that MITF is a positive regulator of miR-579-3p, which is located in the intron 11 of the human gene ZFR (Zink-finger recombinase) and is co-transcribed with its host gene. Moreover, miR-579-3p, by targeting BRAF, is able to stabilize MITF protein thus inducing its own transcription. From biological points of view, early exposure to MAPKi or, alternatively miR-579-3p transfection, induce block of proliferation and trigger senescence programs in BRAF-mutant melanoma cells. Finally, the long-term development of resistance to MAPKi is able to select cells characterized by the loss of both miR-579-3p and MITF and the same down-regulation is also present in patients relapsing after treatments. Altogether these findings suggest that miR-579-3p/MITF interplay potentially governs the balance between proliferation, senescence and resistance to therapies in BRAF-mutant melanomas.

To Be Frail or Not to Be Frail: This Is the Question-A Critical Narrative Review of Frailty.

Many factors have contributed to rendering frailty an emerging, relevant, and very popular concept. First, many pandemics that have affected humanity in history, including COVID-19, most recently, have had more severe effects on frail people compared to non-frail ones. Second, the increase in human life expectancy observed in many developed countries, including Italy has led to a rise in the percentage of the older population that is more likely to be frail, which is why frailty is much a more common concern among geriatricians compared to other the various health-care professionals. Third, the stratification of people according to the occurrence and the degree of frailty allows healthcare decision makers to adequately plan for the allocation of available human professional and economic resources. Since frailty is considered to be fully preventable, there are relevant consequences in terms of potential benefits both in terms of the clinical outcome and healthcare costs. Frailty is becoming a popular, pervasive, and almost omnipresent concept in many different contexts, including clinical medicine, physical health, lifestyle behavior, mental health, health policy, and socio-economic planning sciences. The emergence of the new "science of frailty" has been recently acknowledged. However, there is still debate on the exact definition of frailty, the pathogenic mechanisms involved, the most appropriate method to assess frailty, and consequently, who should be considered frail. This narrative review aims to analyze frailty from many different aspects and points of view, with a special focus on the proposed pathogenic mechanisms, the various factors that have been considered in the assessment of frailty, and the emerging role of biomarkers in the early recognition of frailty, particularly on the role of mitochondria. According to the extensive literature on this topic, it is clear that frailty is a very complex syndrome, involving many different domains and affecting multiple physiological systems. Therefore, its management should be directed towards a comprehensive and multifaceted holistic approach and a personalized intervention strategy to slow down its progression or even to completely reverse the course of this condition.

Longitudinal evaluation of two different COVID-19 vaccination strategies in individuals with and without previous SARS-CoV-2 infection.

Background: We tested the antibody response to SARS-CoV-2 vaccination in individuals with and without previous infection that received different vaccination strategies. Methods: We recruited 203 volunteers. Individuals who have had SARS-CoV-2 infection during the six months preceding vaccination received one dose (group 1), the others received two (group 2). After 3 months, 98 subjects received a booster dose. Anti-SARS-CoV-2 Spike RBD IgG were tested in all subjects before vaccination (T0), and at 15 (T15), 90 (T90), 180 (T180) and 360 (T360) days after second or single dose; additionally, in group 2, IgG were tested 10 days after the vaccination (T10). Results: The difference of IgG concentration between the groups was statistically significant (p<0.05) at T0, T15 and T90, but not at T180 (p=0.713) and T360 (p=0.069). At T0 and T90 the antibody titre was higher in group 1, but it dropped in all volunteers 90 days after vaccination. Most of infections after vaccination occurred between T90 and T180. Conclusions: Antibody titre is significantly associated with a previous SARS-CoV-2 infection. Probability of contracting the infection increases after three months from primary vaccination, even among who had a previous infection, confirming the efficacy of vaccination as a preventive measure against SARS-CoV-2 infections and the need of booster administrations.

Upregulated expression of miR-4443 and miR-4488 in drug resistant melanomas promotes migratory and invasive phenotypes through downregulation of intermediate filament nestin.

BACKGROUND: BRAF-mutant melanoma patients benefit from the combinatorial treatments with BRAF and MEK inhibitors. However, acquired drug resistance strongly limits the efficacy of these targeted therapies in time. Recently, many findings have underscored the involvement of microRNAs as main drivers of drug resistance. In this context, we previously identified a subset of oncomiRs strongly up-regulated in drug-resistant melanomas. In this work, we shed light on the molecular role of two as yet poorly characterized oncomiRs, miR-4443 and miR-4488. METHODS: Invasion and migration have been determined by wound healing, transwell migration/invasion assays and Real Time Cell Analysis (RTCA) technology. miR-4488 and miR-4443 have been measured by qRT-PCR. Nestin levels have been tested by western blot, confocal immunofluorescence, immunohistochemical and flow cytometry analyses. RESULTS: We demonstrate that the two oncomiRs are responsible for the enhanced migratory and invasive phenotypes, that are a hallmark of drug resistant melanoma cells. Moreover, miR-4443 and miR-4488 promote an aberrant cytoskeletal reorganization witnessed by the increased number of stress fibers and cellular protrusions-like cancer cell invadopodia. Mechanistically, we identified the intermediate filament nestin as a molecular target of both oncomiRs. Finally, we have shown that nestin levels are able to predict response to treatments in melanoma patients. CONCLUSIONS: Altogether these findings have profound translational implications in the attempt i) to develop miRNA-targeting therapies to mitigate the metastatic phenotypes of BRAF-mutant melanomas and ii) to identify novel biomarkers able to guide clinical decisions.

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype¼, while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Relationship Between Real-time TDM-guided Pharmacodynamic Target Attainment of Continuous Infusion Beta-lactam Monotherapy and Microbiologic Outcome in the Treatment of Critically Ill Children With Severe Documented Gram-negative Infections.

OBJECTIVES: To explore the relationship between real-time therapeutic drug monitoring (TDM)-guided pharmacodynamic target attainment of continuous infusion (CI) beta-lactam monotherapy and microbiological outcome in the treatment of critically ill children with severe documented Gram-negative infections. METHODS: Observational, monocentric, retrospective study of critically ill patients receiving CI piperacillin-tazobactam, ceftazidime, or meropenem in monotherapy for documented Gram-negative infections optimized by means of a real-time TDM-guided strategy. Average steady-state beta-lactam concentrations (C ss ) were calculated for each patient, and the beta-lactam C ss /minimum inhibitory concentration (MIC) ratio was selected as a pharmacodynamic parameter of efficacy. The C ss /MIC ratio was defined as optimal if ≥4, quasi-optimal if between 1 and 4, and suboptimal if <1. The relationship between C ss /MIC and microbiological outcome was assessed. RESULTS: Forty-six TDM assessments were carried out in 21 patients [median age 2 (interquartile range: 1-8) years]. C ss /MIC ratios were optimal in 76.2% of cases. Patients with optimal C ss /MIC ratios had both a significantly higher microbiological eradication rate (75.0% vs. 0.0%; P = 0.006) and lower resistance development rate (25.0% vs. 80.0%; P = 0.047) than those with quasi-optimal or suboptimal C ss /MIC ratios. Quasi-optimal/suboptimal C ss /MIC ratio occurred more frequently when patients had infections caused by pathogens with MIC values above the European Committee on Antimicrobial Susceptibility Testing clinical breakpoint (100.0% vs. 6.3%; P < 0.001). CONCLUSIONS: Real-time TDM-guided pharmacodynamic target attainment of CI beta-lactam monotherapy allowed to maximize treatment efficacy in most critically ill children with severe Gram-negative infections. Attaining early optimal C ss /MIC ratios of CI beta-lactams could be a key determinant associated with microbiologic eradication during the treatment of Gram-negative infections. Larger prospective studies are warranted for confirming our findings.

Histologic Analysis of Idiopathic Pulmonary Fibrosis by Morphometric and Fractal Analysis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive fibrotic lung disorder, ultimately leading to respiratory failure and death. Despite great research advances in understanding the mechanisms underlying the disease, its diagnosis, and its treatment, IPF still remains idiopathic without known biological or histological markers able to predict disease progression or response to treatment. The histologic hallmark of IPF is usual interstitial pneumonia (UIP), with its intricate architectural distortion and temporal inhomogeneity. We hypothesize that normal lung alveolar architecture can be compared to fractals, such as the Pythagoras tree with its fractal dimension (Df), and every pathological insult, distorting the normal lung structure, could result in Df variations. In this study, we aimed to assess the UIP histologic fractal dimension in relationship to other morphometric parameters in newly diagnosed IPF patients and its possible role in the prognostic stratification of the disease. Clinical data and lung tissue specimens were obtained from twelve patients with IPF, twelve patients with non-specific interstitial pneumonia (NSIP), and age-matched "healthy" control lung tissue from patients undergoing lung surgery for other causes. Histology and histomorphometry were performed to evaluate Df and lacunarity measures, using the box counting method on the FracLac ImageJ plugin. The results showed that Df was significantly higher in IPF patients compared to controls and fibrotic NSIP patients, indicating greater architectural distortion in IPF. Additionally, high Df values were associated with higher fibroblastic foci density and worse prognostic outcomes in IPF, suggesting that Df may serve as a potential novel prognostic marker for IPF. The scalability of Df measurements was demonstrated through repeated measurements on smaller portions from the same surgical biopsies, which were selected to mimic a cryobiopsy. Our study provides further evidence to support the use of fractal morphometry as a tool for quantifying and determining lung tissue remodeling in IPF, and we demonstrated a significant correlation between histological and radiological Df in UIP pattern, as well as a significant association between Df and FF density. Furthermore, our study demonstrates the scalability and self-similarity of Df measurements across different biopsy types, including surgical and smaller specimens.

Evaluation of laboratory parameters in 1587 COVID-19 patients admitted to metropolitan hospital area of Bologna, Italy.

Since the outbreak of the 2019 pandemic coronavirus disease (COVID-19), great attention has been given to identifying the main clinical features of the disease. Identification of laboratory parameters able to classify patients based on their risk is mandatory to improve their clinical management. We retrospectively evaluated twenty-six laboratory tests measured in COVID-19 positive patients admitted to the hospital in March and April 2020 to find any correlation between their changes and the risk of death. We divided them into surviving and non-surviving patients. A total of 1587 patients were recruited, 854 males with median age of 71 (IQR 56-81) and 733 females with median age of 77 (IQR 61-87). On admission, death was found to be positively correlated with age (p=0.001), but not with sex (p=0.640) or with hospitalization in days (p=0.827). Brain natriuretic peptide (BNP), creatinine, C-reactive protein (CRP), INR, leukocyte count, lymphocyte count, neutrophil count, and procalcitonin (PCT) demonstrated a statistically significant difference between the two groups (p<0.001), suggesting their role as markers of disease severity; only lymphocyte count resulted as an independent risk factor for death.

Impact of COVID-19 pandemic on outpatient visit volume in cancer patients: Results of COMETA multicenter retrospective observational study.

Objective: To evaluate the impact of the COVID-19 pandemic on first and follow-up visits for cancer outpatients. Methods: This is a multicenter retrospective observational study involving three Comprehensive Cancer Care Centers (CCCCs): IFO, including IRE and ISG in Rome, AUSL-IRCCS of Reggio Emilia, and IRCCS Giovanni Paolo II in Bari) and one oncology department in a Community Hospital (Saint'Andrea Hospital, Rome). From 1 January 2020 and 31 December 2021, we evaluated the volume of outpatient consultations (first visits and follow-up), comparing them with the pre-pandemic year (2019). Results were analyzed by quarter according to the Rt (real-time indicator used to assess the evolution of the pandemic). IFO and IRCCS Giovanni Paolo II were "COVID-free" while AUSL-IRCCS RE was a "COVID-mixed" Institute. Depending on the Rt, Sain't Andrea Hospital experienced a "swinging" organizational pathway (COVID-free/ COVID-mixed). Results: Regarding the "first appointments", in 2020 the healthcare facilities operating in the North and Center of Italy showed a downward trend. In 2021, only AUSL-IRCCS RE showed an upward trend. Regarding the "follow-up", only AUSL IRCCS RE showed a slight up-trend in 2020. In 2021, IFO showed an increasing trend, while S. Andrea Hospital showed a negative plateau. Surprisingly, IRCCS Giovanni Paolo II in Bari showed an uptrend for both first appointment and follow-ups during pandemic and late pandemic except for the fourth quarter of 2021. Conclusions: During the first pandemic wave, no significant difference was observed amongst COVID-free and COVID-mixed Institutes and between CCCCs and a Community Hospital. In 2021 ("late pandemic year"), it has been more convenient to organize COVID-mixed pathway in the CCCCs rather than to keep the Institutions COVID-free. A swinging modality in the Community Hospital did not offer positive results in term of visit volumes. Our study about the impact of COVID-19 pandemic on visit volume in cancer outpatients may help health systems to optimize the post-pandemic use of resources and improve healthcare policies.

Comparison of Questionnaire and Performance-Based Physical Frailty Scales to Predict Survival and Health-Related Quality of Life in Patients With Heart Failure.

Background Frailty is prevalent in older adults with heart failure and is associated with poor outcomes; however, there remains uncertainty on how to measure frailty in clinical practice. Methods and Results A multicentric prospective cohort study was assembled at 4 heart failure clinics to compare the prognostic value of 3 physical frailty scales in ambulatory patients with heart failure. Outcomes were all-cause death or hospitalization and health-related quality of life using the 36-Item Short Form survey questionnaire (SF-36) at 3 months. Multivariable regression was adjusted for age, sex, Meta-Analysis Global Group in Chronic Heart Failure score, and baseline SF-36 score. The cohort included 215 patients (mean age 77.6 years). All 3 frailty scales were independently associated with death or hospitalization at 3 months; the adjusted odds ratios standardized per 1 SD worsening of the Short Physical Performance Battery; Fried, and strength, assistance with walking, rising from a chair, climbing stairs, and falls scales were 1.67 (95% CI, 1.09-2.55), 1.60 (95% CI, 1.04-2.46), and 1.55 (95% CI, 1.03-2.35), respectively, with C statistics of 0.77 to 0.78. All 3 frailty scales were independently associated with worsening SF-36 scores, especially the Short Physical Performance Battery, for which 1 SD worsening of frailty translated to a decrement of -5.86 (-8.55 to -3.17) and -5.51 (-7.82 to -3.21) points in the Physical Component Score and Mental Component Score. Conclusions All 3 physical frailty scales were associated with death, hospitalization, and reduced health-related quality of life in ambulatory patients with heart failure. Questionnaire or performance-based physical frailty scales can be used to offer prognostic value and a therapeutic target in this vulnerable population. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03887351.

ALDOC- and ENO2- driven glucose metabolism sustains 3D tumor spheroids growth regardless of nutrient environmental conditions: a multi-omics analysis.

BACKGROUND: Metastases are the major cause of cancer-related morbidity and mortality. By the time cancer cells detach from their primary site to eventually spread to distant sites, they need to acquire the ability to survive in non-adherent conditions and to proliferate within a new microenvironment in spite of stressing conditions that may severely constrain the metastatic process. In this study, we gained insight into the molecular mechanisms allowing cancer cells to survive and proliferate in an anchorage-independent manner, regardless of both tumor-intrinsic variables and nutrient culture conditions. METHODS: 3D spheroids derived from lung adenocarcinoma (LUAD) and breast cancer cells were cultured in either nutrient-rich or -restricted culture conditions. A multi-omics approach, including transcriptomics, proteomics, and metabolomics, was used to explore the molecular changes underlying the transition from 2 to 3D cultures. Small interfering RNA-mediated loss of function assays were used to validate the role of the identified differentially expressed genes and proteins in H460 and HCC827 LUAD as well as in MCF7 and T47D breast cancer cell lines. RESULTS: We found that the transition from 2 to 3D cultures of H460 and MCF7 cells is associated with significant changes in the expression of genes and proteins involved in metabolic reprogramming. In particular, we observed that 3D tumor spheroid growth implies the overexpression of ALDOC and ENO2 glycolytic enzymes concomitant with the enhanced consumption of glucose and fructose and the enhanced production of lactate. Transfection with siRNA against both ALDOC and ENO2 determined a significant reduction in lactate production, viability and size of 3D tumor spheroids produced by H460, HCC827, MCF7, and T47D cell lines. CONCLUSIONS: Our results show that anchorage-independent survival and growth of cancer cells are supported by changes in genes and proteins that drive glucose metabolism towards an enhanced lactate production. Notably, this finding is valid for all lung and breast cancer cell lines we have analyzed in different nutrient environmental conditions. broader Validation of this mechanism in other cancer cells of different origin will be necessary to broaden the role of ALDOC and ENO2 to other tumor types. Future in vivo studies will be necessary to assess the role of ALDOC and ENO2 in cancer metastasis.

Oncosuppressive miRNAs loaded in lipid nanoparticles potentiate targeted therapies in BRAF-mutant melanoma by inhibiting core escape pathways of resistance.

BRAF-mutated melanoma relapsing after targeted therapies is an aggressive disease with unmet clinical need. Hence the need to identify novel combination therapies able to overcome drug resistance. miRNAs have emerged as orchestrators of non-genetic mechanisms adopted by melanoma cells to challenge therapies. In this context we previously identified a subset of oncosuppressor miRNAs downregulated in drug-resistant melanomas. Here we demonstrate that lipid nanoparticles co-encapsulating two of them, miR-199-5p and miR-204-5p, inhibit tumor growth both in vitro and in vivo in combination with target therapy and block the development of drug resistance. Mechanistically they act by directly reducing melanoma cell growth and also indirectly by hampering the recruitment and reprogramming of pro-tumoral macrophages. Molecularly, we demonstrate that the effects on macrophages are mediated by the dysregulation of a newly identified miR-204-5p-miR-199b-5p/CCL5 axis. Finally, we unveiled that M2 macrophages programs are molecular signatures of resistance and predict response to therapy in patients. Overall, these findings have strong translational implications to propose new combination therapies making use of RNA therapeutics for metastatic melanoma patients.

Complete Pseudo-Anodontia in an Adult Woman with Pseudo-Hypoparathyroidism Type 1a: A New Additional Nonclassical Feature?

Pseudo-anodontia consists in the clinical, not radiographic, absence of teeth, due to failure in their eruption. It has been reported as part of an extremely rare syndrome, named GAPO syndrome. Pseudo-hypoparathyroidism type 1a (PHPT-1a) is a rare condition, characterized by resistance to the parathyroid hormone (PTH), as well as to many other hormones, and resulting in hypocalcemia, hyperphosphatemia, and elevated PTH. We report here the case of a 32-year-old woman with a long-standing history of non-treated hypocalcemia, in the context of an undiagnosed PHPT-1a. She had an intellectual disability, showed clinical features of the Albright hereditary osteodystrophy (AHO) and presented signs of multiple hormone resistances. She received treatment for seizures since the age of six. Examination of her mouth revealed a complete absence of teeth. Treatment of hypocalcemia and hormone deficiencies were started only at 29 years of age. Genetic testing demonstrated the presence of a frameshift variant in the GNAS gene in the proband as well as in her mother. A Single Nucleotide Polymorphism (SNP) array analysis failed to demonstrate pathogenic copy number variants (CNVs) but showed several regions with loss of heterozygosity (LOHs) for a final percentage of 1.75%, compatible with a fifth degree of relationship. Clinical exome sequencing (CES) ruled out any damaging variants in all the teeth agenesis-related genes. In conclusion, although we performed an extensive genetic analysis in search of possible additional gene alterations that could explain the presence of the peculiar phenotypic characteristics observed in our patient, we could not find any additional genetic defects. Our results suggest that the association of genetically confirmed PHPT-1a and complete pseudo-anodontia associated with persistent patchy alopecia areata is a new additional nonclassical feature related to the GNAS pathogenic variant.

B4GALT1 as a New Biomarker of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by progressive scarring of the lung that involves the pulmonary interstitium. The disease may rapidly progress, leading to respiratory failure, and the long-term survival is poor. There are no accurate biomarkers available so far. Our aim was to evaluate the expression of the B4GALT1 in patients with IPF. Analysis of B4GALT1 gene expression was performed in silico on two gene sets, retrieved from the Gene Expression Omnibus database. Expression of B4GALT1 was then evaluated, both at the mRNA and protein levels, on lung specimens obtained from lung biopsies of 4 IPF patients, on one IPF-derived human primary cell and on 11 cases of IPF associated with cancer. In silico re-analysis demonstrated that the B4GALT1 gene was overexpressed in patients and human cell cultures with IPF (p = 0.03). Network analysis demonstrated that B4GALT1 upregulation was correlated with genes belonging to the EMT pathway (p = 0.01). The overexpression of B4GALT1 was observed, both at mRNA and protein levels, in lung biopsies of our four IPF patients and in the IPF-derived human primary cell, in other fibrotic non-lung tissues, and in IPF associated with cancer. In conclusion, our results indicate that B4GALT1 is overexpressed in IPF and could represent a novel marker of this disease.

Interleukin-6 and Outcome of Chronic Hemodialysis Patients with SARS-CoV-2 Pneumonia.

Background and Objectives: Chronic hemodialysis (CHD) patients are at increased risk of SARS-CoV-2 infection and the related complications and mortality of COVID-19 due to the high rate of comorbidities combined with advanced age. This observational study investigated the clinical manifestations of SARS-CoV-2 infection in CHD and the risk factors for patients' death. Materials and Methods: The study included 26 CHD patients with SARS-CoV-2 pneumonia detected by positive RT-PCR on nasopharyngeal swabs and high-resolution computed tomography at hospital admission, aged 71 + 5.9 years, 14 of which (53.8%) were male, 20 (77%) under hemodiafiltration, and 6 (23%) on standard hemodialysis, with a median follow-up of 30 days. Results: Simple logistic regression analysis revealed that the factors associated with a higher risk of death were older age (OR: 1.133; 95%CI: 1.028−1.326, p = 0.0057), IL-6 levels at admission (OR: 1.014; 95%CI: 1.004−1.028, p = 0.0053), and C-reactive protein (OR: 1.424; 95%CI: 1.158−2.044, p < 0.0001). In the multiple logistic regression model, circulating IL-6 values at admission remained the only significant prognosticator of death. The ROC curve indicated the discriminatory cut-off value of 38.20 pg/mL of blood IL-6 for predicting death in chronic hemodialysis patients with SARS-CoV-2 pneumonia (sensitivity: 100%; specificity: 78%; AUC: 0.8750; p = 0.0027). Conclusions: This study identified a threshold of IL-6 levels at hospital admission for death risk in CHD patients with SARS-CoV-2 pneumonia. This might represent a valuable outcome predictor, feasibly better than other clinical, radiological, or laboratory parameters and preceding the IL-6 peak, which is unpredictable.

Identification of a miRNA-based non-invasive predictive biomarker of response to target therapy in BRAF-mutant melanoma.

Rationale: Metastatic melanoma is the most aggressive and dangerous form of skin cancer. The introduction of immunotherapy with Immune checkpoint Inhibitors (ICI) and of targeted therapy with BRAF and MEK inhibitors for BRAF mutated melanoma, has greatly improved the clinical outcome of these patients. Nevertheless, response to therapy remains highly variable and the development of drug resistance continues to be a daunting challenge. Within this context there is a need to develop diagnostic tools capable of predicting response or resistance to therapy in order to select the best therapeutic approach. Over the years, accumulating evidence brought to light the role of microRNAs (miRNAs) as disease biomarkers. Methods: In particular, the detection of miRNAs in whole blood or specific blood components such as serum or plasma, allows these molecules to be good candidates for diagnosis, prognosis and for monitoring response to anticancer therapy. In this paper, we evaluated circulating basal levels of 6 previously identified miRNAs in serum samples of 70 BRAF-mutant melanoma patients before starting targeted therapy. Results: Results show that the circulating levels of the oncosuppressor miR-579-3p and of the oncomiR miR-4488 are able to predict progression free survival (PFS) but not overall survival (OS). Most importantly, we observed that the best predictor of disease outcome is represented by the ratio of circulating miR-4488 vs. miR-579-3p (miRatio). Finally, the combination of the Lactate dehydrogenase (LDH) blood levels with the two circulating miRNAs alone or together did not produce any improvement in predicting PFS indicating that miR-579-3p and miR-4488 are independent predictors of PFS as compared to LDH. Conclusions: All together these data underscored the relevance of circulating miRNAs as suitable tools to predict therapy response in melanoma and maybe further developed as companion diagnostics in the clinic.

A New SMAD4 Splice Site Variant in a Three-Generation Italian Family with Juvenile Polyposis Syndrome.

Juvenile polyposis syndrome (JPS) is an autosomal dominant disorder characterized by hyperplastic polyps in the upper and lower gastrointestinal (GI) tract with a high risk of developing GI cancers. We have described a three-generation Italian family with all the spectrum of SMAD4 phenotype. A multigene panel test was performed on the genomic DNA of the proband by next-generation sequencing, including genes related to hereditary GI tumor syndromes. Molecular analysis revealed the presence of the c.1140-2A>G substitution in the SMAD4 gene, a novel splice variant that has never been described before. Our family is remarkable in that it illustrates the variable expressivity of the SMAD4 phenotype within the same family. The possibility of phenotype variability should also be considered within family members carrying the same mutation. In JPS, a timely genetic diagnosis allows clinicians to better manage patients and to provide early surveillance and intervention for their asymptomatic mutated relatives in the early decades of life.

"Monkeypox: What Do You Know about That?" Italian Adults' Awareness of a New Epidemic.

In the course of 2022, an epidemic of monkeypox (MPX) arose worldwide. In order to assess the level of knowledge of the Italian adults regarding the new emerging disease, its prevention and their level of worrying and the acceptance of a possible vaccination, a web questionnaire was spread nationwide. A total of 1352 individuals (mean age 54.5 ± 13.4, 52.4% males) completed the questionnaire. Only 26. 7% of the sample wereaware of the burden of the epidemic at the moment of the investigation; 47.1% were not able to identify the transmission route, nor the main symptoms (48.9%); and 54.2% were hesitant toward a possible MPXV vaccination, mainly because of a lack of confident in vaccines (38.5%). A low level of worrying about the disease was registered (mean score 2.3 ± 1.2 on a 5-point scale). In the regression analysis performed considering MPX knowledge as outcome, a lower level of knowledge was associated with higher age (OR 1.378, CI95% 0.998-1.904), working or studying in a nonhealthcare setting (OR 0.046, CI95% 0.033-0.066), being single (OR 0.624, CI95% 0.455-0.856) and having mass media as the main source of information (OR 0.332, CI95% 0.158-0.696). These findings indicate as of the time of this investigation, the communication about the MPXV epidemic was not effective in determining a good level of knowledge about the disease and its transmission among Italian adults. This highlights the need to improve risk communication strategies.