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The BRAF gene is mutated in a plethora of human cancers. The majority of such molecular lesions result in the expression of a constitutively active BRAF variant (BRAF V600E ) which continuously bolsters cell proliferation. Although we recently addressed the early effects triggered by BRAF V600E -activation, the specific contribution of ERK1 and ERK2 in BRAF V600E -driven responses in vivo has never been explored. Here we describe the first murine model suitable for genetically dissecting the ERK1/ERK2 impact in multiple phenotypes induced by ubiquitous BRAF V600E -expression. We unveil that ERK1 is dispensable for BRAF V600E -dependent lifespan shortening and for BRAF V600E -driven tumor growth. We show that BRAF V600E -expression provokes an ERK1-independent lymphocyte depletion which does not rely on p21 CIP1 -induced cell cycle arrest and is unresponsive to ERK-chemical inhibition. Moreover, we also reveal that ERK1 is dispensable for BRAF V600E -triggered cytotoxicity in lungs and that ERK-chemical inhibition abrogates some of these detrimental effects, such as DNA damage, in Club cells but not in pulmonary lymphocytes. Our data suggest that ERK1/ERK2 contribution to BRAF V600E -driven phenotypes is dynamic and varies dependently on cell type, the biological function, and the level of ERK-pathway activation. Our findings also provide useful insights into the comprehension of BRAF V600E -driven malignancies pathophysiology as well as the consequences in vivo of novel ERK pathway-targeted anti-cancer therapies., (© 2024. The Author(s).)

RNA-DNA covalent hybrids (RDHs) are widely employed in biology. Although RDHs can be manufactured, the synthesis of molecules longer than 120 nucleotides is challenging. Here, we present a protocol for the generation and purification of high-grade purified high-molecular-weight 5'-RNA-DNA-3' hybrids. We describe steps for preparing oligos and buffers, ligation reaction, and high-performance liquid chromatography-based RDH purification. This protocol is executable in standard molecular biology laboratories., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Evidence supporting the effectiveness of Antimicrobial Stewardship (AMS) Programs in the emergency department (ED) setting is limited. We conducted a prospective cohort study to assess the efficacy of an AMS program in an ED and a short-stay observation unit. The intervention included periodic prospective audits (twice a week), conducted by four infectious disease consultants. Primary outcomes included the difference in the hospital mortality rate, antibiotic consumption, and the incidence of bloodstream infections (BSI) caused by multidrug resistant (MDR) bacteria, before March 2020-February 2021 and after March 2021-February 2022 when the program was implemented. Interrupted time-series analysis was performed to assess the effect of our program. During the 12-month program, we performed 152 audits and evaluated 366 antibiotic therapies out of a total of 853 patients admitted. In the intervention period, we observed a non-statistically significant decrease in total antibiotic consumption, with a change in level of - 31.2 defined daily dose/100 patient-days (PD) (p = 0.71). Likewise, we found no significant variations in the rate of BSI due to MDR Gram-positive (CT - 0.02 events/PD, p = 0.84), MDR Gram-negative bacteria (CT 0.08, p = 0.71), or Candida spp. (CT 0.008, p = 0.86). Conversely, we found a significant decrease in the mortality rate between the pre- and post-intervention periods (- 1.98 deaths/100 PD, CI - 3.9 to - 0.007, p = 0.049). The Antibiotic Stewardship Program in the ED was associated with a significant decrease in the mortality rate. More high-quality studies are needed to determine the most effective ASP strategies in this unique setting., (© 2023. The Author(s).)

Elevated intracranial pressure (ICP) is defined as a cerebrospinal fluid (CSF) opening pressure (OP) greater than 25 cmH2O. When a diagnostic lumbar puncture is performed it is useful to estimate also intracranial pressure. To do this it is required a presence of pressure gauges, which are currently the gold standard, not available in most resource-constrained contexts. We decided to evaluate whether it is possible to estimate it simply by counting the drops of cerebrospinal liquor, which are collected after lumbar puncture, according to Poiseuille's law. Was examined a sample of 52 patients, aged between 18 and 85 years, belonging to the emergency room of "Santa Maria delle Grazie" Hospital in Pozzuoli (Naples) who needed a diagnostic lumbar puncture (LP). The ICP was initially measured using a standard narrow-gauge manometer by attaching it to the spinal needle. After removing the pressure gauge, the number of drops of cerebrospinal fluid flowing from the spinal needle in 30  seconds was counted. A statistical analysis was made with linear regression and ROC analysis. OP as measured by standard manometry was raised on 17 occasions with CSF drop rate median of 47 drops/30 seconds and range 30-74 drops/30 seconds. OP was normal on 35 occasions with CSF drop rate median of 23 drops/30 seconds  with range of 14-34 drops/30 seconds. A linear regression analysis was performed which resulted in a Pearson correlation of 0.936 an adjusted R square of 0.874 (see Fig. 1). Analysis through ANOVA documented an F of 355.301 with p < 0.01 and Dubin Watson of 1.642. The analysis through ROC showed an AUC of 0.980, with a sensitivity of 100% and a specificity of 91% if chosen as a limit, 29 drops in 30 seconds  (Youden Index of 0.9140). Therefore, we have concluded, that although there are several precautions, like patient's position, it is technically feasible to indirectly estimate cerebrospinal fluid pressure with good accuracy by counting the drops of cerebrospinal fluid flowing from a 22 G spinal needle., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Chromobox Protein 3 (CBX3) overexpression is a common event occurring in cancer, promotes cancer cell proliferation and represents a poor prognosis marker in a plethora of human cancers. Here we describe that a wide spectrum of human cancers harbors a co-amplification of CBX3 gene with either EGFR or RAC1, which yields a statistically significant increase of both mRNA and protein levels of CBX3, EGFR and RAC1. We also reveal that the simultaneous overexpression of CBX3, RAC1 and EGFR gene products correlates with a worse prognosis compared to the condition when CBX3, RAC1 and EGFR are singularly upregulated. Furthermore, we also show that a co-occurrence of low-grade amplification, in addition to high-grade amplification, between CBX3 and EGFR or RAC1 is associated with a reduced patient lifespan. Finally, we find that CBX3 and RAC1/EGFR genetically interact in the model organism Drosophila melanogaster, suggesting that the simultaneous overexpression as well as well the co-occurrence of high- or low-grade copy number alterations in these genes is not accidental and could reflect evolutionarily conserved functional relationships., (© 2023. Cell Death Differentiation Association (ADMC).)

Objectives: An increasing number of COVID-19 patients were treated with continuous positive airways pressure (CPAP). To evaluate the clinical effects of personalized positive end-expiratory pressure (PEEP) compared to standard fixed PEEP in COVID-19 patients requiring CPAP., Methods: This is a single center, prospective, randomized clinical study. Sixty-three COVID-19 patients with hypoxemic respiratory failure and bilateral pneumonia were randomized in two Groups: Group A received CPAP with fixed PEEP of 10 cm H 2 O, Group B performed the "PEEP trial", that consists in the evaluation of best PEEP defined as the PEEP value that precedes the echographic appearance of "lung pulse" determining a PaO 2 /FiO 2 increase. Primary outcome was composite in-hospital mortality + intubation, secondary outcome was the percentage increase of PaO 2 /FiO 2 . As safety indicator, the incidence of pneumothorax was collected., Results: Thirty-two patients were enrolled in Group A and 31 in Group B . The two groups were comparable for clinical characteristics and laboratory parameters. The primary outcome occurred in 36 (57.1 %) patients: 23 (71.8 %) in Group A and 13 (41.9 %) in Group B (p<0.01). Mortality was higher in Group A (53.1 vs. 19.3 %, p<0.01), while intubation rate was comparable between groups. Group B showed a higher PaO 2 /FiO 2 increase than Group A (34.9 vs. 13.1 %, p<0.01). Five cases of pneumothorax were reported in Group A , none in Group B ., Conclusions: Lung ultrasound-guided PEEP trial is associated with lower mortality in COVID-19 patients treated with CPAP. Identifying the best PEEP is useful to increase oxygenation and reduce the incidence of complications., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Background: Type 2 diabetes mellitus is associated with a cluster of lipid and apolipoprotein abnormalities which increase the risk for atherosclerotic cardiovascular disease. The aim of this study was to evaluate the adherence to guidelines-oriented dyslipidemia treatment in diabetic patients and to assess the efficacy of a territorial goal-oriented program., Methods: One thousand seventy-one diabetic patients at very high cardiovascular risk were enrolled in this prospective study. They performed a clinical-laboratory follow-up program, received lifestyle recommendations and optimization of their antihyperlipidemic therapies. At the beginning and the 3-month follow-up visit, LDL-c data were collected, and further therapies were prescribed to the patients that did not reach the target. After 12 month follow-up, LDL-c data were collected again., Results: Diabetic patients significantly improved mean LDL cholesterol levels during one-year follow-up (LDLc mean value 135 mg/dL at baseline, 60 mg/dL at the end of the study), obtaining a greater reduction compared to non-diabetic patients participating in the same program. Accordingly, the percentage of patients that reached the lipid target was significantly higher in diabetic patients after 3-months and 12- follow-ups (P<0.05). Diabetic patients assuming statins, both in monotherapy and in combination with ezetimibe, increased during the follow-up (74.1% at the enrolment vs. 88.2% one year later). GLP1ra-treated patients achieved the greatest reduction in cholesterol levels compared to baseline., Conclusions: The results of the study recommend encouraging strategies and appropriate treatments to achieve a targeted lipid profile in diabetic patients at very high cardiovascular risk.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer death. Tumor progression depends on interactions of cancer cells with the tumor microenvironment. Here, we find increased copy number and mRNA expression of the catalytic subunit of telomerase, TERT, in tumors from NSCLC patients, contributing to a lower survival. Moreover, TERT expression in NSCLC patients from the TCGA cohort is mainly associated to the reduced infiltration of CD8 + T lymphocytes, as well as to increased infiltration of myeloid-derived suppressor cells (MDSCs). We also show that TERT deficiency and dysfunctional telomeres induced by 6-thio-dG treatment in mice reduced lung tumor implantation and vascularization, increased DNA damage response, cell cycle arrest and apoptosis, as well as reduced proliferation, inflammation, lung tumor immunosupression and invasion upon induction of a Lewis lung carcinoma (LLC). Furthermore, 6-thio-dG-treated human NSCLC xenografts exhibited increased telomere damage, cell cycle arrest and apoptosis, as well as reduced proliferation, resulting in a reduced tumor growth. Our results show that targeting telomeres might be an effective therapeutic strategy in NSCLC., (© 2023. The Author(s).)

Background: Epilepsy has been associated with an increased risk of cardiovascular events. Anti-seizure medication (ASM) may contribute to vascular risk by several mechanisms, including increased homocysteine levels. This study aims to assess the global vascular burden in hyperhomocysteinemic people with epilepsy (PWE) on long-term ASM before and after folic acid supplementation and in subgroups of PWE treated with single enzyme-inducing or single non-enzyme inducing ASM., Methods: One hundred and seventy-four hyperhomocysteinemic (HHcy) PWE who met the inclusion criteria were enrolled. Carotid Doppler ultrasonography, FMD and ultrasound assessment of the brachial artery properties at the baseline and after 90 days of folic acid supplementation were performed. The vascular biomarkers MMP-9 and TIMP-1 were also detected., Results: After folic acid supplementation, in HHcy patients homocysteine levels reduced from 26.8 ± 10.5 to 20.2 ± 5.3 μmol/L, carotid Intima-Media-Thickness reduced from 0.83+0.06 mm to 0.79±0.05 mm, and FMD, distensibility coefficient and β-stiffness improved (p < 0.05). Moreover, MMP-9 and TIMP-1 reduced after supplementation (p < 0.05). PWE treated with a single enzyme-inducing ASM showed an impairment of vascular parameters compared to patients treated with non-enzyme inducing ASM., Conclusions: The results highlight the importance of assessing homocysteine levels and estimating the cardiovascular risk of PWE, preferring non-enzyme inducing ASM in high cardiovascular-risk patients. An adequate correction of homocysteine levels with folate supplementation should be considered to improve the cardiovascular profile., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest/competing interests., (Published by Elsevier Ltd.)

TRF1 is an essential component of the telomeric protective complex or shelterin. We previously showed that dysfunctional telomeres in alveolar type II (ATII) cells lead to interstitial lung fibrosis. Here, we study the lung pathologies upon telomere dysfunction in fibroblasts, club and basal cells. TRF1 deficiency in lung fibroblasts, club and basal cells induced telomeric damage, proliferative defects, cell cycle arrest and apoptosis. While Trf1 deletion in fibroblasts does not spontaneously lead to lung pathologies, upon bleomycin challenge exacerbates lung fibrosis. Unlike in females, Trf1 deletion in club and basal cells from male mice resulted in lung inflammation and airway remodeling. Here, we show that depletion of TRF1 in fibroblasts, Club and basal cells does not lead to interstitial lung fibrosis, underscoring ATII cells as the relevant cell type for the origin of interstitial fibrosis. Our findings contribute to a better understanding of proper telomere protection in lung tissue homeostasis., (© 2022. The Author(s).)

The association between LDL-c levels and cardiovascular outcomes suggests tailoring lipid-lowering therapies according to total cardiovascular risk. We aimed to evaluate the adherence to guidelines-oriented dyslipidaemia's treatment in an outpatient population referring to ARCA cardiologists, and assess the efficacy of treatment's optimization for each specific level of risk. Three thousand seventy-five patients enrolled in this prospective study were classified according to cardiovascular risk category, and their therapies were optimized. At the beginning and the 3 month follow-up visit, LDL-c data were collected, and further therapies were prescribed to the patients that did not reach the target. A significant LDL-c reduction was observed in all subgroups at different cardiovascular risk at the end of the study (p < 0.05). The number of patients assuming statins, both in monotherapy and in combination with ezetimibe, increased during the follow-up (63% at the enrollment vs 89% after 12 months). At the enrollment, only 1.4% of patients were treated with PCSK-9 inhibitors while after 12 months the percentage increased both in high (5.8%) and very high-risk (18.4%) patients. At the beginning of the study, only 698/3075 patients (22.7%) reached lipid targets. At the end of the study, carried out by the referring cardiologists in the pertaining healthcare districts and specifically aimed to control the lipid profile, the percentage of patients on target increased in all risk categories (68.5%). Our results suggest carefully implementing measures that encourage outpatients and their cardiologists to achieve the targeted lipid profile according to cardiovascular risk., (© 2021. The Author(s).)

The BRAF gene is frequently mutated in cancer. The most common genetic mutation is a single nucleotide transition which gives rise to a constitutively active BRAF kinase (BRAF V600E ) which in turn sustains continuous cell proliferation. The study of BRAF V600E murine models has been mainly focused on the role of BRAF V600E in tumor development but little is known on the early molecular impact of BRAF V600E expression in vivo. Here, we study the immediate effects of acute ubiquitous BRAF V600E activation in vivo. We find that BRAF V600E elicits a rapid DNA damage response in the liver, spleen, lungs but not in thyroids. This DNA damage response does not occur at telomeres and is accompanied by activation of the senescence marker p21 CIP1 only in lungs but not in liver or spleen. Moreover, in lungs, BRAF V600E provokes an acute inflammatory state with a tissue-specific recruitment of neutrophils in the alveolar parenchyma and macrophages in bronchi/bronchioles, as well as bronchial/bronchiolar epithelium transdifferentiation and development of adenomas. Furthermore, whereas in non-tumor alveolar type II (ATIIs) pneumocytes, acute BRAF V600E induction elicits rapid p53-independent p21 CIP1 activation, adenoma ATIIs express p53 without resulting in p21 CIP1 gene activation. Conversely, albeit in Club cells BRAF V600E -mediated proliferative cue is more exacerbated compared to that occurring in ATIIs, such oncogenic stimulus culminates with p21 CIP1 -mediated cell cycle arrest and apoptosis. Our findings indicate that acute BRAF V600E expression drives an immediate induction of DNA damage response in vivo. More importantly, it also results in rapid differential responses of cell cycle and senescence-associated proteins in lung epithelia, thus revealing the early molecular changes emerging in BRAF V600E -challenged cells during tumorigenesis in vivo., (© 2022. The Author(s).)

Purpose: Coronavirus infection disease 2019 (COVID-19) causes in 10% of patients a severe respiratory distress syndrome managed with invasive mechanical ventilation (IMV), sometimes difficult to wean. The role of tracheotomy is debated for the possible risks for patients and staff. We are going to describe here our experience with surgical tracheotomy in COVID-19 positive patients., Methods: We enrolled all intensive care unit (ICU) patients requiring longer than 10 days of IMV. Demographic, clinical, respiratory, complications, and outcomes data were collected, in a particular length of weaning from sedation and IMV, in-ICU and in-hospital mortality rate. All healthcare operators involved were tested for SARS-CoV2 by pharyngeal swab and blood test (antibody test)., Results: 13 out of 68 ICU patients (19.1%) underwent surgical tracheotomy after a median intubation period of 14 days. The mean age was 60 (56-65) years. 85% were male patients. Postoperative mild bleeding was seen in 30.7%, pneumothorax in 7.7%. Mean weaning from sedation required 3 days, 19 days from IMV. In-ICU and in-hospital COVID-infection-related mortality was 23.1% and 30.7%, respectively. None of the healthcare operators was found SARS-CoV2 positive during the period of the study., Conclusions: In COVID-19 pandemic surgical tracheotomy enables to wean from sedation and subsequently from IMV in a safe way for both patients and personnel., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)