Functional characterization of a CDKN1B mutation in a Sardinian kindred with multiple endocrine neoplasia type 4 (MEN4)

Inactivating germline mutations of the CDKN1B gene, encoding for the nuclear cyclin-dependent kinase inhibitor p27kip1 protein, have been reported in patients with multiple endocrine neoplasia type 4 (MEN4), a MEN1-like phenotype without MEN1 mutations. The aim of this study was to in vitro characterize the germline CDKN1B mutation c.374_375delCT (S125X) we detected in a patient with MEN4. The proband was affected by multiglandular primary hyperparathyroidism and gastro-entero-pancreatic tumors. We carried out subcellular localization experiments transfecting into eukaryotic HeLa and GH3 cell lines plasmid vectors expressing the CDKN1B wild type (wt) or mutant cDNA. Western blot studies showed that fusion proteins were expressed at equal levels. The mutated protein was shorter compared to the wt protein and lacked the highly conserved C-terminal domain, which includes the bipartite nuclear localization signal at amino acids 152/153 and 166/168. In HeLa and GH3 cells wt p27 localized in the nucleus whereas the p27_S125X protein was retained in the cytoplasm predicting the loss of tumor suppressive function. The proband's tumoral parathyroid tissue did not show allelic loss, since wt and mutant alleles were both present by sequencing the somatic DNA. Immunohistochemistry showed a complete loss of nuclear p27 expression in the parathyroid adenoma removed by the patient at the second surgery. In conclusion, our study confirms the pathogenic role of the c.374_375delCT CDKN1B germline mutation in a patient with MEN4

Mutational and large deletion study of genes implicated in hereditary forms of primary hyperparathyroidism and correlation with clinical features

The aim of this study was to carry out genetic screening of the MEN1, CDKN1B and AIP genes, both by direct sequencing of the coding region and multiplex ligation-dependent probe amplification (MLPA) assay in the largest monocentric series of Italian patients with Multiple Endocrine Neoplasia type 1 syndrome (MEN1) and Familial Isolated Hyperparathyroidism (FIHP). The study also aimed to describe and compare the clinical features of MEN1 mutation-negative and mutation-positive patients during long-term follow-up and to correlate the specific types and locations of MEN1 gene mutations with onset and aggressiveness of the main MEN1 manifestations. A total of 69 index cases followed at the Endocrinology Unit in Pisa over a period of 19 years, including 54 MEN1 and 15 FIHP kindreds were enrolled. Seven index cases with MEN1 but MEN1 mutation-negative, followed at the University Hospital of Cagliari, were also investigated. FIHP were also tested for CDC73 and CaSR gene alterations. MEN1 germline mutations were identified in 90% of the index cases of familial MEN1 (F-MEN1) and in 23% of sporadic cases (S-MEN1). MEN1 and CDC73 mutations accounted for 13% and 7% of the FIHP cohort, respectively. A CDKN1B mutation was identified in one F-MEN1. Two AIP variants of unknown significance were detected in two MEN1-negative S-MEN1. A MEN1 positive test best predicted the onset of all three major MEN1-related manifestations or parathyroid and gastro-entero-pancreatic tumors during follow-up. A comparison between the clinical characteristics of F and S-MEN1 showed a higher prevalence of a single parathyroid disease and pituitary tumors in sporadic compared to familial MEN1 patients. No significant correlation was found between the type and location of MEN1 mutations and the clinical phenotype. Since all MEN1 mutation-positive sporadic patients had a phenotype resembling that of familial MEN1 (multiglandular parathyroid hyperplasia, a prevalence of gastro-entero-pancreatic tumors and/or the classic triad) we might hypothesize that a subset of the sporadic MEN1 mutation-negative patients could represent an incidental coexistence of sporadic primary hyperparathyroidism and pituitary tumors or a MEN1 phenocopy, in our cohort, as in most cases described in the literature

A synthetic peptide that prevents cAMP regulation in mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels

Binding of TRIP8b to the cyclic nucleotide binding domain (CNBD) of mammalian hyperpolarization-activated cyclic nucleotide-gated (HCN) channels prevents their regulation by cAMP. Since TRIP8b is expressed exclusively in the brain, we envisage that it can be used for orthogonal control of HCN channels beyond the central nervous system. To this end, we have identified by rational design a 40-aa long peptide (TRIP8bnano) that recapitulates affinity and gating effects of TRIP8b in HCN isoforms (hHCN1, mHCN2, rbHCN4) and in the cardiac current If in rabbit and mouse sinoatrial node cardiomyocytes. Guided by an NMR-derived structural model that identifies the key molecular interactions between TRIP8bnano and the HCN CNBD, we further designed a cell-penetrating peptide (TAT-TRIP8bnano) which successfully prevented β-adrenergic activation of mouse If leaving the stimulation of the L-type calcium current (ICaL) unaffected. TRIP8bnano represents a novel approach to selectively control HCN activation, which yields the promise of a more targeted pharmacology compared to pore blockers

Elective cancer surgery in COVID-19-free surgical pathways during the SARS-CoV-2 pandemic: An international, multicenter, comparative cohort study

PURPOSE As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19–free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19–free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19–free surgical pathways. Patients who underwent surgery within COVID-19–free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19–free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score–matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19–free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION Within available resources, dedicated COVID-19–free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Elective Cancer Surgery in COVID-19-Free Surgical Pathways During the SARS-CoV-2 Pandemic: An International, Multicenter, Comparative Cohort Study.

PURPOSE: As cancer surgery restarts after the first COVID-19 wave, health care providers urgently require data to determine where elective surgery is best performed. This study aimed to determine whether COVID-19-free surgical pathways were associated with lower postoperative pulmonary complication rates compared with hospitals with no defined pathway. PATIENTS AND METHODS: This international, multicenter cohort study included patients who underwent elective surgery for 10 solid cancer types without preoperative suspicion of SARS-CoV-2. Participating hospitals included patients from local emergence of SARS-CoV-2 until April 19, 2020. At the time of surgery, hospitals were defined as having a COVID-19-free surgical pathway (complete segregation of the operating theater, critical care, and inpatient ward areas) or no defined pathway (incomplete or no segregation, areas shared with patients with COVID-19). The primary outcome was 30-day postoperative pulmonary complications (pneumonia, acute respiratory distress syndrome, unexpected ventilation). RESULTS: Of 9,171 patients from 447 hospitals in 55 countries, 2,481 were operated on in COVID-19-free surgical pathways. Patients who underwent surgery within COVID-19-free surgical pathways were younger with fewer comorbidities than those in hospitals with no defined pathway but with similar proportions of major surgery. After adjustment, pulmonary complication rates were lower with COVID-19-free surgical pathways (2.2% v 4.9%; adjusted odds ratio [aOR], 0.62; 95% CI, 0.44 to 0.86). This was consistent in sensitivity analyses for low-risk patients (American Society of Anesthesiologists grade 1/2), propensity score-matched models, and patients with negative SARS-CoV-2 preoperative tests. The postoperative SARS-CoV-2 infection rate was also lower in COVID-19-free surgical pathways (2.1% v 3.6%; aOR, 0.53; 95% CI, 0.36 to 0.76). CONCLUSION: Within available resources, dedicated COVID-19-free surgical pathways should be established to provide safe elective cancer surgery during current and before future SARS-CoV-2 outbreaks

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

The Subtle Balance between Lipolysis and Lipogenesis: A Critical Point in Metabolic Homeostasis

Excessive accumulation of lipids can lead to lipotoxicity, cell dysfunction and alteration in metabolic pathways, both in adipose tissue and peripheral organs, like liver, heart, pancreas and muscle. This is now a recognized risk factor for the development of metabolic disorders, such as obesity, diabetes, fatty liver disease (NAFLD), cardiovascular diseases (CVD) and hepatocellular carcinoma (HCC). The causes for lipotoxicity are not only a high fat diet but also excessive lipolysis, adipogenesis and adipose tissue insulin resistance. The aims of this review are to investigate the subtle balances that underlie lipolytic, lipogenic and oxidative pathways, to evaluate critical points and the complexities of these processes and to better understand which are the metabolic derangements resulting from their imbalance, such as type 2 diabetes and non alcoholic fatty liver disease

ATTIVITA' ANTIATEROGENA DI UNA NUOVA CLASSE DI COMPOSTI: I DERIVATI DEL PIRIDO(1,2-a)PIRIMIDIN-4-ONE.

L’Aterosclerosi è una malattia caratterizzata da placche fibrolipidiche nelle arterie e attualmente rappresenta una delle cause più importanti di malattie cardiovascolari. La comprensione della patogenesi del processo aterosclerotico è dunque di cruciale importanza, al fine di delineare target terapeutici efficaci. In questo processo patogenetico, l’infiammazione gioca un ruolo chiave. Stimoli infiammatori inducono l’“attivazione delle cellule endoteliali” cioè un’alterazione fenotipica che include la neo-espressione di molecole di adesione come VCAM-1 ed il rilascio di mediatori chimici derivanti dal metabolismo dell’ acido arachidonico per mezzo degli enzimi lipossigenasi e ciclossigenasi, eventi accompagnati dalla produzione di radicali liberi dell’ossigeno (ROS). Obiettivo: Lo scopo di questa tesi era la valutazione “in vitro” delle capacità antiinfiammatorie, antiossidanti e antiproliferative di alcune molecole di nuova sintesi, derivati del pirido (1,2-a)pirimidin-4-one, sulle cellule endoteliali. Metodi: Sono state utilizzate cellule endoteliali provenienti da cordoni ombelicali umani su cui sono stati eseguiti i seguenti saggi: Saggio dell’Amido-Black per la valutazione della tossicità cellulare. Saggio immunoenzimatico di superficie cellulare per determinare l’espressione di VCAM-1. Dosaggio immunoenzimatico competitivo per la produzione di prostacicline. Saggio per la valutazione di ROS intracellulari mediante l’uso della carbossi-2’,7’-diclorodidrofluoresceina di acetato bis (acetossimetil)-estere (C-DCFH-DA). Risultati: Noi abbiamo valutato tre diverse concentrazioni (0.1µM, 1µM e 10µM) di 6 derivati del pirido-(1,2-a)pirimidin-4-one da noi siglati: DB87-A, DB92-B, DB94-C, DB96-D, DB99-E, DB103-F. Le concentrazioni da noi valutate di ogni composto sono risultate compatibili con la vitalità cellulare. Dalla valutazione dell’espressione di VCAM-1 indotta dalla citochina infiammatoria TNF-a (10ng/mL) risulta che le sostanze DB87-A, DB92-B e DB99-E non ne inibiscono l’espressione indotta; le sostanze DB94-C e DB96-D la inibiscono debolmente ma solo alla concentrazione più alta (10µM). Invece la sostanza DB103-F ha una significativa capacità di inibire l’espressione di VCAM-1, la produzione di ROS intracellulari e la produzione di prostacicline infiammatorie in modo concentrazione dipendente. Conclusioni: Il derivato DB103-F ha manifestato proprietà antiaterogene, antiinfiammatorie ed antiossidanti a concentrazioni compatibili con un suo utilizzo in vivo