PNPLA3 rs738409 I748M is associated with steatohepatitis in 434 non-obese subjects with hepatitis C

Background The PNPLA3/Adiponutrin rs738409 C/G single nucleotide polymorphism is associated with the severity of steatosis, steatohepatitis and fibrosis in patients with non-alcoholic fatty liver disease, as well as the severity of steatosis and fibrosis in patients with chronic hepatitis C (CHC). Aim To test in genotype 1(G1)-CHC patients, the putative association between the PNPLA3 variant and histological features of steatohepatitis, as well as their impact on the severity of fibrosis. Methods Four hundred and thirty-four consecutively biopsied Caucasian G1-CHC patients were genotyped for PNPLA3 rs738409, its effect evaluated by using an additive model. Histological features of steatohepatitis in CHC were assessed using the Bedossa classification. Hepatic expression of PNPLA3 mRNA was evaluated in 63 patients. Results The prevalence of steatohepatitis increased from 16.5% in patients with PNPLA3 CC, to 23.2% in CG and 29.2% in the GG genotype (P = 0.02). By multiple logistic regression, PNPLA3 genotype (OR 1.54, 95% CI 1.03-2.30, P = 0.03), together with age (OR 1.03, 95% CI 1.00-1.05, P = 0.02), BMI 65 30 (OR 2.06, 95% CI 1.04-4.10, P = 0.03) and homoeostasis model assessment (HOMA, OR 1.18, 95% CI 1.04-1.32, P = 0.006) were independently linked to steatohepatitis. When stratifying for obesity, PNPLA3 was associated with NASH in non-obese patients only (12.0% in CC vs. 18.3% in CG vs. 27.3% in GG, P = 0.01), including after correction for metabolic confounders (OR 2.06, 95% CI 1.26-3.36, P = 0.004). We showed an independent association between steatohepatitis (OR 2.05, 95% CI 1.05-4.02, P = 0.003) and severe fibrosis. Higher liver PNPLA3 mRNA was associated both with the severity of steatosis (adjusted P = 0.03) and steatohepatitis after adjusting for gender, age, BMI and HOMA (P = 0.002). Conclusion In patients with genotype 1 hepatitis C, the PNPLA3 G variant is associated with a higher risk of steatosis severity and steatohepatitis, particularly among non-obese subjects

Expression pattern of perilipins in human brain during aging and in Alzheimer's disease

Aims: Perilipins are conserved proteins that decorate intracellular lipid droplets and are essential for lipid metabolism. To date, there is limited knowledge on their expression in human brain or their involvement in brain aging and neurodegeneration. The aim of this study was to characterise the expression levels of perilipins (Plin1–Plin5) in different cerebral areas from subjects of different age, with or without signs of neurodegeneration. Methods: We performed real-time RT-PCR, western blotting, immunohistochemistry and confocal microscopy analyses in autoptic brain samples of frontal and temporal cortex, cerebellum and hippocampus from subjects ranging from 33 to 104 years of age, with or without histological signs of neurodegeneration. To test the possible relationship between Plins and inflammation, correlation analysis with IL-6 expression was also performed. Results: Plin2, Plin3 and Plin5, but not Plin1 and Plin4, are expressed in the considered brain areas with different intensities. Plin2 appears to be expressed more in grey matter, particularly in neurons in all the areas analysed, whereas Plin3 and Plin5 appear to be expressed more in white matter. Plin3 seems to be expressed more in astrocytes. Only Plin2 expression is higher in old subjects and patients with early tauopathy or Alzheimer's disease and is associated with IL-6 expression. Conclusions: Perilipins are expressed in human brain but only Plin2 appears to be modulated with age and neurodegeneration and linked to an inflammatory state. We propose that the accumulation of lipid droplets decorated with Plin2 occurs during brain aging and that this accumulation may be an early marker and initial step of inflammation and neurodegeneration

Circulating cell-free DNA and circulating tumor cells as prognostic and predictive biomarkers in advanced non-small cell lung cancer patients treated with first-line chemotherapy

Cell-free DNA (cfDNA) and circulating tumor cells (CTCs) are promising prognostic and predictive biomarkers in non-small cell lung cancer (NSCLC). In this study, we examined the prognostic role of cfDNA and CTCs, in separate and joint analyses, in NSCLC patients receiving first line chemotherapy. Seventy-three patients with advanced NSCLC were enrolled in this study. CfDNA and CTC were analyzed at baseline and after two cycles of chemotherapy. Plasma cfDNA quantification was performed by quantitative PCR (qPCR) whereas CTCs were isolated by the ScreenCell Cyto (ScreenCell, Paris, France) device and enumerated according to malignant features. Patients with baseline cfDNA higher than the median value (96.3 hTERT copy number) had a significantly worse overall survival (OS) and double the risk of death (hazard ratio (HR): 2.14; 95% confidence limits (CL) = 1.24\u20133.68; p-value = 0.006). Conversely, an inverse relationship between CTC median baseline number (6 CTC/3 mL of blood) and OS was observed. In addition, we found that in patients reporting stable disease (SD), the baseline cfDNA and CTCs were able to discriminate patients at high risk of poor survival. cfDNA demonstrated a more reliable biomarker than CTCs in the overall population. In the subgroup of SD patients, both biomarkers identified patients at high risk of poor prognosis who might deserve additional/alternative therapeutic interventions

Oropharyngeal Microbiome Profiled at Admission is Predictive of the Need for Respiratory Support Among COVID-19 Patients [preprint]

The clinical course of infection due to respiratory viruses such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2), the causative agent of Coronavirus Disease 2019 (COVID-19) is thought to be influenced by the community of organisms that colonizes the upper respiratory tract, the oropharyngeal microbiome. In this study, we examined the oropharyngeal microbiome of suspected COVID-19 patients presenting to the Emergency Department and an inpatient COVID-19 unit with symptoms of acute COVID-19. Of 115 enrolled patients, 74 were confirmed COVID-19+ and 50 had symptom duration of 14 days or less; 38 acute COVID-19+ patients (76%) went on to require respiratory support. Although no microbiome features were found to be significantly different between COVID-19+ and COVID-19-patients, when we conducted random forest classification modeling (RFC) to predict the need of respiratory support for the COVID-19+ patients our analysis identified a subset of organisms and metabolic pathways whose relative abundance, when combined with clinical factors (such as age and Body Mass Index), was highly predictive of the need for respiratory support (F1 score 0.857). Microbiome Multivariable Association with Linear Models (MaAsLin2) analysis was then applied to the features identified as predicative of the need for respiratory support by the RFC. This analysis revealed reduced abundance of Prevotella salivae and metabolic pathways associated with lipopolysaccharide and mycolic acid biosynthesis to be the strongest predictors of patients requiring respiratory support. These findings suggest that composition of the oropharyngeal microbiome in COVID-19 may play a role in determining who will suffer from severe disease manifestations. Importance: The microbial community that colonizes the upper airway, the oropharyngeal microbiome, has the potential to affect how patients respond to respiratory viruses such as SARS-CoV2, the causative agent of COVID-19. In this study, we investigated the oropharyngeal microbiome of COVID-19 patients using high throughput DNA sequencing performed on oral swabs. We combined patient characteristics available at intake such as medical comorbidities and age, with measured abundance of bacterial species and metabolic pathways and then trained a machine learning model to determine what features are predicative of patients needing respiratory support in the form of supplemental oxygen or mechanical ventilation. We found that decreased abundance of some bacterial species and increased abundance of pathways associated bacterial products biosynthesis was highly predictive of needing respiratory support. This suggests that the oropharyngeal microbiome affects disease course in COVID-19 and could be targeted for diagnostic purposes to determine who may need oxygen, or therapeutic purposes such as probiotics to prevent severe COVID-19 disease manifestations

Predicting Novel Binding Modes of Agonists to β Adrenergic Receptors Using All-Atom Molecular Dynamics Simulations

Understanding the binding mode of agonists to adrenergic receptors is crucial to enabling improved rational design of new therapeutic agents. However, so far the high conformational flexibility of G protein-coupled receptors has been an obstacle to obtaining structural information on agonist binding at atomic resolution. In this study, we report microsecond classical molecular dynamics simulations of β1 and β2 adrenergic receptors bound to the full agonist isoprenaline and in their unliganded form. These simulations show a novel agonist binding mode that differs from the one found for antagonists in the crystal structures and from the docking poses reported by in silico docking studies performed on rigid receptors. Internal water molecules contribute to the stabilization of novel interactions between ligand and receptor, both at the interface of helices V and VI with the catechol group of isoprenaline as well as at the interface of helices III and VII with the ethanolamine moiety of the ligand. Despite the fact that the characteristic N-C-C-OH motif is identical in the co-crystallized ligands and in the full agonist isoprenaline, the interaction network between this group and the anchor site formed by Asp(3.32) and Asn(7.39) is substantially different between agonists and inverse agonists/antagonists due to two water molecules that enter the cavity and contribute to the stabilization of a novel network of interactions. These new binding poses, together with observed conformational changes in the extracellular loops, suggest possible determinants of receptor specificity

Penile cancer: prognostic and predictive factors in clinical decision-making

: Penile cancer (PC) is a typical tumor of non-industrialized countries. The incidence is 20-30 times higher in Africa and South America, considering the elevated prevalence of sexually transmitted diseases. Histologically, PC includes squamous cell carcinoma (SCPC), the most frequent, and nonsquamous carcinoma (NSCPC). Early diagnosis is the goal, whereas later diagnosis relates to poor functional outcomes and worse prognosis. The 5-year survival rate is 85% for patients with histologically regional negative lymph nodes, compared to 29%-40% for those with histologically regional positive lymph nodes. To date no new drugs are approved, and there are few new data about molecular mechanisms underlying tumorigenesis. The SCPC remains a rare tumor and the current therapeutic algorithm is based principally on retrospective analysis and less on prospective trials. In this review article, biomarkers of prognosis and efficacy of current treatments are summarized with a focus on those that have the potential to affect treatment decision-making in SCPC

The coordination of cell growth during fission yeast mating requires Ras1-GTP hydrolysis

The spatial and temporal control of polarity is fundamental to the survival of all organisms. Cells define their polarity using highly conserved mechanisms that frequently rely upon the action of small GTPases, such as Ras and Cdc42. Schizosaccharomyces pombe is an ideal system with which to study the control of cell polarity since it grows from defined tips using Cdc42-mediated actin remodeling. Here we have investigated the importance of Ras1-GTPase activity for the coordination of polarized cell growth during fission yeast mating. Following pheromone stimulation, Ras1 regulates both a MAPK cascade and the activity of Cdc42 to enable uni-directional cell growth towards a potential mating partner. Like all GTPases, when bound to GTP, Ras1 adopts an active conformation returning to an inactive state upon GTP-hydrolysis, a process accelerated through interaction with negative regulators such as GAPs. Here we show that, at low levels of pheromone stimulation, loss of negative regulation of Ras1 increases signal transduction via the MAPK cascade. However, at the higher concentrations observed during mating, hyperactive Ras1 mutations promote cell death. We demonstrate that these cells die due to their failure to coordinate active Cdc42 into a single growth zone resulting in disorganized actin deposition and unsustainable elongation from multiple tips. These results provide a striking demonstration that the deactivation stage of Ras signaling is fundamentally important in modulating cell polarity

The potential role of the extracellular matrix in the activity of trabectedin in UPS and L-sarcoma: evidences from a patient‐derived primary culture case series in tridimensional and zebrafish models

Background: Soft tissue sarcomas (STS) are a rare group of solid neoplasm including among others liposarcoma, leiomyosarcoma (L-sarcoma) and undifferentiated pleomorphic sarcoma (UPS) entities. The current first-line treatment is represented by anthracycline based- regimens, second-line may include trabectedin. Currently the activity of trabectedin and its mechanism of action is not completely elucidated. Methods: Taking the advantages of our 3D patient-derived primary culture translational model we performed genomic-, chemobiogram, proteomic- and in vivo analysis in a UPS culture (S1). Furthermore pharmacological profiling of a UPS and L-sarcoma patient-derived case series and in silico analysis were carried out. Results: Trabectedin exhibited an increased activity in 3D respect to 2D cultures suggesting an extracellular matrix (ECM) and timp1 involvement in its mechanism of action. Moreover 3D S1 xenotranspanted zebrafish model showed an increased sensitivity to trabectedin. Finally the results were further validated in a UPS and L-sarcoma case series. Conclusions: Taken together these results confirmed the activity of trabectedin in these STS histotypes. Moreover the data underline the ECM involvement in the cytotoxic effect mediated by trabectedin and could open the door for researches aimed to focus on the patient setting that could benefit from this agent