Mechanotransduction in human and mouse beta cell lines: reliable models to characterize novel signaling pathways controlling beta cell fate

Background and aims: Attempts to influence \u3b2-cell differentiation by engineering substrates that mimic appropriate extracellular matrix (ECM) topographies are hampered by the fact that profound details of mechanosensing/transduction complexity remain elusive. We recently demonstrated that human islets of Langerhans sense the ECM nanotopography and activate a mechanotransductive pathway, which is essential for preserving long-term \u3b2-cell differentiation and function in vitro. However, human islets of Langerhans are extremely heterogeneous and their availability for research purpose is limited. Therefore, aim of the proposed research was to investigate whether mouse and human \u3b2-cell lines might sense changes innthe ECM topography and might be used as a simplified model to dissect the molecular pathways involved in mechanotransduction. Materials and methods: We used supersonic cluster beam deposition to fabricate nanostructured substrates characterized by a quantitatively controllable ECM-like nanoroughness. Mouse \u3b2TC3 and human 1.1B4 cells were seeded on these substrates and after five days in culture, the activation of the mechanotransductive pathway was verified by means of morphological (super-resolution fluorescence microscopy), functional and proteomic techniques. Results: Quantitative immunofluorescence studies demonstrated that the cell-nanotopography interaction affects the focal adhesion structures (smaller vinculin clusters), the organization of the actin cytoskeleton (shorter actin fiber) and the nuclear architecture. Functional studies revealed that nanostructured surfaces improve the \u3b2-cell mitochondrial activity and increase the glucose-stimulated Ca2+currents and insulin release. Label-free shotgun proteomics broadly confirmed the morphological and functional studies and showed the upregulation of a number of mechanosensors and transcription factors involved in \u3b2-cell differentiation in cells grown on nanostructured substrates compared to those grown on flat standard control surfaces. Conclusion: Our data reveal that mouse and human \u3b2-cell lines sense changes in extracellular mechanical forces and activate a mechanotransductive pathway. The findings from this study will be useful to clarify the link between mechanotransduction and cell fate and to successfully engineer scaffolds in order to have functional beta cells

COVID-19 and surgical training in Italy: Residents and young consultants perspectives from the battlefield

COVID-19 is seriously affecting Italy, putting the health system under extreme pressure. Training of medical students and residents is also suffering from this with the suspension of lectures and clinical rotations. What solutions have been taken to deal with the issue

Proteomic Analysis Reveals a Mitochondrial Remodeling of βTC3 Cells in Response to Nanotopography

Recently, using cluster-assembled zirconia substrates with tailored roughness produced by supersonic cluster beam deposition, we demonstrated that \u3b2 cells can sense nanoscale features of the substrate and can translate these stimuli into a mechanotransductive pathway capable of preserveing \u3b2-cell differentiation and function in vitro in long-term cultures of human islets. Using the same proteomic approach, we now focused on the mitochondrial fraction of \u3b2TC3 cells grown on the same zirconia substrates and characterized the morphological and proteomic modifications induced by the nanostructure. The results suggest that, in \u3b2TC3 cells, mitochondria are perturbed by the nanotopography and activate a program involving metabolism modification and modulation of their interplay with other organelles. Data were confirmed in INS1E, a different \u3b2-cell model. The change induced by the nanostructure can be pro-survival and prime mitochondria for a metabolic switch to match the new cell needs

Acute environmental temperature variation affects brain protein expression, anxiety and explorative behaviour in adult zebrafish

This study investigated the effect of 4-d acute thermal treatments at 18 \ub0C, 26 \ub0C (control) and 34 \ub0C on the nervous system of adult zebrafish (Danio rerio) using a multidisciplinary approach based on behavioural tests and brain proteomic analysis. The behavioural variations induced by thermal treatment were investigated using five different tests, the novel tank diving, light and dark preference, social preference, mirror biting, and Y-Maze tests, which are standard paradigms specifically tailored for zebrafish to assess their anxiety-like behaviour, boldness, social preference, aggressiveness, and explorative behaviour, respectively. Proteomic data revealed that several proteins involved in energy metabolism, messenger RNA translation, protein synthesis, folding and degradation, cytoskeleton organisation and synaptic vesiculation are regulated differently at extreme temperatures. The results showed that anxiety-like behaviours increase in zebrafish at 18 \ub0C compared to those at 26 \ub0C or 34 \ub0C, whereas anxiety-related protein signalling pathways are downregulated. Moreover, treatments at both 18 \ub0C and 34 \ub0C affect the exploratory behaviour that appears not to be modulated by past experiences, suggesting the impairment of fish cognitive abilities. This study is the continuation of our previous work on the effect of 21-d chronic treatment at the same constant temperature level and will enable the comparison of acute and chronic treatment effects on the nervous system function in adult zebrafish

Environmental temperature variation affects brain protein expression and cognitive abilities in adult zebrafish (Danio rerio): A proteomic and behavioural study.

Water temperature is an important environmental parameter influencing the distribution and the health of fishes and it plays a central role in ectothermic animals. The aim of this study is to determine the effects of environmental temperature on the brain proteome and the behavioural responses in zebrafish, a widely used animal model for environmental "omics" studies. Adult specimens of wild-type zebrafish were kept at 18 °C, 34 °C and 26 °C (control) for 21 days. Proteomic data revealed that several proteins involved in cytoskeletal organization, mitochondrial regulation and energy metabolism are differently regulated at the extreme temperatures. In particular, the expression of proteins associated to synapses and neurotransmitter release is down-regulated at 18 °C and 34 °C. In both thermal conditions, fish exhibited a reduced interest for the novel environment and an impairment of cognitive abilities during Y-Maze behavioural tests. The observed pathways of protein expression are possibly associated to functional alterations of the synaptic transmission that may result in cognitive functions impairment at central nervous system level as those revealed by behavioural tests. This study indicates that temperature variations can elicit biochemical changes that may affect fish health and behaviour. This combined approach provides insights into mechanisms supporting thermal acclimation and plasticity in fishes. SIGNIFICANCE: Environmental temperature variation may impact on all levels of biological life. Understanding the impact of thermal variation on the nervous system and animal behaviour is of primary importance since the results obtained can be applied from the ecological to the biomedical fields

Brain Proteome and Behavioural Analysis in Wild Type, BDNF+/− and BDNF−/− Adult Zebrafish (Danio rerio) Exposed to Two Different Temperatures

Experimental evidence suggests that environmental stress conditions can alter the expression of BDNF and that the expression of this neurotrophin influences behavioural responses in mammalian models. It has been recently demonstrated that exposure to 34 degrees C for 21 days alters the brain proteome and behaviour in zebrafish. The aim of this work was to investigate the role of BDNF in the nervous system of adult zebrafish under control and heat treatment conditions. For this purpose, zebrafish from three different genotypes (wild type, heterozygous BDNF+/- and knock out BDNF-/-) were kept for 21 days at 26 degrees C or 34 degrees C and then euthanized for brain molecular analyses or subjected to behavioural tests (Y-maze test, novel tank test, light and dark test, social preference test, mirror biting test) for assessing behavioural aspects such as boldness, anxiety, social preference, aggressive behaviour, interest for the novel environment and exploration. qRT-PCR analysis showed the reduction of gene expression of BDNF and its receptors after heat treatment in wild type zebrafish. Moreover, proteomic analysis and behavioural tests showed genotype- and temperature-dependent effects on brain proteome and behavioural responding. Overall, the absent expression of BDNF in KO alters (1) the brain proteome by reducing the expression of proteins involved in synapse functioning and neurotransmitter-mediated transduction; (2) the behaviour, which can be interpreted as bolder and less anxious and (3) the cellular and behavioural response to thermal treatment

Comparing the safety and efficacy of ruxolitinib in patients with Dynamic International Prognostic Scoring System low-, intermediate-1-, intermediate-2-, and high-risk myelofibrosis in JUMP, a Phase 3b, expanded-access study

Ruxolitinib, a potent Janus kinase 1/2 inhibitor, has demonstrated durable improvements in patients with myelofibrosis. In this analysis of the Phase 3b JUMP study, which included patients aged =18 years with a diagnosis of primary or secondary myelofibrosis, we assessed the safety and efficacy of ruxolitinib in patients stratified by Dynamic International Prognostic Scoring System (DIPSS) risk categories. Baseline characteristic data were available to assess DIPSS status for 1844 of the 2233 enrolled patients; 60, 835, 755, and 194 in the low-, intermediate (Int)-1-, Int-2-, and high-risk groups, respectively. Ruxolitinib was generally well tolerated across all risk groups, with an adverse-event (AE) profile consistent with previous reports. The most common hematologic AEs were thrombocytopenia and anemia, with highest rates of Grade =3 events in high-risk patients. Approximately, 73% of patients experienced =50% reductions in palpable spleen length at any point in the =24-month treatment period, with highest rates in lower-risk categories (low, 82.1%; Int-1, 79.3%; Int-2, 67.1%; high risk, 61.6%). Median time to spleen length reduction was 5.1 weeks and was shortest in lower-risk patients. Across measures, 40%–57% of patients showed clinically meaningful symptom improvements, which were observed from 4 weeks after treatment initiation and maintained throughout the study. Overall survival (OS) was 92% at Week 72 and 75% at Week 240 (4.6 years). Median OS was longer for Int-2-risk than high-risk patients (253.6 vs. 147.3 weeks), but not evaluable in low-/Int-1-risk patients. By Week 240, progression-free survival (PFS) and leukemia-free survival (LFS) rates were higher in lower-risk patients (PFS: low, 90%; Int-1, 82%; Int-2, 46%; high risk, 15%; LFS: low, 92%; Int-1, 86%; Int-2, 58%; high risk, 19%). Clinical benefit was seen across risk groups, with more rapid improvements in lower risk patients. Overall, this analysis indicates that ruxolitinib benefits lower-risk DIPSS patients in addition to higher risk