Transcriptomic Analysis Unveils Correlations between Regulative Apoptotic Caspases and Genes of Cholesterol Homeostasis in Human Brain

Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.     

Seasonal Variation in Female Mate Choice and Operational Sex Ratio in Wild Populations of an Annual Fish,Austrolebias reicherti

The intensity of mating competition and the potential benefits for female of mating with certain males can be influenced by several extrinsic factors, such that behavioral decisions can be highly context-dependent. Short-lived species with a single reproductive season are a unique model to study context-sensitive mating decisions. Through exhaustive sampling in the field and simultaneous choice tests in the laboratory, we evaluated operational sex ratio (OSR) and female mate choice at the beginning and end of the reproductive season in the annual killifish Austrolebias reicherti. We found seasonal change in both OSR and female mate choice. At the start of the reproductive season the OSR did not deviate from parity, and females preferred larger males. Later in the reproductive season, while the proportion of males in the ponds decreased, females became unselective with respect to male size. The particular biological cycle of annual killifish, where both life expectancy and mating opportunities decline sharply over a short timescale, could account for the seasonal change in female choice. Reduction in choosiness could arise from diminished reproductive prospects due to a decline in male availability. Moreover, as the end of the season approaches, any benefits of choosiness are presumably reduced: a female’s fitness will be higher if she mates with any male than if she forgoes reproduction and dies. Future work will disentangle the mechanisms underlying seasonal changes in mating preferences, notably direct responses to demographic factors, environmental cues, or intrinsic changes during development.     

Isolation,Characterization and Potential Role in Beta Cell-Endothelium Cross-Talk of Extracellular Vesicles Released from Human Pancreatic Islets

The cross-talk between beta cells and endothelium plays a key role in islet physiopathology and in the revascularization process after islet transplantation. However, the molecular mechanisms involved in this cross-talk are not fully elucidated. Extracellular vesicles (EVs) are secreted membrane nanoparticles involved in inter-cellular communication through the transfer of proteins and nucleic acids. The aims of this study were: 1) isolation and characterization of EVs from human islets; 2) evaluation of the pro-angiogenic effect of islet-derived EVs on human islet endothelial cells (IECs). EVs were isolated by ultracentrifugation from conditioned medium of human islets and characterized by nanotrack analysis (Nanosight), FACS, western blot, bioanalyzer, mRNA/microRNA RT-PCR array. On IECs, we evaluated EV-induced insulin mRNA transfer, proliferation, resistance to apoptosis, in vitro angiogenesis, migration, gene and protein profiling. EVs sized 236±54 nm, expressed different surface molecules and islet-specific proteins (insulin, C-peptide, GLP1R) and carried several mRNAs (VEGFa, eNOS) and microRNAs (miR-27b, miR-126, miR-130 and miR-296) involved in beta cell function, insulin secretion and angiogenesis. Purified EVs were internalized into IECs inducing insulin mRNA expression, protection from apoptosis and enhancement of angiogenesis. Human islets release biologically active EVs able to shuttle specific mRNAs and microRNAs (miRNAs) into target endothelial cells. These results suggest a putative role for islet-derived EVs in beta cell-endothelium cross-talk and in the neoangiogenesis process which is critical for engraftment of transplanted islets.     

Autonomic Dysfunction in Mild Cognitive Impairment: Evidence from Power Spectral Analysis of Heart Rate Variability in a Cross-Sectional Case-Control Study

Background

Mild cognitive impairment (MCI) is set to become a major health problem with the exponential ageing of the world''s population. The association between MCI and autonomic dysfunction, supported by indirect evidence and rich with clinical implications in terms of progression to dementia and increased risk of mortality and falls, has never been specifically demonstrated.

Aim

To conduct a comprehensive assessment of autonomic function in subjects with MCI by means of power spectral analysis (PSA) of heart rate variability (HRV) at rest and during provocative manoeuvres.

Methods

This cross-sectional study involved 80 older outpatients (aged ≥65) consecutively referred to a geriatric unit and diagnosed with MCI or normal cognition (controls) based on neuropsychological testing. PSA was performed on 5-minute electrocardiographic recordings under three conditions—supine rest with free breathing (baseline), supine rest with paced breathing at 12 breaths/minute (parasympathetic stimulation), and active standing (orthosympathetic stimulation)—with particular focus on the changes from baseline to stimulation of indices of sympathovagal balance: normalized low frequency (LFn) and high frequency (HFn) powers and the LF/HF ratio. Blood pressure (BP) was measured at baseline and during standing. Given its exploratory nature in a clinical population the study included subjects on medications with a potential to affect HRV.

Results

There were no significant differences in HRV indices between the two groups at baseline. MCI subjects exhibited smaller physiological changes in all three HRV indices during active standing, consistently with a dysfunction of the orthosympathetic system. Systolic BP after 10 minutes of standing was lower in MCI subjects, suggesting dysautonomia-related orthostatic BP dysregulation.

Conclusions

Our study is novel in providing evidence of autonomic dysfunction in MCI. This is associated with orthostatic BP dysregulation and the ongoing follow-up of the study population will determine its prognostic relevance as a predictor of adverse health outcomes.     

From a 2DE-Gel Spot to Protein Function: Lesson Learned From HS1 in Chronic Lymphocytic Leukemia

The identification of molecules involved in tumor initiation and progression is fundamental for understanding disease’s biology and, as a consequence, for the clinical management of patients. In the present work we will describe an optimized proteomic approach for the identification of molecules involved in the progression of Chronic Lymphocytic Leukemia (CLL). In detail, leukemic cell lysates are resolved by 2-dimensional Electrophoresis (2DE) and visualized as “spots” on the 2DE gels. Comparative analysis of proteomic maps allows the identification of differentially expressed proteins (in terms of abundance and post-translational modifications) that are picked, isolated and identified by Mass Spectrometry (MS). The biological function of the identified candidates can be tested by different assays (i.e. migration, adhesion and F-actin polymerization), that we have optimized for primary leukemic cells.     

Tetrahydroquinazole-based secondary sulphonamides as carbonic anhydrase inhibitors: synthesis,biological evaluation against isoforms I,II, IV,and IX,and computational studies

A library of variously decorated N-phenyl secondary sulphonamides featuring the bicyclic tetrahydroquinazole scaffold was synthesised and biologically evaluated for their inhibitory activity against human carbonic anhydrase (hCA) I, II, IV, and IX. Of note, several compounds were identified showing submicromolar potency and excellent selectivity for the tumour-related hCA IX isoform. Structure–activity relationship data attained for various substitutions were rationalised by molecular modelling studies in terms of both inhibitory activity and selectivity.     

Cell cycle and cell fate interactions in neural development

Mechanisms coupling cell cycle and cell fate operate at different steps during neural development. Intrinsic factors control the cell proliferation of distinct brain regions and changes of cell fate competence, whereas components of the cell cycle machinery could play a major role in setting the appropriate timing of the generation of different cell types.     

Anthranilic acid derivatives: a new class of non-peptide CCK1 receptor antagonists

Having successfully obtained new CCK(1) ligands holding appropriate groups on the anthranilic acid dimer used as molecular scaffold we were interested in increasing their micromolar affinity for the CCK(1) receptors by modifying the spatial relationship of the main pharmacophoric groups. Since, we have proposed simplified analogues reducing the anthranilic acid dimer to a monomer. In this stage of our research program we have prepared and tested on CCK receptors a series of N-substituted anthranilic acid derivatives keeping a Phe residue at the C-terminal site. The indole-2-carbonyl group imparts the best CCK(1) receptor binding affinity (compound 1: IC(50)=197.5 nM) while a sharp decrease in binding affinity is observed for the other indole containing derivatives. Moreover, in order to support the different binding behaviour observed for the synthesized compounds, a conformational investigation was carried out. Finally, on the basis of the main pharmacophoric groups of the obtained new lead compound (1) (coded VL-0395) a receptor binding hypothesis has been provided.