The Thermal Structural Transition of Alpha-Crystallin Modulates Subunit Interactions and Increases Protein Solubility

Background: Alpha crystallin is an oligomer composed of two types of subunits, alpha-A and alpha-B crystallin, and is the major constituent of human lens. The temperature induced condensation of alpha-crystallin, the main cause for eye lens opacification (cataract), is a two step-process, a nucleation followed by an aggregation phase, and a protective effect towards the aggregation is exhibited over the alpha crystallin phase transition temperature (Tc = 318.16 K). Methods/Results: To investigate if a modulation of the subunit interactions over Tc could trigger the protective mechanism towards the aggregation, we followed, by using simultaneously static and dynamic light scattering, the temperature induced condensation of alpha-crystallin. By developing a mathematical model able to uncouple the nucleation and aggregation processes, we find a previously unobserved transition in the nucleation rate constant. Its temperature dependence allows to determine fundamental structural parameters, the chemical potential (Dm) and the interfacial tension (c) of the aggregating phase, that characterize subunit interactions. Conclusions/General Significance: The decrease of both Dm and c at Tc, and a relative increase in solubility, reveal a significative decrease in the strenght of alpha-crystallin subunits interactions, which protects from supramolecolar condensation in hypertermic conditions. On the whole, we suggest a general approach able to understand the structural and kinetic mechanisms involved in aggregation-related diseases and in drugs development and testing

Automatic Detection of Aerobic Threshold through Recurrence Quantification Analysis of Heart Rate Time Series

During exercise with increasing intensity, the human body transforms energy with mechanisms dependent upon actual requirements. Three phases of the body’s energy utilization are recognized, characterized by different metabolic processes, and separated by two threshold points, called aerobic (AerT) and anaerobic threshold (AnT). These thresholds occur at determined values of exercise intensity(workload) and can change among individuals. They are considered indicators of exercise capacities and are useful in the personalization of physical activity plans. They are usually detected by ventilatory or metabolic variables and require expensive equipment and invasive measurements. Recently, particular attention has focused on AerT, which is a parameter especially useful in the overweight and obese population to determine the best amount of exercise intensity for weight loss and increasing physical fitness. The aim of study is to propose a new procedure to automatically identify AerT using the analysis of recurrences (RQA) relying only on Heart rate time series, acquired from a cohort of young athletes during a sub-maximal incremental exercise test (Cardiopulmonary Exercise Test, CPET) on a cycle ergometer. We found that the minima of determinism, an RQA feature calculated from the Recurrence Quantification by Epochs (RQE) approach, identify the time points where generic metabolic transitions occur. Among these transitions, a criterion based on the maximum convexity of the determinism minima allows to detect the first metabolic threshold. The ordinary least products regression analysis shows that values of the oxygen consumption VO2 , heart rate (HR), and Workload correspondent to the AerT estimated by RQA are strongly correlated with the one estimated by CPET (r > 0.64). Mean percentage differences are <2% for both HR and VO2 and <11% for Workload. The Technical Error for HR at AerT is <8%; intraclass correlation coefficients values are moderate (≥0.66) for all variables at AerT. This system thus represents a useful method to detect AerT relying only on heart rate time series, and once validated for different activities, in future, can be easily implemented in applications acquiring data from portable heart rate monitors

Plant-derived extracellular nanovesicles: a promising biomedical approach for effective targeting of triple negative breast cancer cells

Introduction: Triple negative breast cancer (TNBC), a highly aggressive subtype accounting for 15–20% of all breast cancer cases, faces limited treatment options often accompanied by severe side effects. In recent years, natural extracellular nanovesicles derived from plants have emerged as promising candidates for cancer therapy, given their safety profile marked by non-immunogenicity and absence of inflammatory responses. Nevertheless, the potential anti-cancer effects of Citrus limonL.-derived extracellular nanovesicles (CLENs) for breast cancer treatment is still unexplored.Methods: In this study, we investigated the anti-cancer effects of CLENs on two TNBC cell lines (4T1 and HCC-1806 cells) under growth conditions in 2D and 3D culture environments. The cellular uptake efficiency of CLENs and their internalization mechanism were evaluated in both cells using confocal microscopy. Thereafter, we assessed the effect of different concentrations of CLENs on cell viability over time using a dual approach of Calcein-AM PI live-dead assay and CellTiter-Glo bioluminescence assay. We also examined the influence of CLENs on the migratory and evasion abilities of TNBC cells through wound healing and 3D Matrigel drop evasion assays. Furthermore, Western blot analysis was employed to investigate the effects of CLENs on the phosphorylation levels of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal- regulated kinase (ERK) expression.Results: We found that CLENs were internalized by the cells via endocytosis, leading to decreased cell viability, in a dose- and time-dependent manner. Additionally, the migration and evasion abilities of TNBC cells were significantly inhibited under exposed to 40 and 80 μg/mL CLENs. Furthermore, down-regulated expression levels of phosphorylated phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), and extracellular signal-regulated kinase (ERK), suggesting that the inhibition of cancer cell proliferation, migration, and evasion is driven by the inhibition of the PI3K/AKT and MAPK/ERK signaling pathways.Discussion: Overall, our results demonstrate the anti-tumor efficiency of CLENs against TNBC cells, highlighting their potential as promising natural anti-cancer agents for clinical applications in cancer treatment

INSIDIA:a FIJI macro delivering high-throughput and high-content spheroid invasion analysis

Time-series image capture of in vitro 3D spheroidal cancer models embedded within an extracellular matrix affords examination of spheroid growth and cancer cell invasion. However, a customizable, comprehensive and open source solution for the quantitative analysis of such spheroid images is lacking. Here, the authors describe INSIDIA (INvasion SpheroID ImageJ Analysis), an open-source macro implemented as a customizable software algorithm running on the FIJI platform, that enables high-throughput high-content quantitative analysis of spheroid images (both bright-field gray and fluorescent images) with the output of a range of parameters defining the spheroid “tumor” core and its invasive characteristics

Mycobacterium tuberculosis may escape helper T cell recognition by infecting human fibroblasts

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Red blood cells membrane micropolarity as a novel diagnostic indicator of type 1 and type 2 diabetes

Classification of the category of diabetes is extremely important for clinicians to diagnose and select the correct treatment plan. Glycosylation, oxidation and other post-translational modifications of membrane and transmembrane proteins, as well as impairment in cholesterol homeostasis, can alter lipid density, packing, and interactions of Red blood cells (RBC) plasma membranes in type 1 and type 2 diabetes, thus varying their membrane micropolarity. This can be estimated, at a submicrometric scale, by determining the membrane relative permittivity, which is the factor by which the electric field between the charges is decreased relative to vacuum. Here, we employed a membrane micropolarity sensitive probe to monitor variations in red blood cells of healthy subjects (n=16) and patients affected by type 1 (T1DM, n=10) and type 2 diabetes mellitus (T2DM, n=24) to provide a cost-effective and supplementary indicator for diabetes classification. We find a less polar membrane microenvironment in T2DM patients, and a more polar membrane microenvironment in T1DM patients compared to control healthy patients. The differences in micropolarity are statistically significant among the three groups (p<0.01). The role of serum cholesterol pool in determining these differences was investigated, and other factors potentially altering the response of the probe were considered in view of developing a clinical assay based on RBC membrane micropolarity. These preliminary data pave the way for the development of an innovative assay which could become a tool for diagnosis and progression monitoring of type 1 and type 2 diabetes. Keywords: Diabetes mellitus, Membrane micropolarity, Red blood cells, Fluorescence lifetime microscopy, Metabolic imaging, Personalized medicin

Nutrient withdrawal rescues growth factor-deprived cells from mTOR-dependent damage

Deregulated nutrient signaling plays pivotal roles in body ageing and in diabetic complications; biochemical cascades linking energy dysmetabolism to cell damage and loss are still incompletely clarified, and novel molecular paradigms and pharmacological targets critically needed. We provide evidence that in the retrovirus-packaging cell line HEK293-T Phoenix, massive cell death in serum-free medium is remarkably prevented or attenuated by either glucose or aminoacid withdrawal, and by the glycolysis inhibitor 2-deoxy-glucose. A similar protection was also elicited by interference with mitochondrial function, clearly suggesting involvement of energy metabolism in increased cell survival. Oxidative stress did not account for nutrient toxicity on serum-starved cells. Instead, nutrient restriction was associated with reduced activity of the mTOR/S6 Kinase cascade. Moreover, pharmacological and genetic manipulation of the mTOR pathway modulated in an opposite fashion signaling to S6K/S6 and cell viability in nutrient-repleted medium. Additionally, stimulation of the AMP-activated Protein Kinase concomitantly inhibited mTOR signaling and cell death, while neither event was affected by overexpression of the NAD+ dependent deacetylase Sirt-1, another cellular sensor of nutrient scarcity. Finally, blockade of the mTOR cascade reduced hyperglycemic damage also in a more pathophysiologically relevant model, i.e. in human umbilical vein endothelial cells (HUVEC) exposed to hyperglycemia. Taken together these findings point to a key role of the mTOR/S6K cascade in cell damage by excess nutrients and scarcity of growth-factors, a condition shared by diabetes and other ageing-related pathologies

3D graphene scaffolds for skeletal muscle regeneration: future perspectives

Although skeletal muscle can regenerate after injury, in chronic damages or in traumatic injuries its endogenous self-regeneration is impaired. Consequently, tissue engineering approaches are promising tools for improving skeletal muscle cells proliferation and engraftment. In the last decade, graphene and its derivates are being explored as novel biomaterials for scaffolds production for skeletal muscle repair. This review describes 3D graphene-based materials that are currently used to generate complex structures able not only to guide cell alignment and fusion but also to stimulate muscle contraction thanks to their electrical conductivity. Graphene is an allotrope of carbon that has indeed unique mechanical, electrical and surface properties and has been functionalized to interact with a wide range of synthetic and natural polymers resembling native musculoskeletal tissue. More importantly, graphene can stimulate stem cell differentiation and has been studied for cardiac, neuronal, bone, skin, adipose, and cartilage tissue regeneration. Here we recapitulate recent \ufb01ndings on 3D scaffolds for skeletal muscle repairing and give some hints for future research in multifunctional graphene implants

Investigation of DHA-Induced Regulation of Redox Homeostasis in Retinal Pigment Epithelium Cells through the Combination of Metabolic Imaging and Molecular Biology

Diabetes-induced oxidative stress leads to the onset of vascular complications, which are major causes of disability and death in diabetic patients. Among these, diabetic retinopathy (DR) often arises from functional alterations of the blood-retinal barrier (BRB) due to damaging oxidative stress reactions in lipids, proteins, and DNA. This study aimed to investigate the impact of the ω3-polyunsaturated docosahexaenoic acid (DHA) on the regulation of redox homeostasis in the human retinal pigment epithelial (RPE) cell line (ARPE-19) under hyperglycemic-like conditions. The present results show that the treatment with DHA under high-glucose conditions activated erythroid 2-related factor Nrf2, which orchestrates the activation of cellular antioxidant pathways and ultimately inhibits apoptosis. This process was accompanied by a marked increase in the expression of NADH (Nicotinamide Adenine Dinucleotide plus Hydrogen) Quinone Oxidoreductase 1 (Nqo1), which is correlated with a contextual modulation and intracellular re-organization of the NAD+/NADH redox balance. This investigation of the mechanisms underlying the impairment induced by high levels of glucose on redox homeostasis of the BRB and the subsequent recovery provided by DHA provides both a powerful indicator for the detection of RPE cell impairment as well as a potential metabolic therapeutic target for the early intervention in its treatment