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The rapid spread of SARS-COV-2 and the millions of worldwide deaths and hospitalizations have prompted an urgent need for the development of screening tests capable of rapidly and accurately detecting the virus, even in asymptomatic people. The easy collection and the biomarker content of saliva, together with the label-free and informative power of surface-enhanced Raman spectroscopy (SERS) analysis have driven the creation of point-of-care platforms capable of identifying people with COVID-19. Indeed, different salivary fingerprints were observed between uninfected and infected people. Hence, we performed a retrospective analysis of SERS spectra from salivary samples of COVID-19-infected and -vaccinated subjects to understand if viral components and/or the immune response are implicated in spectral variations. The high sensitivity of the proposed SERS-based method highlighted the persistence of molecular alterations in saliva up to one month after the first positive swab, even when the subject tested negative for the rapid antigenic test. Nevertheless, no specific spectral patterns attributable to some viral proteins and immunoglobulins involved in COVID-19 infection and its progression were found, even if differences in peak intensity, presence, and position were observed in the salivary SERS fingerprint.

The antimicrobial peptide LRGG (LLRLLRRGGRRLLRLL-NH2) was designed and chemically synthesized in a study conducted by Jia et al. Gram-negative bacteria were found to be sensitive to LRGG and exhibited a high therapeutic index. Genetic engineering methods were used to create the prokaryotic fusion expression vector pQE-GFP-LRGG, and the resulting corresponding fusion protein GFP-LRGG was subsequently expressed and purified. The precursor GFP was then removed by TEV proteolysis, and pure LRGG was obtained after another round of purification and endotoxin removal. The prokaryotic-expressed antimicrobial peptide LRGG displays a broad-spectrum antibacterial effect on Gram-negative bacteria, and its minimum inhibitory activity (MIC) against Escherichia coli can reach 2 μg/mL. Compared to the chemically synthesized LRGG, the prokaryotic-expressed LRGG exhibits similar temperature, pH, salt ion, serum stability, and cell selectivity. Furthermore, prokaryotic-expressed LRGG showed excellent therapeutic effects in both the infection model of cell selectivity and no embryotoxicity in a Galleria mellonella infection model. The mechanism by which LRGG causes bacterial death was found to be the disruption of the Gram-negative cell membrane.

IntroductionMicroglia and macrophages can influence the evolution of myelin lesions through the production of extracellular vesicles (EVs). While microglial EVs promote in vitro differentiation of oligodendrocyte precursor cells (OPCs), whether EVs derived from macrophages aid or limit OPC maturation is unknown.MethodsImmunofluorescence analysis for the myelin protein MBP was employed to evaluate the impact of EVs from primary rat macrophages on cultured OPC differentiation. Raman spectroscopy and liquid chromatography-mass spectrometry was used to define the promyelinating lipid components of myelin EVs obtained in vitro and isolated from human plasma.Results and discussionHere we show that macrophage-derived EVs do not promote OPC differentiation, and those released from macrophages polarized towards an inflammatory state inhibit OPC maturation. However, their lipid cargo promotes OPC maturation in a similar manner to microglial EVs. We identify the promyelinating endocannabinoids anandamide and 2-arachidonoylglycerol in EVs released by both macrophages and microglia in vitro and circulating in human plasma. Analysis of OPC differentiation in the presence of the endocannabinoid receptor antagonists SR141716A and AM630 reveals a key role of vesicular endocannabinoids in OPC maturation. From this study, EV-associated endocannabinoids emerge as important mediators in microglia/macrophage-oligodendrocyte crosstalk, which may be exploited to enhance myelin repair.

BackgroundIntensive treadmill training (TT) has been documented to improve gait parameters and functional independence in Parkinson’s Disease (PD), but the optimal intervention protocol and the criteria for tailoring the intervention to patients’ performances are lacking. TT may be integrated with augmented virtual reality (AVR), however, evidence of the effectiveness of this combined treatment is still limited. Moreover, prognostic biomarkers of rehabilitation, potentially useful to customize the treatment, are currently missing. The primary aim of this study is to compare the effects on gait performances of TT + AVR versus TT alone in II-III stage PD patients with gait disturbance. Secondary aims are to assess the effects on balance, gait parameters and other motor and non-motor symptoms, and patient’s satisfaction and adherence to the treatment. As an exploratory aim, the study attempts to identify biomarkers of neuroplasticity detecting changes in Neurofilament Light Chain concentration T0-T1 and to identify prognostic biomarkers associated to blood-derived Extracellular Vesicles.MethodsSingle-center, randomized controlled single-blind trial comparing TT + AVR vs. TT in II-III stage PD patients with gait disturbances. Assessment will be performed at baseline (T0), end of training (T1), 3 (T2) and 6 months (T3, phone interview) from T1. The primary outcome is difference in gait performance assessed with the Tinetti Performance-Oriented Mobility Assessment gait scale at T1. Secondary outcomes are differences in gait performance at T2, in balance and spatial–temporal gait parameters at T1 and T2, patients’ satisfaction and adherence. Changes in falls, functional mobility, functional autonomy, cognition, mood, and quality of life will be also assessed at different timepoints. The G*Power software was used to estimate a sample size of 20 subjects per group (power 0.95, α

Abstract Blood is the most commonly used body fluid for extracellular vesicle (EV) research. The composition of a blood sample and its derivatives (i.e., plasma and serum) are not only donor‐dependent but also influenced by collection and preparation protocols. Since there are hundreds of pre‐analytical protocols and over forty variables, the development of standard operating procedures for EV research is very challenging. To improve the reproducibility of blood EV research, the International Society for Extracellular Vesicles (ISEV) Blood EV Task Force proposes standardized reporting of (i) the applied blood collection and preparation protocol and (ii) the quality of the prepared plasma and serum samples. Gathering detailed information will provide insight into the performance of the protocols and more effectively identify potential confounders in the prepared plasma and serum samples. To collect this information, the ISEV Blood EV Task Force created the Minimal Information for Blood EV research (MIBlood‐EV), a tool to record and report information about pre‐analytical protocols used for plasma and serum preparation as well as assays used to assess the quality of these preparations. This tool does not require modifications of established local pre‐analytical protocols and can be easily implemented to enhance existing databases thereby enabling evidence‐based optimization of pre‐analytical protocols through meta‐analysis. Taken together, insight into the quality of prepared plasma and serum samples will (i) improve the quality of biobanks for EV research, (ii) guide the exchange of plasma and serum samples between biobanks and laboratories, (iii) facilitate inter‐laboratory comparative EV studies, and (iv) improve the peer review process.

Cardiovascular diseases (CVD) can cause various conditions, including an increase in reactive oxygen species (ROS) levels that can decrease nitric oxide (NO) availability and promote vasoconstriction, leading to arterial hypertension. Physical exercise (PE) has been found to be protective against CVD by helping to maintain redox homeostasis through a decrease in ROS levels, achieved by increased expression of antioxidant enzymes (AOEs) and modulation of heat shock proteins (HSPs). Extracellular vesicles (EVs) circulating in the body are a major source of regulatory signals, including proteins and nucleic acids. Interestingly, the cardioprotective role of EVs released after PE has not been fully described.The aim of this study was to investigate the role of circulating EVs, obtained through Size Exclusion Chromatography (SEC) of plasma samples from healthy young males (age: 26.95 ± 3.07; estimated maximum oxygen consumption rate (VO2max): 51.22 ± 4.85 (mL/kg/min)) at basal level (Pre_EVs) and immediately after a single bout of endurance exercise (30’ treadmill, 70% heart rate (HR) -Post_EVs). Gene ontology (GO) analysis of proteomic data from isolated EVs, revealed enrichment in proteins endowed with catalytic activity in Post_EVs, compare to Pre_EVs, with MAP2K1 being the most significantly upregulated protein. Enzymatic assays on EVs derived from Pre and Post samples showed increment in Glutathione Reductase (GR) and Catalase (CAT) activity in Post_EVs. At functional level, Post_EVs, but not Pre_EVs, enhanced the activity of antioxidant enzymes (AOEs) and reduced oxidative damage accumulation in treated human iPS-derived cardiomyocytes (hCM) at basal level and under stress conditions (Hydrogen Peroxide (H2O2) treatment), resulting in a global cardioprotective effect.In conclusion, our data demonstrated, for the first time, that a single 30-min endurance exercise is able to alter the cargo of circulating EVs, resulting in cardioprotective effect through antioxidant activity.

Exercise promotes healthy aging of skeletal muscle. This benefit may be mediated by youthful factors in the circulation released in response to an exercise protocol. While numerous studies to date have explored soluble proteins as systemic mediators of rejuvenating effect of exercise on tissue function, here we showed that the beneficial effect of skeletal muscle contractile activity on aged muscle function is mediated, at least in part, by regenerative properties of circulating extracellular vesicles (EVs). Muscle contractile activity elicited by neuromuscular electrical stimulation (NMES) decreased intensity of expression of the tetraspanin surface marker, CD63, on circulating EVs. Moreover, NMES shifted the biochemical Raman fingerprint of circulating EVs in aged animals with significant changes in lipid and sugar content in response to NMES when compared to controls. As a demonstration of the physiological relevance of these EV changes, we showed that intramuscular administration of EVs derived from aged animals subjected to NMES enhanced aged skeletal muscle healing after injury. These studies suggest that repetitive muscle contractile activity enhances the regenerative properties of circulating EVs in aged animals.

Parkinson’s disease (PD) is a neurodegenerative disorder affecting about 10 million people worldwide with a prevalence of about 2% in the over-80 population. The disease brings in also a huge annual economic burden, recently estimated by the Michael J Fox Foundation for Parkinson’s Research to be USD 52 billion in the United States alone. Currently, no effective cure exists, but available PD medical treatments are based on symptomatic prescriptions that include drugs, surgical approaches and rehabilitation treatment. Due to the complex biology of a PD brain, the design of clinical trials and the personalization of treatment strategies require the identification of accessible and measurable biomarkers to monitor the events induced by treatment and disease progression and to predict patients’ responsiveness. In the present review, we strive to briefly summarize current knowledge about PD biomarkers, focusing on the role of extracellular vesicles as active or involuntary carriers of disease-associated proteins, with particular attention to those research works that envision possible clinical applications.

Previous studies demonstrated sex-related differences in several areas of the human brain, including patterns of brain activation in males and females when observing their own bodies and faces (versus other bodies/faces or morphed versions of themselves), but a complex paradigm touching multiple aspects of embodied self-identity is still lacking. We enrolled 24 healthy individuals (12 M, 12 F) in 3 different fMRI experiments: the vision of prototypical body silhouettes, the vision of static images of the face of the participants morphed with prototypical male and female faces, the vision of short videos showing the dynamic transformation of the morphing. We found differential sexual activations in areas linked to self-identity and to the ability to attribute mental states: In Experiment 1, the male group activated more the bilateral thalamus when looking at sex congruent body images, while the female group activated more the middle and inferior temporal gyrus. In Experiment 2, the male group activated more the supplementary motor area when looking at their faces; the female group activated more the dorsomedial prefrontal cortex (dmPFC). In Experiment 3, the female group activated more the dmPFC when observing either the feminization or the masculinization of their face. The defeminization produced more activations in females in the left superior parietal lobule and middle occipital gyrus. The performance of all classifiers built using single ROIs exceeded chance level, reaching an area under the ROC curves > 0.85 in some cases (notably, for Experiment 2 using the V1 ROI). The results of the fMRI tasks showed good agreement with previously published studies, even if our sample size was small. Therefore, our functional MRI protocol showed significantly different patterns of activation in males and females, but further research is needed both to investigate the gender-related differences in activation when observing a morphing of their face/body, and to validate our paradigm using a larger sample.

The progression of Alzheimer’s disease (AD) correlates with the propagation of hyperphosphorylated tau (pTau) from the entorhinal cortex to the hippocampus and neocortex. Neutral sphingomyelinase2 (nSMase2) is critical in the biosynthesis of extracellular vesicles (EVs), which play a role in pTau propagation. We recently conjugated DPTIP, a potent nSMase2 inhibitor, to hydroxyl-PAMAM-dendrimer nanoparticles that can improve brain delivery. We showed that dendrimer-conjugated DPTIP (D–DPTIP) robustly inhibited the spread of pTau in an AAV-pTau propagation model. To further evaluate its efficacy, we tested D-DPTIP in the PS19 transgenic mouse model. Unexpectantly, D-DPTIP showed no beneficial effect. To understand this discrepancy, we assessed D-DPTIP’s brain localization. Using immunofluorescence and fluorescence-activated cell-sorting, D-DPTIP was found to be primarily internalized by microglia, where it selectively inhibited microglial nSMase2 activity with no effect on other cell types. Furthermore, D-DPTIP inhibited microglia-derived EV release into plasma without affecting other brain-derived EVs. We hypothesize that microglial targeting allowed D-DPTIP to inhibit tau propagation in the AAV-hTau model, where microglial EVs play a central role in propagation. However, in PS19 mice, where tau propagation is independent of microglial EVs, it had a limited effect. Our findings confirm microglial targeting with hydroxyl-PAMAM dendrimers and highlight the importance of understanding cell-specific mechanisms when designing targeted AD therapies.

Abstract The pandemic of COVID-19 is continuously spreading, becoming a worldwide emergency. Early and fast identification of subjects with a current or past infection must be achieved to slow down the epidemiological widening. Here we report a Raman-based approach for the analysis of saliva, able to significantly discriminate the signal of patients with a current infection by COVID-19 from healthy subjects and/or subjects with a past infection. Our results demonstrated the differences in saliva biochemical composition of the three experimental groups, with modifications grouped in specific attributable spectral regions. The Raman-based classification model was able to discriminate the signal collected from COVID-19 patients with accuracy, precision, sensitivity and specificity of more than 95%. In order to translate this discrimination from the signal-level to the patient-level, we developed a Deep Learning model obtaining accuracy in the range 89–92%. These findings have implications for the creation of a potential Raman-based diagnostic tool, using saliva as minimal invasive and highly informative biofluid, demonstrating the efficacy of the classification model.

The inflammatory, reparative and regenerative mechanisms activated in ischemic stroke patients immediately after the event cooperate in the response to injury, in the restoration of functions and in brain remodeling even weeks after the event and can be sustained by the rehabilitation treatment. Nonetheless, patients’ response to treatments is difficult to predict because of the lack of specific measurable markers of recovery, which could be complementary to clinical scales in the evaluation of patients. Considering that Extracellular Vesicles (EVs) are carriers of multiple molecules involved in the response to stroke injury, in the present study, we have identified a panel of EV-associated molecules that (i) confirm the crucial involvement of EVs in the processes that follow ischemic stroke, (ii) could possibly profile ischemic stroke patients at the beginning of the rehabilitation program, (iii) could be used in predicting patients’ response to treatment. By means of a multiplexing Surface Plasmon Resonance imaging biosensor, subacute ischemic stroke patients were proven to have increased expression of vascular endothelial growth factor receptor 2 (VEGFR2) and translocator protein (TSPO) on the surface of small EVs in blood. Besides, microglia EVs and endothelial EVs were shown to be significantly involved in the intercellular communications that occur more than 10 days after ischemic stroke, thus being potential tools for the profiling of patients in the subacute phase after ischemic stroke and in the prediction of their recovery.

The rapid spread of SARS-COV-2 and the millions of worldwide deaths and hospitalizations have prompted an urgent need for the development of screening tests capable of rapidly and accurately detecting the virus, even in asymptomatic people. The easy collection and the biomarker content of saliva, together with the label-free and informative power of surface-enhanced Raman spectroscopy (SERS) analysis have driven the creation of point-of-care platforms capable of identifying people with COVID-19. Indeed, different salivary fingerprints were observed between uninfected and infected people. Hence, we performed a retrospective analysis of SERS spectra from salivary samples of COVID-19-infected and -vaccinated subjects to understand if viral components and/or the immune response are implicated in spectral variations. The high sensitivity of the proposed SERS-based method highlighted the persistence of molecular alterations in saliva up to one month after the first positive swab, even when the subject tested negative for the rapid antigenic test. Nevertheless, no specific spectral patterns attributable to some viral proteins and immunoglobulins involved in COVID-19 infection and its progression were found, even if differences in peak intensity, presence, and position were observed in the salivary SERS fingerprint. [ABSTRACT FROM AUTHOR]

The article aims at integrating Schumpeter's focus on innovation, entrepreneurship, and qualitative change with a theory of the demand side. It argues that, by remaining close to Schumpeter's "methodological core", it is possible to go forward with Schumpeter's seminal contribution. The new perspective deals with innovation and demand together, therefore, laying out the basics for a theory of the demand side, a goal that despite the numerous attempts has remained elusive to Neo-Schumpeterian\Evolutionist theory. Its recent development highlights a "micro" orientation that does not facilitate that task. The suggested approach in this article focuses on the exploitation of the potential implicit in current consumption patterns by means of innovation and new products. Its main goal is to provide a theoretical framework for the large number of case studies and empirical analyses of new products and innovation that constitutes the bulk of research on demand in the Neo-Schumpeterian\Evolutionist literature. That should also contribute to analyzing the structural aspects of the "demand generating" process. [ABSTRACT FROM AUTHOR]

The characterization of nanoparticle-based drug-delivery systems represents a crucial step in achieving a comprehensive overview of their physical, chemical, and biological features and evaluating their efficacy and safety in biological systems. We propose Raman Spectroscopy (RS) for the characterization of liposomes (LPs) to be tested for the control of neuroinflammation and microglial dysfunctions in Glioblastoma multiforme and Alzheimer’s disease. Drug-loaded LPs were functionalized to cross the blood–brain barrier and to guarantee localized and controlled drug release. The Raman spectra of each LP component were used to evaluate their contribution in the LP Raman fingerprint. Raman data analysis made it possible to statistically discriminate LPs with different functionalization patterns, showing that each molecular component has an influence in the Raman spectrum of the final LP formulation. Moreover, CLS analysis on Raman data revealed a good level of synthetic reproducibility of the formulations and confirmed their stability within one month from their synthesis, demonstrating the ability of the technique to evaluate the efficacy of LP synthesis using small amount of sample. RS represents a valuable tool for a fast, sensitive and label free biochemical characterization of LPs that could be used for quality control of nanoparticle-based therapeutics.

Extracellular vesicles (EVs) are natural nanoparticles secreted under physiological and pathological conditions. Thanks to their diagnostic potential, EVs are increasingly being studied as biomarkers of a variety of diseases, including neurological disorders. To date, most studies on EV biomarkers use blood as the source, despite different disadvantages that may cause an impure isolation of the EVs. In the present article, we propose the use of saliva as a valuable source of EVs that could be studied as biomarkers in an easily accessible biofluid. Using a comparable protocol for the isolation of EVs from both liquid biopsies, salivary EVs showed greater purity in terms of co-isolates (evaluated by nanoparticle tracking analysis and Conan test). In addition, Raman spectroscopy was used for the identification of the overall biochemical composition of EVs coming from the two different biofluids. Even considering the limited amount of EVs that can be isolated from saliva, the use of Raman spectroscopy was not hampered, and it was able to provide a comprehensive characterization of EVs in a high throughput and repeatable manner. Raman spectroscopy can thus represent a turning point in the application of salivary EVs in clinics, taking advantage of the simple method of collection of the liquid biopsy and of the quick, sensitive and label-free biophotonics-based approach.

Despite the wide range of proposed biomarkers for Parkinson’s disease (PD), there are no specific molecules or signals able to early and uniquely identify the pathology onset, progression and stratification. Saliva is a complex biofluid, containing a wide range of biological molecules shared with blood and cerebrospinal fluid. By means of an optimized Raman spectroscopy procedure, the salivary Raman signature of PD can be characterized and used to create a classification model. Raman analysis was applied to collect the global signal from the saliva of 23 PD patients and related pathological and healthy controls. The acquired spectra were computed using machine and deep learning approaches. The Raman database was used to create a classification model able to discriminate each spectrum to the correct belonging group, with accuracy, specificity, and sensitivity of more than 97% for the single spectra attribution. Similarly, each patient was correctly assigned with discriminatory power of more than 90%. Moreover, the extracted data were significantly correlated with clinical data used nowadays for the PD diagnosis and monitoring. The preliminary data reported highlight the potentialities of the proposed methodology that, once validated in larger cohorts and with multi-centered studies, could represent an innovative minimally invasive and accurate procedure to determine the PD onset, progression and to monitor therapies and rehabilitation efficacy.

Abstract This study aimed to investigate the efficacy and safety of sofosbuvir‐based therapies for the treatment of cirrhosis from hepatitis C virus (HCV) genotype 2 infection. Data of all consecutive HCV genotype 2 cirrhotic patients who started sofosbuvir‐based treatments between January 2015 and March 2017 in eight Italian tertiary hospitals were collected retrospectively. Overall, 273 patients (Child A: 94.5%) were enrolled. In the 194 subjects treated with sofosbuvir/ribavirin, median initial ribavirin dosage was 13.9 mg/kg/day, and therapy duration was 16 weeks. Sustained virological response (SVR) rates were 93.8% in intention‐to‐treat (ITT) and 95.3% in per‐protocol (PP) analyses for the 129 treatment‐naïve patients, and 96.9% (ITT) and 98.4% (PP) for the 65 treatment‐experienced subjects. Adverse events were reported in 142 patients (73.2%), but only 1.5% discontinued treatment. Eighty‐eight subjects with treatment‐induced anemia (mild: 34.5%, moderate: 7.7%, severe: 3.1%) had to reduce ribavirin dosage, but SVR rates were comparable to the weight‐based dose group, both in ITT (95.4% and 94.3%) and PP (97.7% and 95.2%) analyses, respectively. Moreover, ITT and PP SVR rates were similar between shorter (

Background: Benzodiazepines have been widely used in clinical practice for over four decades and continue to be one of the most consumed and highly prescribed class of drugs available in the treatment of anxiety, depression, and insomnia. The literature indicates that Benzodiazepine users at a significantly increased risk of Motor Vehicle accidents compared to non-users but the impact on injuries at workplace is not well-defined. We aimed to investigate whether use of benzodiazepine is associated with increased risk of occupational injuries (OI).Methods: PubMed, Embase, and Scopus databases were searched. A meta-analysis was performed to calculate odds ratio (OR) and 95% confidence interval (CI) among case controls, cross-sectional studies, either questionnaire or laboratory exams based.Results: A total of 13 studies met inclusion criteria, involving 324,168 OI from seven different countries, with an estimated occurrence of benzodiazepine positivity of 2.71% (95% CI 1.45–4.98). A total of 14 estimates were retrieved. Of them, 10 were based on laboratory analyses, three on institutional databases, while one study was based on questionnaires. Regarding the occupational groups, three estimates focused on commercial drivers (0.73%, 95% CI 0.12–4.30), that exhibited a reduced risk ratio for benzodiazepine positivity compared to other occupational groups (RR 0.109, 95% CI 0.063–0.187). Eventually, no increased risk for benzodiazepine positivity was identified, either from case control studies (OR 1.520, 95% CI 0.801–2.885, I2 76%), or cross sectional studies, when only laboratory based estimates were taken in account (OR 0.590, 95% CI 0.253–1.377, I2 63%).Conclusions: Even though benzodiazepines have the potential to increase injury rates among casual and chronic users, available evidence are insufficient to sustain this hypothesis, particularly when focusing on laboratory-based studies (i.e., studies the characterized the benzodiazepine immediately before the event).

Extracellular Vesicles (EVs) and Conditioned Medium (CM) are promising cell-free approaches to repair damaged and diseased tissues for regenerative rehabilitation purposes. They both entail several advantages, mostly in terms of safety and handling, compared to the cell-based treatment. Despite the growing interest in both EVs and CM preparations, in the light of a clinical translation, a number of aspects still need to be addressed mainly because of limits in the reproducibility and reliability of the proposed protocols. Raman spectroscopy (RS) is a non-destructive vibrational investigation method that provides detailed information about the biochemical composition of a sample, with reported ability in bulk characterization of clusters of EVs from different cell types. In the present brief report, we acquired and compared the Raman spectra of the two most promising cell-free therapeutics, i.e., EVs and CM, derived from two cytotypes with a history in the field of regenerative medicine, adipose-derived mesenchymal stem/stromal cells (ASCs) and dermal fibroblasts (DFs). Our results show how RS can verify the reproducibility not only of EV isolation, but also of the whole CM, thus accounting for both the soluble and the vesicular components of cell secretion. RS can provide hints for the identification of the soluble factors that synergistically cooperate with EVs in the regenerative effect of CM. Still, we believe that the application of RS in the pipeline of cell-free products preparation for therapeutic purposes could help in accelerating translation to clinics and regulatory approval.

As free-living organisms, bacteria are subject to continuous, numerous and occasionally drastic environmental changes to which they respond with various mechanisms which enable them to adapt to the new conditions so as to survive. Here we describe three situations in which the ribosome and its functions represent the sensor or the target of the stress and play a key role in the subsequent cellular response. The three stress conditions which are described are those ensuing upon: a) zinc starvation; b) nutritional deprivation, and c) temperature downshift.

The therapeutic potential of the conditioned medium (CM) derived from MSCs (mesenchymal stem/stromal cells) in disparate medical fields, from immunology to orthopedics, has been widely suggested by in vitro and in vivo evidences. Prior to MSC-CM use in clinical applications, appropriate quality controls are needed in order to assess its reproducibility. Here, we evaluated different CM characteristics, including general features and precise protein and lipid concentrations, in 3 representative samples from adipose-derived MSCs (ASCs). In details, we first investigated the size and distribution of the contained extracellular vesicles (EVs), lipid bilayer-delimited particles whose pivotal role in intercellular communication has been extensively demonstrated. Then, we acquired Raman signatures, providing an overlook of ASC-CM composition in terms of proteins, lipids, and nucleic acids. At last, we analyzed a panel of 200 molecules including chemokines, cytokines, receptors, and inflammatory and growth factors and searched for 32 lipids involved in cell signalling and inflammation. All ASC-CM contained a homogeneous and relevant number of EVs (1.0×109±1.1×108 particles per million donor ASCs) with a mean size of 190±5.2 nm, suggesting the appropriateness of the method for EV retaining and concentration. Furthermore, also Raman spectra confirmed a high homogeneity among samples, allowing the visualization of specific peaks for nucleic acids, proteins, and lipids. An in depth investigation that focused on 200 proteins involved in relevant biological pathways revealed the presence in all specimens of 104 factors. Of these, 26 analytes presented a high degree of uniformity, suggesting that the samples were particularly homogenous for a quarter of the quantified molecules. At last, lipidomic analysis allowed the quantification of 7 lipids and indicated prostaglandin-E2 and N-stearoylethanolamide as the most homogenous factors. In this study, we assessed that ASC-CM samples obtained with a standardized protocol present stable features spanning from Raman fingerprint to specific marker concentrations. In conclusion, we identified key ingredients that may be involved in ASC-CM therapeutic action and whose consistent levels may represent a promising quality control in the pipeline of its preparation for clinical applications.

Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ability of endosomal escape and release DNA into the nucleus, whereas, poorly efficient binary lipoplexes exhibited minor, if any, endosomal rupture ability and remained confined in perinuclear late endosomes. Stopped-flow mixing measurements showed that the fusion rates of multicomponent cationic liposomes with anionic vesicles, used as model systems of cell membranes, were definitely shorter than those of binary liposomes. As either lipoplex uptake and endosomal escape involve fusion between lipoplex and cellular membranes, we suggest that a mechanism of lipoplex-cellular membrane interaction, driven by lipid mixing between cationic and anionic cellular lipids, does explain the TE boost of multicomponent lipoplexes.

Extracellular vesicles (EVs) from a variety of stem cell sources are believed to harbour regenerative capacity, which may be exploited for therapeutic purposes. Because of EV interaction with other soluble secreted factors, EV activity may depend on the employed purification method, which limits cross-study comparisons and therapeutic development. Raman spectroscopy (RS) is a quick and easy method to assess EV purity and composition, giving in-depth biochemical overview on EV preparation. Hereby, we show how this method can be used to characterise EVs isolated from human liver stem cells and bone marrow mesenchymal stem/stromal cells by means of conventional ultracentrifugation (UC) and size exclusion chromatography (SEC) protocols. The obtained EV preparations were demonstrated to be characterised by different degrees of purity and a specific Raman fingerprint that represents both the cell source and the isolation procedure used. Moreover, RS provided useful hints to explore the factors underlying the functional diversity of EV preparations from the same cell source, thus representing a valuable tool to assess EV quality prior to functional assays or therapeutic application.

There is an increasing interest in exploring clinically relevant information that is present in body fluids, and extracellular vesicles (EVs) are intrinsic components of body fluids (“liquid biopsies”). In this report, we will focus on blood. Blood contains not only EVs but also cells, and non-EV particles including lipoproteins. Due to the high concentration of soluble proteins and lipoproteins, blood, plasma and serum have a high viscosity and density, which hampers the concentration, isolation and detection of EVs. Because most if not all studies on EVs are single-centre studies, their clinical relevance remains limited. Therefore, there is an urgent need to improve standardization and reproducibility of EV research. As a first step, the International Society on Extracellular Vesicles organized a biomarker workshop in Birmingham (UK) in November 2017, and during that workshop several working groups were created to focus on a particular body fluid. This report is the first output of the blood EV work group and is based on responses by work group members to a questionnaire in order to discover the contours of a roadmap. From the answers it is clear that most respondents are in favour of evidence-based research, education, quality control procedures, and physical models to improve our understanding and comparison of concentration, isolation and detection methods. Since blood is such a complex body fluid, we assume that the outcome of the survey may also be valuable for exploring body fluids other than blood.

Substitution of the conserved Histidine 448 present in one of the three consensus elements characterizing the guanosine nucleotide binding domain (IF2 G2) of Escherichia coli translation initiation factor IF2 resulted in impaired ribosome-dependent GTPase activity which prevented IF2 dissociation from the ribosome, caused a severe protein synthesis inhibition, and yielded a dominant lethal phenotype. A reduced IF2 affinity for the ribosome was previously shown to suppress this lethality. Here, we demonstrate that also a reduced IF2 affinity for fMet-tRNA can suppress this dominant lethal phenotype and allows IF2 to support faithful translation in the complete absence of GTP hydrolysis. These results strengthen the premise that the conformational changes of ribosome, IF2, and fMet-tRNA occurring during the late stages of translation initiation are thermally driven and that the energy generated by IF2-dependent GTP hydrolysis is not required for successful translation initiation and that the dissociation of the interaction between IF2 C2 and the acceptor end of fMet-tRNA, which represents the last tie anchoring the factor to the ribosome before the formation of an elongation-competent 70S complex, is rate limiting for both the adjustment of fMet-tRNA in a productive P site and the IF2 release from the ribosome.

Extracellular Vesicles (EVs) are naturally secreted nanoparticles with a plethora of functions in the human body and remarkable potential as diagnostic and therapeutic tools. Starting from their discovery, EV nanoscale dimensions have hampered and slowed new discoveries in the field, sometimes generating confusion and controversies among experts. Microtechnological and especially nanotechnological advances have sped up biomedical research dealing with EVs, but efforts are needed to further clarify doubts and knowledge gaps. In the present review, we summarize some of the most interesting data presented in the Annual Meeting of the International Society for Extracellular Vesicles (ISEV), ISEV2021, to stimulate discussion and to share knowledge with experts from all fields of research. Indeed, EV research requires a multidisciplinary knowledge exchange and effort. EVs have demonstrated their importance and significant biological role; still, further technological achievements are crucial to avoid artifacts and misleading conclusions in order to enable outstanding discoveries.