Minimal information for studies of extracellular vesicles (MISEV2023): From basic to advanced approaches

Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its 'Minimal Information for Studies of Extracellular Vesicles', which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly

Towards new insights in psychiatric disorders: expression profiles of inflammation-related genes in murine models of sickness behaviour

Studi recenti suggeriscono che la depressione maggiore, la cui eziologia \ue8 ancora parzialmente ignota, possa essere determinata da processi infiammatori e interazioni tra il sistema neuroendocrino ed il sistema immunitario. L\u2019interleuchina-1\u3b2 (IL-1\u3b2) \ue8 una componente chiave dei processi infiammatori ed \ue8 stata recentemente correlata all\u2019insorgenza di sindromi depressive maggiori. E\u2019 stato inoltre ipotizzato che possa agire a livello del sistema nervoso centrale (SNC), dove sarebbe responsabile dell\u2019induzione dei sintomi osservati nel cosiddetto sickness behavior \u2013 che riproduce le condizioni psicologiche dei sintomi depressivi \u2013 e di una diminuzione nella neurogenesi ippocampale. Abbiamo ipotizzato che diversi geni legati ai processi infiammatori, interagendo con l\u2019IL-1\u3b2, possano essere direttamente coinvolti nella patofisiologia della depressione. Al fine di confermare tale ipotesi, abbiamo utilizzato topi con genotipo wild-type (WT) e topi deprivati dei principali componenti della risposta immunitaria innata, come topi knock-out per l\u2019enzima ossido nitrico sintasi (NOS2-/-) e topi knock-out per l\u2019enzima caspasi 1 (casp1-/-). I topi NOS2-/- sono privi di un potente mediatore infiammatorio, la forma inducibile dell\u2019enzima ossido nitrico sintasi (NOS2), mentre i topi knock-out per l\u2019enzima casp1 sono privi della forma matura e biologicamente attiva dell\u2019IL-1\u3b2. Tutti i nostri topi sono stati trattati con lipopolisaccaride (LPS), una potente endotossina batterica in grado di indurre il sickness behavior in modelli murini. Tramite l\u2019uso di Real-Time PCR quantitativa abbiamo caratterizzato per tutti i gruppi di topi i profili di espressione di nove geni coinvolti nei processi infiammatori \u2013 a disintegrin and metalloproteinase with thrombospondin repeats (ADAMTs1), cyclooxigenase 2 (COX2), nitric oxide synthase 2 (NOS2), chemoattractant cytokines 1 and 10 (CXCL1 and CXCL10), lipocalin 2 (LCN2), T-cell specific GTPase (TGTP), guanylate binding protein 2 (GBP2), e IL-1 receptor antagonist (IL1RA) \u2013 e di due geni coinvolti in disordini psichiatrici di diversa natura \u2013 latrophilin 3 (LPHN3, precedentemente associato al disordine da iperattivit\ue0/deficit dell\u2019attenzione) e disrupted in schizophrenia 1 (DISC1, precedentemente associato a schizofrenia). I profili di espressione di questi 11 geni sono stati investigati nell\u2019ippocampo, una regione del SNC coinvolta nel controllo dell\u2019umore e nella formazione della memoria, ed in due organi periferici che rappresentano il sistema immunitario (la milza) ed il sistema endocrino (le ghiandole surrenali). Dopo il trattamento dei modelli murini con LPS, abbiamo osservato un\u2019esacerbata insorgenza dei sintomi del sickness behaviour con una letalit\ue0 del 100% nei topi WT e NOS2-/- entro 12-24 ore. Al contrario i topi casp1-/-, sebbene anch\u2019essi abbiano sviluppato una risposta infiammatoria, hanno mostrato una sopravvivenza del 100% alla letalit\ue0 indotta dall\u2019LPS a 24 ore dal trattamento. In modo significativo, abbiamo osservato una forte risposta infiammatoria nell\u2019ippocampo di tutti i topi e ci\uf2 indica che gli effetti dell\u2019IL-1\u3b2, a sua volta indotta dall\u2019LPS, sono effettivamente molto potenti anche a livello cerebrale, e non solo negli organi periferici. Inoltre, \ue8 stato osservato un decremento pari a circa il 30% nell\u2019attivazione ippocampale tardiva dei geni COX2, NOS2, IL-1RA e LCN2 in topi casp1-/-, oltre a differenze significative nei pattern di espressione dei vari geni in studio tra diversi tessuti. Nel complesso, i nostri risultati suggeriscono che i topi casp1-/- sono pi\uf9 protetti dagli effetti neuro-infiammatori indotti dall\u2019LPS rispetto agli altri topi, e questo fortifica ulteriormente l\u2019ipotesi che l\u2019IL-1\u3b2 possa giocare il pi\uf9 importante ruolo come fattore di stimolo e di regolazione dei geni correlati all\u2019infiammazione. Inoltre, il fatto che ci siano dei profili di espressione diversi tra i vari tessuti in analisi suggerisce che i processi neuro-infiammatori possano essere temporalmente successivi all\u2019instaurazione di una risposta infiammatoria periferica indotta dall\u2019LPS. Grazie ai nostri risultati, abbiamo potuto ipotizzare un meccanismo patofisiologico che rappresenta il ruolo giocato da ognuno dei geni in studio nella complessa correlazione tra sistema nervoso, endocrino ed immune, fino all\u2019insorgenza del sickness behaviour o dei sintomi depressivi. Questi meccanismi coinvolgono ipoteticamente l\u2019attivazione di processi infiammatori in aree cerebrali, con un conseguente deficit della neurogenesi e modificazioni in tessuti cerebrali deputati al controllo dell\u2019umore.Recent studies suggest that major depressive disorder (MDD), whose etiology is still unknown, might be determined by inflammatory processes and interactions among the neuroendocrine and the immune system. Interleukin-1\u3b2 (IL-1\u3b2) is a key component of inflammatory processes and it has recently been shown to be involved in MDD. It has been suggested to act in the central nervous system (CNS) both by inducing the symptoms observed in sickness behaviour \u2013 that mimic the psychological condition of depressive symptoms \u2013 and by decreasing neurogenesis in the hippocampus. We hypothesized that pro-inflammatory genes, interplaying with IL-1\u3b2, are directly involved in the pathophysiology of MDD. To test this hypothesis, we used wild-type (WT) mice and mice genetically deprived of major components of the innate immune response inflammatory arm, such as NOS2-/- and casp1-/- mice. NOS2 knockout mice lacked a potent pro-inflammatory mediator, the inducible form of nitric oxide synthase (NOS2), whereas casp1 knockout mice lacked the mature and biologically active form of IL-1\u3b2. All these mice have been treated with lypopolysaccharide (LPS), which is a potent bacterial endotoxin that induces sickness behavior in murine models. Using quantitative Real-Time PCR we have characterized for all these mice groups the spatial-temporal expression of nine inflammatory-related genes \u2013 a disintegrin and metalloproteinase with thrombospondin repeats (ADAMTs1), cyclooxigenase 2 (COX2), nitric oxide synthase 2 (NOS2), chemoattractant cytokines 1 and 10 (CXCL1 and CXCL10), lipocalin 2 (LCN2), T-cell specific GTPase (TGTP), guanylate binding protein 2 (GBP2), and IL-1 receptor antagonist (IL1RA) \u2013 and of two genes associated with other psychiatric disorders \u2013 latrophilin 3 (LPHN3, previously associated with attention deficit/hyperactivity disorder) and disrupted in schizophrenia 1 (DISC1, previously associated with schizophrenia). The profiles of expression of these 11 genes have been investigated within the hippocampus, a region of the CNS associated with mood control and memory formation, and in two peripheral organs representing the immune (spleen) and the endocrine system (adrenal glands). After challenging the murine models with LPS, we observed exacerbated sickness behavior that led to 100% lethality in WT and NOS2-/- mice between 12-24 hs. On the other hand, although casp1-/- models displayed an inflammatory response, 100% of these mice survived the LPS-induced lethality after 24h. Notably, we detected a high inflammatory response in the hippocampus of all mice, indicating that the effects of LPS-induced IL-1\u3b2 are indeed strongly activated in the brain, as well as in peripheral organs. Moreover, we observed a nearly 30% decrease in late hippocampal activation of COX2, NOS2, IL-1RA, and LCN2 genes in casp1-/- mice, and differences in patterns of expression of the genes in study among different tissues, as well. Taken together, our results suggest that casp1-/- mice are more protected against neuro-inflammation induced by LPS than other mice, and this strengthens the hypothesis that IL-1\u3b2 might play the most important stimulant role in the regulation of inflammation-related genes. Also, differential patterns of expression among tissues suggested that neuro-inflammatory processes might be temporally consequent to LPS-induced peripheral inflammation. Through our results, we were able to hypothesize a pathophysiological mechanism depicting the role played by each of the genes in study in the complex inflammatory, endocrine and neural networks finally converging into a state of sickness behaviour or depression. These processes hypothetically involve the activation of inflammation pathways in cerebral areas, with a consequent impairment of neurogenesis and changes in brain areas involved in mood regulation

The role of non-verbal communication on the evaluation of ingroup members who adopt egalitarian vs. discriminatory behaviors towards a minority group: A study on a sample of 4-6 years old children

reservedLa letteratura evidenzia come i bambini mostrino generalmente una preferenza per gli atteggiamenti egalitari, tuttavia questa preferenza può essere modulata da diversi fattori, tra cui l’appartenenza di gruppo (Fehr, Bernhard & Rockenbach 2008; Carraro e Castelli, 2015). Gli atteggiamenti interetnici nei bambini infatti vengono osservati fin dall’età di 3-4 anni (Aboud, 2003) e la letteratura evidenzia come i bambini mostrino una preferenza per il proprio gruppo di appartenenza. L’obiettivo di questo studio è quello di indagare il ruolo degli agenti di socializzazione, ossia il ruolo di figure coinvolte nel contesto sociale dei bambini, come gli insegnanti. In particolare lo studio si concentra sul ruolo della comunicazione non verbale degli adulti, che risulta avere un ruolo importante per quanto riguarda gli atteggiamenti intergruppi nei bambini (Brey & Pauker, 2019). Attraverso la rilevazione di misure implicite (Child-IAT) ed esplicite in bambini di età compresa tra i 4 e i 6 anni, lo studio si pone l’obiettivo di indagare il ruolo della comunicazione non verbale degli adulti di riferimento sugli atteggiamenti dei bambini nei confronti dei membri del proprio gruppo che mettono in atto comportamenti egalitari o discriminatori verso un gruppo di minoranza

Development and Characterization of Astaxanthin-Containing Whey Protein-Based Nanoparticles

Astaxanthin (ASX) is a carotenoid of great interest due to its potential health benefits. However, its use in the food, feed, and pharmaceutical fields is limited due to low bioavailability, poor stability during thermochemical treatments, susceptibility to oxidation, and poor organoleptic characteristics. The aim of this work was to develop a method to stabilize astaxanthin extracted from the microalgae Haematococcus pluvialis (H.p.) and to improve its nutritional and functional properties through nanoencapsulation. Nanoparticles (NPs) were produced by emulsification-solvent evaporation technique starting from H.p. oleoresin using whey proteins concentrate (WPC) as stabilizer. The efficiency of encapsulation was 96%. The particle size (Z-average) was in the range of 80-130 nm and the superficial charge (measured as zeta-potential) was negative (-20 to -30 mV). The stability of the NPs upon resuspension in water was assayed through a panel of stress tests, i.e., extreme pH, UV radiation, Fe3+ exposition, and heating at 65 \ub0C, that always showed a superior performance of encapsulated ASX in comparison to oleoresin, even if NPs tended to precipitate at pH 3.5-5.5. Simulated gastroenteric digestion was conducted to study the release of ASX in physiological conditions, and showed a maximum bioaccessibility of 76%, with 75% ASX converted into the more bioavailable free form. The collected data suggest that NPs might have possible future applications as supplements for human and animal diets

Microencapsulation by spray-drying of polyphenols extracted from red chicory and red cabbage: Effects on stability and color properties

The research of antioxidants and natural pigments to replace synthetic molecules is increasingly considering wastes from plant food supply chains. Red chicory (RCH) and red cabbage (RCA) are rich sources of polyphenols (PP), especially anthocyanins, well know natural pigments possessing strong antioxidant capacity and beneficial health effects. The aim of this work was to compare different solvents for PP extraction and to evaluate the effect of spray-drying encapsulation using modified starch on PP, antioxidant capacity (AOC) and color properties.Methanol:water (70:30) showed the best extraction capacity, while ethanol:water (70:30) extracts displayed the highest thermal stability. Ethanol:water extracts were spray-dried with a yield of 95-99% for both crops, while the efficiency of PP encapsulation was 79% (RCA) and 88% (RCH). Encapsulation improved retention of PP and AOC upon thermal treatment (RCH: 20-30%, RCA: 44-55%) without altering color properties. This process can be employed for the development of functional foods and supplements

COMT, neuropsychological function and brain structure in schizophrenia:A systematic review and neurobiological interpretation

BACKGROUND: Endophenotypes in genetic psychiatry may increase our understanding of the molecular mechanisms underlying disease risk and its manifestations. We sought to investigate the link between neuropsychological impairments and brain structural abnormalities associated with the COMT Val(158)Met polymorphism in patients with schizophrenia to improve understanding of the pathophysiology of this disorder. METHODS: We performed a systematic review using studies identified in PubMed and MEDLINE (from the date of the first available article to July 2012). Our review examined evidence of an association between the COMT Val(158)Met polymorphism and both neuropsychological performance and brain structure in patients with psychosis, in their relatives and in healthy individuals (step 1). The review also explored whether the neuropsychological tasks and brain structures identified in step 1 met the criteria for an endophenotype (step 2). Then we evaluated evidence that the neuropsychological endophenotypes identified in step 2 are associated with the brain structure endophenotypes identified in that step (step 3). Finally, we propose a neurobiological interpretation for this evidence. RESULTS: A poorer performance on the n-back task and the Continuous Performance Test (CPT) and smaller temporal and frontal brain areas were associated with the COMT Val allele in patients with schizophrenia and their relatives and met most of the criteria for an endophenotype. It is possible that the COMT Val(158)Met polymorphism therefore contributes to the development of these neuropsychological and brain structural endophenotypes of schizophrenia, in which the prefrontal cortex may represent the neural substrate underlying both n-back and CPT performances. LIMITATIONS: The association between a single genetic variant and an endophenotype does not necessarily imply a causal relationship between them. CONCLUSION: This evidence and the proposed interpretation contribute to explain, at least in part, the biological substrate of 4 important endophenotypes that characterize schizophrenia

Microencapsulation of astaxanthin by ionic gelation: effect of different gelling polymers on the carotenoid load, stability and bioaccessibility

Astaxanthin (ASX) is a carotenoid characterised by numerous health-promoting properties but biased by scarce chemical stability. Our aim was to microencapsulate ASX through ionic gelation to improve its stability and bioaccessibility, evaluating the performance of low-methoxyl pectin (LMP) and chitosan, besides the most common alginate. Three formulations were tested: 3% pectin (Pec), 3% pectin + chitosan shell (PecChi), and 1.5% alginate - 1.5% LMP + chitosan shell. The results showed that chitosan deposited onto PecAlChi and PecChi beads improved particle sphericity and limited oil oxidation during processing. PecAlChi provided the highest encapsulation efficiency (87%) and bioaccessibility (58%). The stability test at 65 °C x 24 days showed different polymer-dependent degradation kinetics, with PecChi providing the highest stability (48%). In conclusion, encapsulation performance strongly depended on the chitosan shell and the gelling polymers. This suggests that tuning the alginate/pectin ratio may lead to the best compromise between stability and bioaccessibilit

Virological and phylogenetic characterization of attenuated small ruminant lentivirus isolates eluding efficient serological detection

Three field isolates of small ruminant lentiviruses (SRLVs) were derived from a mixed flock of goats and sheep certified for many years as free of caprine arthritis encephalitis virus (CAEV). The phylogenetic analysis of pol sequences permitted to classify these isolates as A4 subtype. None of the animals showed clinical signs of SRLV infection, confirming previous observations which had suggested that this particular subtype is highly attenuated, at least for goats. A quantitative real time PCR strategy based on primers and probes derived from a highly variable env region permitted us to classify the animals as uninfected, singly or doubly infected. The performance of different serological tools based on this classification revealed their profound inadequacy in monitoring animals infected with this particular SRLV subtype. In vitro, the isolates showed differences in their cytopathicity and a tendency to replicate more efficiently in goat than sheep cells, especially in goat macrophages. By contrast, in vivo, these viruses reached significantly higher viral loads in sheep than in goats. Both env subtypes infected goats and sheep with equal efficiency. One of these, however, reached significantly higher viral loads in both species. In conclusion, we characterized three isolates of the SRLV subtype A4 that efficiently circulate in a mixed herd of goats and sheep in spite of their apparent attenuation and a strict physical separation between goats and sheep. The poor performance of the serological tools applied indicates that, to support an SRLV eradication campaign, it is imperative to develop novel, subtype specific tools