A peptide strategy for inhibiting different protein aggregation pathways.

Protein aggregation correlates with many human diseases. Protein aggregates differ in structure and shape. Strategies to develop effective aggregation inhibitors that reach the clinic failed so far. Here, we developed a family of peptides targeting early aggregation stages for both amorphous and fibrillar aggregates of proteins unrelated in sequence and structure. They act on dynamic precursors before mechanistic differentiation takes place. Using peptide arrays, we first identified peptides inhibiting the amorphous aggregation of a molten globular, aggregation-prone mutant of the Axin tumor suppressor. Optimization revealed that the peptides activity did not depend on their sequences but rather on their molecular determinants: a composition of 20-30% flexible, 30-40% aliphatic and 20-30% aromatic residues, a hydrophobicity/hydrophilicity ratio close to 1, and an even distribution of residues of different nature throughout the sequence. The peptides also suppressed fibrillation of Tau, a disordered protein that forms amyloids in Alzheimer's disease, and slowed down that of Huntingtin Exon1, an amyloidogenic protein in Huntington's disease, both entirely unrelated to Axin. Our compounds thus target early aggregation stages of different aggregation mechanisms, inhibiting both amorphous and amyloid aggregation. Such cross-mechanistic, multi-targeting aggregation inhibitors may be lead compounds for developing drug candidates against various protein aggregation diseases.

Tau fibrils evade autophagy by excessive p62 coating and TAX1BP1 exclusion.

The accumulation of protein aggregates is a hallmark of many diseases, including Alzheimer's disease. As a major pillar of the proteostasis network, autophagy mediates the degradation of protein aggregates. The autophagy cargo receptor p62 recognizes ubiquitin on proteins and cooperates with TAX1BP1 to recruit the autophagy machinery. Paradoxically, protein aggregates are not degraded in various diseases despite p62 association. Here, we reconstituted the recognition by the autophagy receptors of physiological and pathological Tau forms. Monomeric Tau recruits p62 and TAX1BP1 via the sequential actions of the chaperone and ubiquitylation machineries. In contrast, Tau fibrils from Alzheimer's disease brains are recognized by p62 but fail to recruit TAX1BP1. This failure is due to the masking of fibrils ubiquitin moieties by p62. Tau fibrils are resistant to deubiquitylation, and, thus, this nonproductive interaction of p62 with the fibrils is irreversible. Our results shed light on the mechanism underlying autophagy evasion by protein aggregates and their consequent accumulation in disease.

2023 White Paper on Recent Issues in Bioanalysis: Deuterated Drugs; LNP; Tumor/FFPE Biopsy; Targeted Proteomics; Small Molecule Covalent Inhibitors; Chiral Bioanalysis; Remote Regulatory Assessments; Sample Reconciliation/Chain of Custody (PART 1A - Recommendations on Mass Spectrometry, Chromatography, Sample Preparation Latest Developments, Challenges, and Solutions and BMV/Regulated Bioanalysis PART 1B - Regulatory Agencies' Inputs on Regulated Bioanalysis/BMV, Biomarkers/IVD/CDx/BAV, Immunogenicity, Gene & Cell Therapy and Vaccine).

The 17th Workshop on Recent Issues in Bioanalysis (17th WRIB) took place in Orlando, FL, USA on June 19-23, 2023. Over 1000 professionals representing pharma/biotech companies, CROs, and multiple regulatory agencies convened to actively discuss the most current topics of interest in bioanalysis. The 17th WRIB included 3 Main Workshops and 7 Specialized Workshops that together spanned 1 week to allow an exhaustive and thorough coverage of all major issues in bioanalysis of biomarkers, immunogenicity, gene therapy, cell therapy and vaccines.Moreover, in-depth workshops on "EU IVDR 2017/746 Implementation and impact for the Global Biomarker Community: How to Comply with this NEW Regulation" and on "US FDA/OSIS Remote Regulatory Assessments (RRAs)" were the special features of the 17th edition.As in previous years, WRIB continued to gather a wide diversity of international, industry opinion leaders and regulatory authority experts working on both small and large molecules as well as gene, cell therapies and vaccines to facilitate sharing and discussions focused on improving quality, increasing regulatory compliance, and achieving scientific excellence on bioanalytical issues.This 2023 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2023 edition of this comprehensive White Paper has been divided into three parts for editorial reasons.This publication covers the recommendations on Mass Spectrometry Assays, Regulated Bioanalysis/BMV (Part 1A) and Regulatory Inputs (Part 1B). Part 2 (Biomarkers, IVD/CDx, LBA and Cell-Based Assays) and Part 3 (Gene Therapy, Cell therapy, Vaccines and Biotherapeutics Immunogenicity) are published in volume 16 of Bioanalysis, issues 7 and 8 (2024), respectively.

Myocarditis in an FIP-Diseased Cat with FCoV M1058L Mutation: Clinical and Pathological Changes.

An 8-month-old intact male domestic shorthair cat was referred to the Emergency Service of the Veterinary Teaching Hospital (VTH) of the Department of Veterinary Science of the University of Parma (Italy) from the Parma municipal multi-cat shelter, during the winter season (January 2023), for lethargy, anorexia, hypothermia, and hypoglycemia. At the VTH, upon cardiologic examination, an increase in heart rate, under normal blood pressure conditions, was detected. Signalment, clinical history, basal metabolic panel (BMP), ultrasound investigations, and cytological findings were all consistent with a diagnosis of feline infectious peritonitis (FIP). FIP was confirmed in the effusive abdominal fluid by a molecular genetic test (real-time PCR for feline coronavirus RNA). The molecular genetic investigation also detected an FCoV S gene single-nucleotide mutation: biotype M1058L. At necropsy, an effusive collection was recorded in the abdomen, thoracic cavity, and pericardium sac. White parenchymal nodules, of about 1 mm diameter, were found on the surface and deep in the lungs, liver, kidneys, and heart. Histopathology revealed the typical FIP pyogranulomatous vasculitis and IHC confirmed the presence of the FIP virus (FIPV) antigen. The most relevant histopathological finding was the myocarditis/myocardial necrosis associated with the presence of the S gene-mutated FCoV (M1058L biotype). This is the first case of myocarditis in a cat positive for the FCoV/FIP M1058L biotype. Further studies are necessary to support the mutated FCoV M1058L biotype, as an uncommon, but possible, causative pathogen of myocarditis in FCoV/FIP-positive cats. Studies including several FCoV/FIP M1058L-positive cases could allow us to make a correlation with heart gross pathology, histopathology, and immunolocalization of the FCoV/FIP M1058L biotype in the myocardium. The investigation will potentially allow us to determine the effective tropism of the FCoV/FIP M1058L biotype for myocardiocytes or whether myocardiocyte lesions are evident in the presence of concomitant causes related to the patient, its poor condition, or external environmental distress such as cold season, and whether the aforementioned concomitant events are correlated.

The effects of work on cognitive functions: a systematic review.

Introduction: Cognitive functions play a crucial role in individual's life since they represent the mental abilities necessary to perform any activity. During working life, having healthy cognitive functioning is essential for the proper performance of work, but it is especially crucial for preserving cognitive abilities and thus ensuring healthy cognitive aging after retirement. The aim of this paper was to systematically review the scientific literature related to the effects of work on cognitive functions to assess which work-related factors most adversely affect them. Method: We queried the PubMed and Scopus electronic databases, in February 2023, according to the PRISMA guidelines (PROSPERO ID number = CRD42023439172), and articles were included if they met all the inclusion criteria and survived a quality assessment. From an initial pool of 61,781 papers, we retained a final sample of 64 articles, which were divided into 5 categories based on work-related factors: shift work (n = 39), sedentary work (n = 7), occupational stress (n = 12), prolonged working hours (n = 3), and expertise (n = 3). Results: The results showed that shift work, occupational stress, and, probably, prolonged working hours have detrimental effects on cognitive functioning; instead, results related to sedentary work and expertise on cognitive functions are inconclusive and extremely miscellaneous. Discussion: Therefore, workplace health and well-being promotion should consider reducing or rescheduling night shift, the creation of less demanding and more resourceful work environments and the use of micro-breaks to preserve workers' cognitive functioning both before and after retirement. Systematic review registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023439172, identifier CRD42023439172.

Exploring workability in an older working population: associations with cognitive functioning, sleep quality, and technostress.

Objective: This observational study investigates workability and its associations with cognitive functioning, sleep quality and technostress among an older working population, also shedding light on potential differences between two occupational categories with different work schedules. Methods: Workers aged over 50, employed in different working sectors (banking/finance, chemical and metal-mechanic industry) were administered a self- report questionnaire including Work Ability Index (WAI), cognitive tests (Stroop Color Task, Corsi Blocks, Digit Span), sleep quality questionnaires (Pittsburgh Sleep Quality Index-PSQI; Insomnia Severity Index-ISI; Ford Insomnia Response to Stress Test-FIRST) and technostress scale. Linear regression models evaluated associations among variables, interaction effects investigated potential moderators. Results: A total of 468 aged workers categorized as white (WCWs; N = 289, 62%) or blue collars (BCWs; N = 179, 38%) were enrolled; most BCWs (N = 104; 58%) were night shift workers. WCWs reported higher workability, cognitive functioning, sleep quality and lower technostress (except for invasion and privacy subscales) than BCWs. Associations between cognitive functioning and workability were statistically significant only for BCWs [slopes equal to 0.2 (0.33), 0.8 (0.34), -0.02 (0.001) for Memory Span Corsi, Block Span Digit and Interference Speed respectively]; additionally, sleep quality significantly moderated this association (p = 0.007). Higher levels of technostress were associated with lower workability, and this relationship was stronger for BCWs. Conclusion: The aging of the workforce has important implications for occupational health and safety. Our findings suggest potential interventions and protective measures to promote older workers' wellbeing; blue-collar workers particularly should benefit from tailored intervention to sustain workability and prevent technostress, considering the role of healthy sleep habits promotion.

Large-scale chemoproteomics expedites ligand discovery and predicts ligand behavior in cells.

Chemical modulation of proteins enables a mechanistic understanding of biology and represents the foundation of most therapeutics. However, despite decades of research, 80% of the human proteome lacks functional ligands. Chemical proteomics has advanced fragment-based ligand discovery toward cellular systems, but throughput limitations have stymied the scalable identification of fragment-protein interactions. We report proteome-wide maps of protein-binding propensity for 407 structurally diverse small-molecule fragments. We verified that identified interactions can be advanced to active chemical probes of E3 ubiquitin ligases, transporters, and kinases. Integrating machine learning binary classifiers further enabled interpretable predictions of fragment behavior in cells. The resulting resource of fragment-protein interactions and predictive models will help to elucidate principles of molecular recognition and expedite ligand discovery efforts for hitherto undrugged proteins.

Short-chain fatty acids modulate the IPEC-J2 cell response to pathogenic E. coli LPS-activated PBMC.

Intestinal disorders can affect pigs of any age, especially when animals are young and more susceptible to infections and environmental stressors. For instance, pathogenic E. coli can alter intestinal functions, thus leading to altered nutrient adsorption by interacting with local cells through lipopolysaccharide (LPS). Among several compounds studied to counteract the negative effects on the intestine, short-chain fatty acids (SCFA) were demonstrated to exert beneficial effects on gut epithelial cells and resident immune cells. In this study, acetate and propionate were tested for their beneficial effects in a co-culture model of IPEC-J2 and porcine PBMC pre-stimulated with LPS from E. coli 0111:B4 aimed at mimicking the interaction between intestinal cells and immune cells in an inflammatory/activated status. IPEC-J2 viability was partially reduced when co-cultured with activated PBMC and nitric oxide concentration increased. IPEC-J2 up-regulated innate and inflammatory markers, namely BD-1, TLR-4, IL-8, TNF-α, NF-κB, and TGF-ß. Acetate and propionate positively modulated the inflammatory condition by sustaining cell viability, reducing the oxidative stress, and down-regulating the expression of inflammatory mediators. TNF-α expression and secretion showed an opposite effect in IPEC-J2 depending on the extent of LPS stimulation of PBMC and TGF-ß modulation. Therefore, SCFA proved to mediate a differential effect depending on the degree and duration of inflammation. The expression of the tight junction proteins (TJp) claudin-4 and zonula occludens-1 was up-regulated by LPS while SCFA influenced TJp with a different kinetics depending on PBMC stimulation. The co-culture model of IPEC-J2 and LPS-activated PBMC proved to be feasible to address the modulation of markers related to anti-bacterial immunity and inflammation, and intestinal epithelial barrier integrity, which are involved in the in vivo responsiveness and plasticity to infections.

Short-term exposure to fine particulate matter exposure impairs innate immune and inflammatory responses to a pathogen stimulus: A functional study in the zebrafish model.

Short-term exposure to fine particulate matter (PM2.5) is associated with the activation of adverse inflammatory responses, increasing the risk of developing acute respiratory diseases, such as those caused by pathogen infections. However, the functional mechanisms underlying this evidence remain unclear. In the present study, we generated a zebrafish model of short-term exposure to a specific PM2.5, collected in the northern metropolitan area of Milan, Italy. First, we assessed the immunomodulatory effects of short-term PM2.5 exposure and observed that it elicited pro-inflammatory effects by inducing the expression of cytokines and triggering hyper-activation of both neutrophil and macrophage cell populations. Moreover, we examined the impact of a secondary infectious pro-inflammatory stimulus induced through the injection of Pseudomonas aeruginosa lipopolysaccharide (Pa-LPS) molecules after exposure to short-term PM2.5. In this model, we demonstrated that the innate immune response was less responsive to a second pro-inflammatory infectious stimulus. Indeed, larvae exhibited dampened leukocyte activation and impaired production of reactive oxygen species. The obtained results indicate that short-term PM2.5 exposure alters the immune microenvironment and affects the inflammatory processes, thus potentially weakening the resistance to pathogen infections.

Determination of four aminoglycoside antibiotics and spectinomycin in feed at cross-contamination level: Development and in-house validation of a LC-MS/MS method for enforcing EU Regulation.

Combating antimicrobial resistance is a top priority worldwide involving a concerted action by several high-level institutions and organisations in the health sector. To ensure that a meaningful progress is achieved, several campaigns and political initiatives have been launched targeting the health professionals, the industry, the farmers, and the general public. The Regulation (EU) 2019/4 on medicated feed contains provisions for the limitation and control of the contamination of non-target compound feed with 24 antimicrobials. The purpose of this work was to develop a reliable and effective method for the determination of four aminoglycoside antibiotics (apramycin, paromomycin, tobramycin and neomycin) and spectinomycin in feed at cross-contamination level, where an absolute lack of suitable methods was identified. Four candidate methods described in the literature failed to provide adequate recoveries of all analytes. Therefore, an in-depth investigation was carried out to identify the bottleneck variable. The optimised method was then in-house validated and showed performance features appropriate for the intended purpose. The selected compounds could be analysed by LC-MS/MS in five animal feeds with LOQs between 2.6 and 9.2 µg kg-1 for the AGs and between 28 and 86 µg kg-1 for spectinomycin. Using isotopically labelled internal standards, the recovery rates varied from 63 % to 103 % and the intermediate precision (RSDip) varied from 1.1 % to 14 %. This work represents a step forward in the reliable determination of antibiotics in compound feed as the developed method has shown to be precise and sensitive. It is expected that this method gains wide acceptance and can supplement the legislation with effective control tools for antibiotic residues.

Recommendations and feedback from the European Bioanalysis Forum Workshop: 1 year into ICH M10 - keeping our finger on the pulse.

The ICH M10 guideline on bioanalytical method validation and sample analysis is being adopted since 2023. However, and inevitably, some paragraphs or requirements remain ambiguous and are open for different interpretations. In support of a harmonized interpretation by the industry and health authorities, the European Bioanalysis Forum organized a workshop on 14 November 2023 in Barcelona, Spain, to discuss unclear and/or ambiguous paragraphs which were identified by the European Bioanalysis Forum community and delegates of the workshop prior to the workshop. This manuscript reports back from the workshop with recommendations and aims at continuing an open scientific discussion within the industry and with regulators in support of a science-driven guideline for the bioanalytical community and in line with the ICH mission - that is, achieve greater harmonization worldwide to ensure that safe, effective and high-quality medicines are developed and registered in the most resource-efficient manner.

Environmental and Lifestyle Cancer Risk Factors: Shaping Extracellular Vesicle OncomiRs and Paving the Path to Cancer Development.

Intercellular communication has been transformed by the discovery of extracellular vesicles (EVs) and their cargo, including microRNAs (miRNAs), which play crucial roles in intercellular signaling. These EVs were previously disregarded as cellular debris but are now recognized as vital mediators of biological information transfer between cells. Furthermore, they respond not only to internal stimuli but also to environmental and lifestyle factors. Identifying EV-borne oncomiRs, a subset of miRNAs implicated in cancer development, could revolutionize our understanding of how environmental and lifestyle exposures contribute to oncogenesis. To investigate this, we studied the plasma levels of EV-borne oncomiRs in a population of 673 women and 238 men with a body mass index > 25 kg/m2 (SPHERE population). The top fifty oncomiRs associated with the three most common cancers in women (breast, colorectal, and lung carcinomas) and men (lung, prostate, and colorectal carcinomas) were selected from the OncomiR database. Only oncomiRs expressed in more than 20% of the population were considered for statistical analysis. Using a Multivariate Adaptive Regression Splines (MARS) model, we explored the interactions between environmental/lifestyle exposures and EV oncomiRs to develop optimized predictor combinations for each EV oncomiR. This innovative approach allowed us to better understand miRNA regulation in response to multiple environmental and lifestyle influences. By uncovering non-linear relationships among variables, we gained valuable insights into the complexity of miRNA regulatory networks. Ultimately, this research paves the way for comprehensive exposome studies in the future.

Advances in selenium supplementation: From selenium-enriched yeast to potential selenium-enriched insects, and selenium nanoparticles.

Selenium (Se) is an essential micronutrient that plays an important role in animal and human development and physiological homoeostasis. This review surveys the role of Se in the environment, plants and animal bodies, and discusses data on Se biofortification with different sources of supplementation, from inorganic to organic forms, with special focus on Se-enriched yeast (Se-yeast). Although Se-yeast remains one of the main sources of organic Se, other emerging and innovative sources are reviewed, such as Se-enriched insects and Se-nanoparticles and their potential use in animal nutrition. Se-enriched insects are discussed as an option for supplying Se in organic form to livestock diets. Se-nanoparticles are also discussed, as they represent a more biocompatible and less toxic source of inorganic Se for animal organisms, compared to selenite and selenate. We also provide up to date information on the legal framework in the EU, USA, and Canada of Se that is contained in feed additives. From the scientific evidence available in the literature, it can be concluded that among the inorganic forms, sodium selenite is still one of the main options, whereas Se-yeast remains the primary organic form. However, other potential sources such as Se-enriched insects and Se-nanoparticles are being investigated as they could potentially combine a high bioavailability and reduced Se emissions in the environment.

Temporal, Kinematic and Kinetic Variables Derived from a Wearable 3D Inertial Sensor to Estimate Muscle Power during the 5 Sit to Stand Test in Older Individuals: A Validation Study.

The 5-Sit-to-stand test (5STS) is widely used to estimate lower limb muscle power (MP). An Inertial Measurement Unit (IMU) could be used to obtain objective, accurate and automatic measures of lower limb MP. In 62 older adults (30 F, 66 ± 6 years) we compared (paired t-test, Pearson's correlation coefficient, and Bland-Altman analysis) IMU-based estimates of total trial time (totT), mean concentric time (McT), velocity (McV), force (McF), and MP against laboratory equipment (Lab). While significantly different, Lab vs. IMU measures of totT (8.97 ± 2.44 vs. 8.86 ± 2.45 s, p = 0.003), McV (0.35 ± 0.09 vs. 0.27 ± 0.10 m∙s-1, p < 0.001), McF (673.13 ± 146.43 vs. 653.41 ± 144.58 N, p < 0.001) and MP (233.00 ± 70.83 vs. 174.84 ± 71.16 W, p < 0.001) had a very large to extremely large correlation (r = 0.99, r = 0.93, and r = 0.97 r = 0.76 and r = 0.79, respectively, for totT, McT, McF, McV and MP). Bland-Altman analysis showed a small, significant bias and good precision for all the variables, but McT. A sensor-based 5STS evaluation appears to be a promising objective and digitalized measure of MP. This approach could offer a practical alternative to the gold standard methods used to measure MP.

Promoting health and productivity among ageing workers: a longitudinal study on work ability, biological and cognitive age in modern workplaces (PROAGEING study).

BACKGROUND: Large changes in ageing population and in retirement age are increasing the number of older people in the workforce, raising many challenges for policymakers in promoting employment opportunities and health for older workers. In this respect, longitudinal assessments of workability, well-being perception and cognitive skills over time may allow to detect factors influencing workers' health. Moreover, new available molecular markers permit the measurement of biological age and age-related changes. Most studies analysed one aspect at time (psychological, biological, labour productivity), without considering their interaction. Aims of the study are to evaluate the relationship between workability, cognitive skills, and biological age in a population of ageing workers; to conduct a cross-sectional analysis to assess the impact of occupational exposures on workability, cognitive skills, and biological age; to evaluate inter-individuals changes in a prospective analysis with a re-evaluation of each worker. METHODS: Our study plans to enrol 1000 full-time workers, aged over 50, undergoing the medical surveillance required by the current Italian Legislation. Data collection includes information about: (a) work ability and psychosocial risk factors (work ability index, HSE Management Standard-21 item, Utrecht Work Engagement Scale, World Health Organisation-Five, Well-Being Index, job satisfaction, general well-being, technostress); (b) cognitive skills (Stroop Color and Word test, Simon task, Corsi's block-tapping test, Digit span test); (c) sleep habits and psychological well-being (Pittsburgh Sleep Quality Index, Insomnia Severity Index, Ford Insomnia Response to Stress Test; Symptom Check List 90, Psychological Well-Being Index, Profile of Mood State, Beck Depression Inventory, Beck Anxiety Inventory, Perceived Stress Scale, Brief COPE); (d) biological age (telomere length, DNA methylation) for 500 workers. All workers will repeat the evaluation after one year. DISCUSSION: This study aims to increase our knowledge about interactions between work ability, cognitive ability, well-being perception and psychological status also by including molecular markers, with a longitudinal and multidisciplinary approach. By bringing better insights into the relationship between risk factors and their impact on perceived and biological health, this study also aims at identifying possible interventions and protective measures to ensure aged workers' well-being, consistent with all the eminent calls for actions promoted by key International and European labour organizations.

Acetate and propionate effects in response to LPS in a porcine intestinal co-culture model.

BACKGROUND: The interest in acetate and propionate as short chain fatty acids (SCFA) derives from research on alternative strategies to the utilization of antibiotics in pig farms. SCFA have a protective role on the intestinal epithelial barrier and improve intestinal immunity by regulating the inflammatory and immune response. This regulation is associated with an increase in intestinal barrier integrity, mediated by the enhancement of tight junction protein (TJp) functions, which prevent the passage of pathogens through the paracellular space. The purpose of this study was to evaluate the effect of in vitro supplementation with SCFA (5 mM acetate and 1 mM propionate) on viability, nitric oxide (NO) release (oxidative stress), NF-κB gene expression, and gene and protein expression of major TJp (occludin [OCLN], zonula occludens-1 [ZO-1], and claudin-4 [CLDN4]) in a porcine intestinal epithelial cell (IPEC-J2) and peripheral blood mononuclear cell (PBMC) co-culture model upon LPS stimulation, through which an acute inflammatory state was simulated. RESULTS: Firstly, the inflammatory stimulus induced by LPS evaluated in the IPEC-J2 monoculture was characterized by a reduction of viability, gene expression of TJp and OCLN protein synthesis, and an increase of NO release. The response evaluated in the co-culture showed that acetate positively stimulated the viability of both untreated and LPS-stimulated IPEC-J2 and reduced the release of NO in LPS-stimulated cells. Acetate also promoted an increase of gene expression of CLDN4, ZO-1, and OCLN, and protein synthesis of CLDN4, OCLN and ZO-1 in untreated and LPS-stimulated cells. Propionate induced a reduction of NO release in both untreated and LPS-stimulated IPEC-J2. In untreated cells, propionate induced an increase of TJp gene expression and of CLDN4 and OCLN protein synthesis. Contrarily, propionate in LPS-stimulated cells induced an increase of CLDN4 and OCLN gene expression and protein synthesis. PBMC were influenced by acetate and propionate supplementation, in that NF-κB expression was strongly downregulated in LPS-stimulated cells. CONCLUSIONS: The present study demonstrates the protective effect of acetate and propionate upon acute inflammation by regulating epithelial tight junction expression and protein synthesis in a co-culture model, which simulates the in vivo interaction between epithelial intestinal cells and local immune cells.

Osmolarity modulates the de-differentiation of horse articular chondrocytes during cell expansion in vitro: implications for tissue engineering in cartilage repair.

Due to the importance of joint disease and ostearthritis (OA) in equine athletes, new regenerative treatments to improve articular cartilage repair after damage are gaining relevance. Chondrocyte de-differentiation, an important pathogenetic mechanism in OA, is a limiting factor when differentiated articular chondrocytes are used for cell-based therapies. Current research focuses on the prevention of this de-differentiation and/or on the re-differentiation of chondrocytes by employing different strategies in vitro and in vivo. Articular chondrocytes normally live in a condition of higher osmolarity (350-450 mOsm/L) compared to normal physiological fluids (~ 300 mOsm/L) and some studies have demonstrated that osmolarity has a chondroprotective effect in vitro and in vivo. Therefore, the response of horse articular chondrocytes to osmolarity changes (280, 380, and 480 mOsm/L) was studied both in proliferating, de-differentiated chondrocytes grown in adhesion, and in differentiated chondrocytes grown in a 3D culture system. To this aim, cell proliferation (cell counting), morphology (optical microscopy), and differentiation (gene expression of specific markers) were monitored along with the expression of osmolyte transporters involved in volume regulation [betaine-GABA transporter (BGT-1), taurine transporter (SLC6A6), and neutral amino acid transporter (SNAT)] real-time qPCR. Proliferating chondrocytes cultured under hyperosmolar conditions showed low proliferation, spheroidal morphology, a significant reduction of de-differentiation markers [collagen type I (Col1) and RUNX2] and an increase of differentiation markers [collagen type II (Col2) and aggrecan]. Notably, a persistently high level of BGT-1 gene expression was maintained in chondrocyte cultures at 380 mOsm/L, and particularly at 480 mOsm/L both in proliferating and differentiated chondrocytes. These preliminary data encourage the study of osmolarity as a microenvironmental co-factor to promote/maintain chondrocyte differentiation in both 2D and 3D in vitro culture systems.

Aggrephagy at a glance.

Cells keep their proteome functional by the action of the proteostasis network, composed of the chaperones, the ubiquitin-proteasome system and autophagy. The decline of this network results in the accumulation of protein aggregates and is associated with aging and disease. In this Cell Science at a Glance and accompanying poster, we provide an overview of the molecular mechanisms of the removal of protein aggregates by a selective autophagy pathway, termed aggrephagy. We outline how aggrephagy is regulated by post-translational modifications and via auxiliary proteins. We further describe alternative aggrephagy pathways in physiology and their disruption in pathology. In particular, we discuss aggrephagy pathways in neurons and accumulation of protein aggregates in a wide range of diseases. Finally, we highlight strategies to reprogram aggrephagy to treat protein aggregation diseases.

Easy Prediction of the Maximal Lactate Steady-State in Young and Older Men and Women.

Maximal Lactate steady-state (MLSS) demarcates sustainable from unsustainable exercise and is used for evaluation/monitoring of exercise capacity. Still, its determination is physically challenging and time-consuming. This investigation aimed at validating a simple, submaximal approach based on blood lactate accumulation ([Δlactate]) at the third minute of cycling in a large cohort of men and women of different ages. 68 healthy adults (40♂, 28♀, 43 ± 17 years (range 19-78), VO2max 45 ± 11 ml-1·kg-1·min-1 (25-68)) performed 3-5 constant power output (PO) trials with a target duration of 30 minutes to determine the PO corresponding to MLSS. During each trial, [Δlactate] was calculated as the difference between the third minute and baseline. A multiple linear regression was computed to estimate MLSS based on [Δlactate], subjects` gender, age and the trial PO. The estimated MLSS was compared to the measured value by paired t-test, correlation, and Bland-Altman analysis. The group mean value of estimated MLSS was 180 ± 51 W, not significantly different from (p = 0.98) and highly correlated with (R2 = 0.89) measured MLSS (180 ± 54 watts). The bias between values was 0.17 watts, and imprecision 18.2 watts. This simple, submaximal, time- and cost-efficient test accurately and precisely predicts MLSS across different samples of healthy individuals (adjusted R2 = 0.88) and offers a practical and valid alternative to the traditional MLSS determination.

Nano-adjuvanted dry powder vaccine for the mucosal immunization against airways pathogens.

Nasal vaccination has been shown to provide optimal protection against respiratory pathogens. However, mucosal vaccination requires the implementation of specific immunization strategies to improve its effectiveness. Nanotechnology appears a key approach to improve the effectiveness of mucosal vaccines, since several nanomaterials provide mucoadhesion, enhance mucosal permeability, control antigen release and possess adjuvant properties. Mycoplasma hyopneumoniae is the main causative agent of enzootic pneumonia in pigs, a respiratory disease responsible for considerable economic losses in the pig farming worldwide. The present work developed, characterized, and tested in vivo an innovative dry powder nasal vaccine, obtained from the deposition on a solid carrier of an inactivated antigen and a chitosan-coated nanoemulsion, as an adjuvant. The nanoemulsion was obtained through a low-energy emulsification technique, a method that allowed to achieve nano droplets in the order of 200 nm. The oil phase selected was alpha-tocopherol, sunflower oil, and poly(ethylene glycol) hydroxystearate used as non-ionic tensioactive. The aqueous phase contained chitosan, which provides a positive charge to the emulsion, conferring mucoadhesive properties and favoring interactions with inactivated M. hyopneumoniae. Finally, the nanoemulsion was layered with a mild and scalable process onto a suitable solid carrier (i.e., lactose, mannitol, or calcium carbonate) to be transformed into a solid dosage form for administration as dry powder. In the experimental study, the nasal vaccine formulation with calcium carbonate was administered to piglets and compared to intramuscular administration of a commercial vaccine and of the dry powder without antigen, aimed at evaluating the ability of IN vaccination to elicit an in vivo local immune response and a systemic immune response. Intranasal vaccination was characterized by a significantly higher immune response in the nasal mucosa at 7 days post-vaccination, elicited comparable levels of Mycoplasma-specific IFN-γ secreting cells and comparable, if not higher, responsiveness of B cells expressing IgA and IgG in peripheral blood mononuclear cells, with those detected upon a conventional intramuscular immunization. In conclusion, this study illustrates a simple and effective strategy for the development of a dry powder vaccine formulation for nasal administration which could be used as alternative to current parenteral commercial vaccines.