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.

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.

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.

Traffic-Related Air Pollution and Ground-Level Ozone Associated Global DNA Hypomethylation and Bulky DNA Adduct Formation.

Studies have indicated that air pollution, including surface-level ozone (O3), can significantly influence the risk of chronic diseases. To better understand the carcinogenic mechanisms of air pollutants and identify predictive disease biomarkers, we examined the association between traffic-related pollutants with DNA methylation alterations and bulky DNA adducts, two biomarkers of carcinogen exposure and cancer risk, in the peripheral blood of 140 volunteers-95 traffic police officers, and 45 unexposed subjects. The DNA methylation and adduct measurements were performed by bisulfite-PCR and pyrosequencing and 32P-postlabeling assay. Airborne levels of benzo(a)pyrene [B(a)P], carbon monoxide, and tropospheric O3 were determined by personal exposure biomonitoring or by fixed monitoring stations. Overall, air pollution exposure was associated with a significant reduction (1.41 units) in global DNA methylation (95% C.I. -2.65-0.04, p = 0.026). The decrement in ALU repetitive elements was greatest in the policemen working downtown (95% C.I. -3.23--0.49, p = 0.008). The DNA adducts were found to be significantly increased (0.45 units) in the municipal officers with respect to unexposed subjects (95% C.I. 0.02-0.88, p = 0.039), mainly in those who were controlling traffic in downtown areas (95% C.I. 0.39-1.29, p < 0.001). Regression models indicated an increment of ALU methylation at higher B(a)P concentrations (95% C.I. 0.03-0.60, p = 0.032). Moreover, statistical models showed a decrement in ALU methylation and an increment of DNA damage only above the cut-off value of 30 µg/m3 O3. A significant increment of 0.73 units of IL-6 gene methylation was also found in smokers with respect to non-smokers. Our results highlighted the role of air pollution on epigenetic alterations and genotoxic effects, especially above the target value of 30 µg/m3 surface-level O3, supporting the necessity for developing public health strategies aimed to reduce traffic-related air pollution molecular alterations.

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.

PM2 .5, PM10 and bronchiolitis severity: A cohort study.

BACKGROUND: A few studies suggest that particulate matter (PM) exposure might play a role in bronchiolitis. However, available data are mostly focused on the risk of hospitalization and come from retrospective studies that provided conflicting results. This prospective study investigated the association between PM (PM2.5 and PM10 ) exposure and the severity of bronchiolitis. METHODS: This prospective cohort study was conducted between November 2019 and February 2020 at the pediatric emergency department of the Fondazione IRCCS Ca' Ospedale Maggiore Policlinico, Milan, Italy. Infants <1 year of age with bronchiolitis were eligible. The bronchiolitis severity score was assessed in each infant and a nasal swab was collected to detect respiratory viruses. The daily PM10 and PM2.5 exposure in the 29 preceding days were considered. Adjusted regression models were employed to evaluate the association between the severity score and PM10 and PM2.5 exposure. RESULTS: A positive association between the PM2.5 levels and the severity score was found at day-2 (ß 0.0214, 95% CI 0.0011-0.0417, p = .0386), day-5 (ß 0.0313, 95% CI 0.0054-0.0572, p = .0179), day-14 (ß 0.0284, 95% CI 0.0078-0.0490, p = .0069), day-15 (ß 0.0496, 95% CI 0.0242-0.0750, p = .0001) and day-16 (ß 0.0327, 95% CI 0.0080-0.0574, p = .0093).Similar figures were observed considering the PM10 exposure and limiting the analyses to infants with respiratory syncytial virus. CONCLUSION: This study shows for the first time a direct association between PM2.5 and PM10 levels and the severity of bronchiolitis.

Maternal air pollution exposure during the first trimester of pregnancy and markers of inflammation and endothelial dysfunction.

BACKGROUND: Maternal exposure to air pollutants has been associated with pregnancy complications and adverse birth outcomes. Endothelial dysfunction, an imbalance in vascular function, during pregnancy is considered a key element in the development of pre-eclampsia. Environmental exposure to particulate matter (PM) during the first trimester of pregnancy might increase maternal inflammatory status thus affecting fetal growth, possibly leading to preterm delivery. OBJECTIVES: The purpose of the study was to evaluate possible effects of PM10 and PM2.5 exposure on fetal growth in healthy pregnant women at the end of the first trimester of pregnancy by investigating the relationship between circulating biomarkers of inflammation (IL-6), early systemic prothrombotic effects (CRP, plasma fibrinogen, PAI-1) and endothelial dysfunction (sICAM-1 and sVCAM-1). METHODS: 295 pregnant women were recruited. Individual PM exposure was assigned to each subject by calculating the mean of PM10 and PM2.5 daily values observed during the 30, 60, and 90 days preceding enrolment (long-term) and single lag days back to fourteen days (short-term), and circulating plasma biomarkers were determined. RESULTS: For long-term exposure, we observed an increase in sVCAM-1 and a decrease of PAI-1 levels for each 10 µg/m3 increase in PM10 concentration. Decreases in IL-6 and CRP levels were associated with each 10 µg/m3 PM2.5 increase. For short-term exposure, the levels of sVCAM-1 and PAI-1 were found to be associated with PM10 exposure, whereas fibrinogen levels were associated with PM2.5 exposure. Maternal plasmatic fibrinogen levels were negatively associated with the crown-rump length (p-value = 0.008). DISCUSSION: The present study showed that both long- and short-term exposures to PM are associated with changes in circulating levels of biomarkers in pregnant women reflecting systemic inflammation and endothelial dysfunction/activation. Our findings support the hypothesis that inflammation and endothelial dysfunction might have a central role in modulating the detrimental effects of air pollution exposure during pregnancy.

Performance and Anthropometrics of Classic Powerlifters: Which Characteristics Matter?

ABSTRACT: Ferrari, L, Colosio, AL, Teso, M, and Pogliaghi, S. Performance and anthropometrics of classic powerlifters: Which characteristics matter? J Strength Cond Res 36(4): 1003-1010, 2022-The purpose of this study is: (a) provide normative performance and anthropometric data of Southern European classic powerlifters of both sexes; (b) determine the possible relationships between these variables and performance; and (c) develop population-specific predictive equations for single lifts and overall powerlifting performance. During an unofficial national-level competition, we recruited 74 athletes (51 men and 23 women) and recorded their individual, anthropometric, and performance characteristics and divided them into sex and 2 performance categories based on their Wilks points. Weaker (<370 Wilks points) and stronger (>370 Wilks points) athletes of both sexes were compared by two-way analysis of variance. Simple correlation and multiple linear regression between individual/anthropometric characteristics and performance were modeled. We applied a step-forward multiple linear regression model to predict single lifts and overall performance. All parameters significantly differed between sexes (p < 0.05 for all comparisons). Stronger male athletes had a significantly larger neck (42 ± 2.8 cm; effect size [ES] = 0.59), and flexed (40.6 ± 3.3 cm; ES = 1.18) and relaxed upper-arm (37.5 ± 3.1 cm; ES = 1.34) and thigh girths (63.6 ± 7.0 cm; ES = 0.77) compared to weaker male athletes. Furthermore, stronger women had significantly larger flexed (32.6 ± 3.3 cm; ES = 0.88) and relaxed upper-arm (33 ± 1.5 cm; ES = 2.28) and chest girths (99.3 ± 9.2 cm; ES = 1.10) compared to weaker female athletes. A combination of experience, fat mass, and upper-limb and lower-limb muscle mass indexes can accurately and precisely predict overall and individual lift performance (r2 ≥ 0.83 for all the predictions). This is the first study to provide normative performance and anthropometric data in Southern European male and female powerlifters.

Identification of a new potential plasmatic biomarker panel for the diagnosis of malignant pleural mesothelioma.

BACKGROUND: Malignant pleural mesothelioma (MPM) is a rare highly aggressive tumor strongly associated with asbestos exposure and characterized by poor prognosis. Currently, diagnosis is based on invasive techniques, thus there is a need of identifying non-invasive biomarkers for early detection of the disease among asbestos-exposed subjects. In the present study, we measured the plasmatic concentrations of Mesothelin, Fibulin-3, and HMGB1 protein biomarkers, and of hsa-miR-30e-3p and hsa-miR-103a-3p Extracellular-Vesicles- embedded micro RNAs (EV-miRNAs). We tested the ability of these biomarkers to discriminate between MPM and PAE subjects alone and in combination. METHODS: the study was conducted on a population of 26 patients with MPM and 54 healthy subjects with previous asbestos exposure (PAE). Mesothelin, Fibulin-3, and HMGB1 protein biomarkers were measured by the enzyme-linked immunosorbent assay (ELISA) technique; the levels of hsa-miR-30e-3p and hsa-miR-103a-3p EV-miRNAs was assessed by quantitative real-time PCR (qPCR). RESULTS: the most discriminating single biomarker resulted to be Fibulin-3 (AUC 0.94 CI 95% 0.88-1.0; Sensitivity 88%; Specificity 87%). After investigating the different possible combinations, the best performance was obtained by the three protein biomarkers Mesothelin, Fibulin-3, and HMGB1 (AUC 0.99 CI 95% 0.97-1.0; Sensitivity 96%; Specificity 93%). CONCLUSIONS: the results obtained contribute to identifying new potential non-invasive biomarkers for the early diagnosis of MPM in high-risk asbestos-exposed subjects. Further studies are needed to validate the evidence obtained, in order to assess the reliability of the proposed biomarker panel.

Adoptive transfer of neoantigen-specific T-cell therapy is feasible in older patients with higher-risk myelodysplastic syndrome.

BACKGROUND: Myelodysplastic syndromes (MDS) represent the most common type of acquired bone marrow failure in adults and is characterized by ineffective maturation of myeloid precursor cells and peripheral cytopenias associated with higher rates of infection, bleeding and transfusion dependence. In higher-risk patients with MDS who relapse or do not respond after standard hypomethylating agent (HMA) therapy, the 2-year survival rate is 15%. METHODS: Here the authors report the feasibility and safety of a novel experimental T-cell therapy called personalized adoptive cell therapy, which selects, immunizes and expands T cells against MDS-specific mutations and is targeted to patient-specific tumor cell neoantigens. Somatic mutations serve as the pathogenic drivers of cancer, including MDS, as these transformative genetic mutations may generate novel immunogenic proteins (i.e., neopeptides and possible neoantigens) that may be targeted therapeutically. RESULTS: The authors demonstrate that the adaptive immune system can be trained ex vivo to recognize neopeptides as neoantigens and that the infusion of culture-expanded, neoantigen-immunized autologous T cells has been feasible and safe in the three patients treated to date. DISCUSSION: The authors report on early results from their first-in-human phase 1 clinical trial that aims to assess the safety and tolerability of this novel form of adoptive T-cell immunotherapy for HMA-refractory patients with higher-risk MDS.

In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome, a disease with low mutational burden.

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34+) and normal (CD3+) cells isolated from the patients' blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4+ response and 11 (34%) induced a CD8+ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.

SARS-CoV-2 tests in occupational settings: what you look for is what you get.

During the last month of 2019, a new Coronavirus from China started to spread all around the world causing a pandemic emergency still ongoing. The outbreak made imperative the need for diagnostic and screening tests that could identify the current and past infection state of an individual. Occupational medicine is facing a very demanding challenge, as the pandemic set off the need to re-evaluate many aspects of workplace safety. A fundamental role has been played by tests used to diagnose COVID-19 and to isolate infected asymptomatic subjects, with a view to the viral evolution and the emerging variants. However, the need for the urgent set-up of new methods for assessing both new and past infections has resulted in a large number of methods, not always comparable with each other, in terms of laboratory techniques, viral antigens used for detection, and class of antibodies detected. These factors make it difficult to understand the serological test results and their possible application. In this paper, we reviewed the types of assays currently available, to address some key aspects that characterize each technique, and might have an impact on results interpretation.

Dysregulation of NIPBL leads to impaired RUNX1 expression and haematopoietic defects.

The transcription factor RUNX1, a pivotal regulator of HSCs and haematopoiesis, is a frequent target of chromosomal translocations, point mutations or altered gene/protein dosage. These modifications lead or contribute to the development of myelodysplasia, leukaemia or platelet disorders. A better understanding of how regulatory elements contribute to fine-tune the RUNX1 expression in haematopoietic tissues could improve our knowledge of the mechanisms responsible for normal haematopoiesis and malignancy insurgence. The cohesin RAD21 was reported to be a regulator of RUNX1 expression in the human myeloid HL60 cell line and during primitive haematopoiesis in zebrafish. In our study, we demonstrate that another cohesin, NIPBL, exerts positive regulation of RUNX1 in three different contexts in which RUNX1 displays important functions: in megakaryocytes derived from healthy donors, in bone marrow samples obtained from adult patients with acute myeloid leukaemia and during zebrafish haematopoiesis. In this model, we demonstrate that alterations in the zebrafish orthologue nipblb reduce runx1 expression with consequent defects in its erythroid and myeloid targets such as gata1a and spi1b in an opposite way to rad21. Thus, also in the absence of RUNX1 translocation or mutations, additional factors such as defects in the expression of NIPBL might induce haematological diseases.

Toward Accurate and Robust Liquid Chromatography-Mass Spectrometry-Based Quantification of Antibody Biotherapeutics in Tissues.

Liquid chromatography-mass spectrometry (LC-MS) affords a highly promising solution for absolute quantification of biotherapeutics/targets in tissues, which is critical for drug development. Nonetheless, accurate/robust tissue quantification remains challenging largely owing to the lack of optimal approaches to address the following fundamental prerequisites: (i) efficient removal of residual blood without losing tissue-associated biotherapeutics; (ii) an optimal method to exhaustively/quantitatively recover target proteins from tissues; and (iii) an appropriate strategy to prepare calibration/quality-control samples to ensure accurate tissue analysis. Here, we devised novel analytical procedures enabling extensive and systematic investigation of the above issues and thereby development of optimal strategies for accurate tissue analysis. Key discoveries include: first, using a novel procedure of sequential administration of nonlabeled and then stable-isotope-labeled monoclonal antibody (mAb); it was determined that perfusion with three blood volumes of heparinized saline is optimal, achieving efficient blood removal (95-99%) and low quantitative bias (0.5-13%); second, a reference sample set established by mass-balanced, exhaustive extraction, permitted accurate measurement of absolute protein recovery from tissues of dosed animals; with this method, we found mAb biotherapeutics present in free-(49.3-75.4%) and bound-forms (24.6-50.7%) in tissues, even without a target; therefore, a denaturing detergent buffer is necessary for exhaustive extraction (recovery>90%); third, overnight-incubation of calibration samples after spiking mAb to tissue was found to improve quantitative accuracy, especially for nondenaturing buffer extraction. These investigations established the critical parameters and optimal protocols that can be universally applied to achieve accurate and robust quantification of biotherapeutics/targets in tissues. As a proof of concept, we conducted the first-ever extensive pharmacokinetics measurement of mAb in major tissues with a LC-MS-based method, where interesting features of mAb tissue disposition were observed.

Profile of gamma-delta (γδ) T lymphocytes in the peripheral blood of crossbreed dogs during stages of life and implication in aging.

BACKGROUND: Data on gamma-delta (γδ) T lymphocytes in the peripheral blood of dogs are scant, related only to healthy pure breed dogs and limited to a restricted age range. The aim of the study was to investigate the modulation of the γδ T lymphocyte (TCRγδ+) subpopulation in peripheral blood of crossbreed healthy dogs according to five identified stages of life: Puppy, Junior, Adult, Mature, Senior and to determine its implication in aging. A rigorous method of recruitment was used to minimize the influence of internal or external pressure on the immune response. Twenty-three intact female and twenty-four intact male dogs were enrolled. Blood samples were collected and immunophenotyping of peripheral blood T lymphocytes and γδ T cell subpopulations was performed. RESULTS: The percentage of γδ T cells in peripheral blood lymphocytes was comparable with the value of 2.5% published by Faldyna and co-workers (2001), despite the percentage reported was investigated in less arranged age range groups and coming from four different dog pure breeds, whereas our data were recorded on wider age range groups and coming from crossbreed dogs. Therefore, the γδ T cell percentage (2.5%) is consistent and points out that such value is breed-independent. Statistical analysis highlighted differences in both percentage and absolute γδ T cells according to the stage of life. γδ T cells decreased significantly in the peripheral blood of elder dogs (Senior group) in comparison with previous stages of life (Puppy, Junior, and Adult groups). Differences in γδ T cells are significant and they are reported, for the first time, related to dog aging. CONCLUSIONS: The study confirms dogs to be among the animals with a low TCRγδ+ cell profile. A decrease of the TCRγδ+ subpopulation percentage was observed in elder dogs. TCRγδ+ cells of group S were different from those of groups P, J, and A. The differences are reported for the first time in dog aging. Identifying the stage of life when the decrease of γδ T lymphocytes starts can be useful for providing a rationale for drafting a wellness plan trial to support thymus immune functions and mitigate its functional exhaustion.

Particulate Air Pollution, Clock Gene Methylation, and Stroke: Effects on Stroke Severity and Disability.

Circadian rhythm disturbances have been consistently associated with the development of several diseases, particularly cardiovascular diseases (CVDs). A central clock in the brain maintains the daily rhythm in accordance with the external environment. At the molecular level, the clock is maintained by "clock genes", the regulation of which is mainly due to DNA methylation, a molecular mechanism of gene expression regulation, able to react to and be reprogrammed by environmental exposure such as exposure to particulate matter (PM). In 55 patients with a diagnosis of acute ischemic stroke, we showed that PM2.5 exposure experienced before the event influenced clock genes methylation (i.e., circadian locomotor output cycles protein kaput CLOCK, period 2 PER2, cryprochrome 1 CRY1, Neuronal PAS Domain Protein 2 NPAS2), possibly modulating the patient prognosis after the event, as cryptochrome 1 CRY1 and period 1 PER1 methylation levels were associated with the Rankin score. Moreover, if PM2.5 annual average was low, CRY1/CRY2 methylation levels were positively associated with the National Institutes of Health Stroke Scale (NIHSS) score, whereas they were negatively associated if PM2.5 exposure was high. Whether epigenetic changes in clock genes need to be considered as a prognostic marker of stroke or rather a causal agent in stroke development remains to be determined. Further studies are needed to determine the role of clock gene methylation in regulating the response to and recovery after a stroke event.

Personalised Medicine: implication and perspectives in the field of occupational health.

"Personalised medicine" relies on identifying and integrating individual variability in genomic, biological, and physiological parameters, as well as in environmental and lifestyle factors, to define "individually" targeted disease prevention and treatment. Although innovative "omic" technologies supported the application of personalised medicine in clinical, oncological, and pharmacological settings, its role in occupational health practice and research is still in a developing phase. Occupational personalised approaches have been currently applied in experimental settings and in conditions of unpredictable risks, e.g.. war missions and space flights, where it is essential to avoid disease manifestations and therapy failure. However, a debate is necessary as to whether personalized medicine may be even more important to support a redefinition of the risk assessment processes taking into consideration the complex interaction between occupational and individual factors. Indeed, "omic" techniques can be helpful to understand the hazardous properties of the xenobiotics, dose-response relationships through a deeper elucidation of the exposure-disease pathways and internal doses of exposure. Overall, this may guide the adoption/implementation of primary preventive measures protective for the vast majority of the population, including most susceptible subgroups. However, the application of personalised medicine into occupational health requires overcoming some practical, ethical, legal, economical, and socio-political issues, particularly concerning the protection of privacy, and the risk of discrimination that the workers may experience. In this scenario, the concerted action of academic, industry, governmental, and stakeholder representatives should be encouraged to improve research aimed to guide effective and sustainable implementation of personalised medicine in occupational health fields.

HDAC8 regulates canonical Wnt pathway to promote differentiation in skeletal muscles.

Histone deacetylase 8 (HDAC8) is a class 1 histone deacetylase and a member of the cohesin complex. HDAC8 is expressed in smooth muscles, but its expression in skeletal muscle has not been described. We have shown for the first time that HDAC8 is expressed in human and zebrafish skeletal muscles. Using RD/12 and RD/18 rhabdomyosarcoma cells with low and high differentiation potency, respectively, we highlighted a specific correlation with HDAC8 expression and an advanced stage of muscle differentiation. We inhibited HDAC8 activity through a specific PCI-34051 inhibitor in murine C2C12 myoblasts and zebrafish embryos, and we observed skeletal muscles differentiation impairment. We also found a positive regulation of the canonical Wnt signaling by HDAC8 that might explain muscle differentiation defects. These findings suggest a novel mechanism through which HDAC8 expression, in a specific time window of skeletal muscle development, positively regulates canonical Wnt pathway that is necessary for muscle differentiation.

High-Throughput, Sensitive LC-MS Quantification of Biotherapeutics and Biomarkers Using Antibody-Free, Peptide-Level, Multiple-Mechanism Enrichment via Strategic Regulation of pH and Ionic and Solvent Strengths.

Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths. Multiple-mechanism washing and elution achieved highly selective separation despite the low plate number of the SPE cartridge. We first investigated the adsorption-desorption behaviors of peptides on CX-RP sorbent and the coexisting, perplexing effects of pH, and ionic and organic strengths on the selectivity for SP enrichment, which has not been previously characterized. We demonstrated that the selectivity for separating target SPs from matrix peptides was closely associated with buffer pH relative to the pI of the SP, and pH values of pI - 2, pI, and pI + 2 respectively provided exceptional specificity for the ionic wash, the hydrophobic wash, and selective elution. Furthermore, desorption of peptides from the mixed-mode sorbent showed exponential and linear dependence, respectively, on organic-solvent percentage and salt percentage. On the basis of these findings, we established a streamlined procedure for rapid and robust method development. Quantification of biotherapeutics, targets, and biomarkers in plasma and tissues was used as the model system. Selective enrichment of target SPs was achieved along with elimination of 87-95% of matrix peptides, which improved the LOQ by 20-fold (e.g., 2 ng per gram of tissue). Application was demonstrated by sensitive quantification of time courses of mAb (T84.66) and target (CEA) in plasma and tumor tissues from a low-dose mouse PK study. For the first time, down-regulation of membrane-associated antigen following mAb treatment was observed. The CX-RP enrichment is robust, high-throughput, and universally applicable and thus is highly valuable for ultrasensitive, large-scale measurement of target protein in plasma and tissues.

High-efficiency cellular reprogramming with microfluidics.

We report that the efficiency of reprogramming human somatic cells to induced pluripotent stem cells (hiPSCs) can be dramatically improved in a microfluidic environment. Microliter-volume confinement resulted in a 50-fold increase in efficiency over traditional reprogramming by delivery of synthetic mRNAs encoding transcription factors. In these small volumes, extracellular components of the TGF-ß and other signaling pathways exhibited temporal regulation that appears critical to acquisition of pluripotency. The high quality and purity of the resulting hiPSCs (µ-hiPSCs) allowed direct differentiation into functional hepatocyte- and cardiomyocyte-like cells in the same platform without additional expansion.