SARS-CoV-2 S protein activates NLRP3 inflammasome and deregulates coagulation factors in endothelial and immune cells.

BACKGROUND: Hyperinflammation, hypercoagulation and endothelial injury are major findings in acute and post-COVID-19. The SARS-CoV-2 S protein has been detected as an isolated element in human tissues reservoirs and is the main product of mRNA COVID-19 vaccines. We investigated whether the S protein alone triggers pro-inflammatory and pro-coagulant responses in primary cultures of two cell types deeply affected by SARS-CoV-2, such are monocytes and endothelial cells. METHODS: In human umbilical vein endothelial cells (HUVEC) and monocytes, the components of NF-κB and the NLRP3 inflammasome system, as well as coagulation regulators, were assessed by qRT-PCR, Western blot, flow cytometry, or indirect immunofluorescence. RESULTS: S protein activated NF-κB, promoted pro-inflammatory cytokines release, and triggered the priming and activation of the NLRP3 inflammasome system resulting in mature IL-1ß formation in both cell types. This was paralleled by enhanced production of coagulation factors such as von Willebrand factor (vWF), factor VIII or tissue factor, that was mediated, at least in part, by IL-1ß. Additionally, S protein failed to enhance ADAMTS-13 levels to counteract the pro-coagulant activity of vWF multimers. Monocytes and HUVEC barely expressed angiotensin-converting enzyme-2. Pharmacological approaches and gene silencing showed that TLR4 receptors mediated the effects of S protein in monocytes, but not in HUVEC. CONCLUSION: S protein behaves both as a pro-inflammatory and pro-coagulant stimulus in human monocytes and endothelial cells. Interfering with the receptors or signaling pathways evoked by the S protein may help preventing immune and vascular complications driven by such an isolated viral element. Video Abstract.

Validation of the medium and short version of CENSOPAS-COPSOQ: a psychometric study in the Peruvian population.

BACKGROUND: The presence of psychosocial risks at work are associated with mental and physical health issues in workers. The study aim was to adapt the COPSOQ-ISTAS21 (Spanish version of the Copenhagen Psychosocial Questionnaire and Union Institute of Work, Environment and Health) Medium-Version to the Peruvian context and to develop a Short-Version of the instrument. METHOD: Cross-sectional design study. The COPSOQ-ISTAS21 Medium Version was used. A confirmatory factor analysis was performed to determine the internal structure of each subdimension (first-order) and dimension (second-order) using the Robust Maximum Likelihood estimation method, and classic fit indices in the literature (CFI, SRMR, RMSEA). Internal consistency was evaluated using the alpha and omega coefficients. A short version was developed based on the items with the highest factorial load and that reduce the factorial complexity. RESULTS: A total of 1707 participants were evaluated. In the confirmatory factor analysis, the goodness-of-fit indices for seventeen of the 20 one-dimensional models (subdimensions) were identified; two subdimensions could not be evaluated because they presented only two items. When conducting a multidimensional analysis, we identified that all second-order models presented optimal goodness-of-fit indices, except "psychological demands at work". Finally, a short version of only 31 items was designed from the items with optimal fit indices. CONCLUSIONS: The new adapted versions of COPSOQ-ISTAS21 were renamed CENSOPAS-COPSOQ (National center of occupational health and environment protection for health -in Spanish- and Copenhagen Psychosocial Questionnaire). The CENSOPAS-COPSOQ is an instrument with sufficient evidence of validity and reliability in its medium and short version, which is why its use is recommended in Peruvian work centers to identify the evaluation and prevention of psychosocial risks at work in Peru.

Persistent inequality: evolution of psychosocial exposures at work among the salaried population in Spain between 2005 and 2016.

PURPOSE: To assess the prevalence of poor mental health and of exposure to psychosocial risks among the working population in Spain in 2005, 2010 and 2016; to analyse the associations between workplace psychosocial exposures and mental health problems according to gender and occupation. METHODS: Three representative samples of the Spanish working population were analysed, in 2005 (n = 7,023), 2010 (n = 4,979), and 2016 (n = 1,807). Prevalence ratios between mental health and the five dimensions - job demands, job control, social support, employment insecurity and insecurity over working conditions-were estimated using multilevel mixed-effects Poisson regressions. All the analyses were separated by gender and occupation. RESULTS: In 2016, there were improvements in job control, job demands and social support, and deteriorations in employment insecurity and insecurity over working conditions. The risk of poor mental health among manual workers rose if they were exposed to high demands, low social support and high employment insecurity; among non-manual workers, the risk increased if they were exposed to high demands, low control, low social support and high insecurity over working conditions. There were no differences according to gender. CONCLUSION: The new findings shed light on the evolution of the working conditions and health of the wage-earning population in Spain over the last 11 years. The stratification by gender and occupational group is relevant, since it allows a detailed analysis of the social disparities in the associations between psychosocial risks and mental health. The most vulnerable groups can be identified and preventive measures developed at source.

Sickness presenteeism: Are we sure about what we are studying? A research based on a literature review and an empirical illustration.

BACKGROUND: There has been an increasing interest in studying sickness presenteeism (SP). An ever-increasing amount of scientific literature is published using this term, yet there appears to be considerable heterogeneity in how it is assessed, which could result in substantial differences in the definition and interpretation of the phenomenon really being studied. We aim to discuss what really is being studied, depending on how the phenomenon is operationalized, measured, and analyzed. METHODS: A study based on a literature review and an empirical illustration using data of the third Spanish Psychosocial Risks Survey (2016). RESULTS: Differences are observed based on the population in which SP is measured, the cut-off points used to define a worker as presenteeist, the reasons for an SP episode and even an analysis of the phenomenon treated as a count or as a dichotomous. CONCLUSIONS: Without being completely exclusive, it seems that restricting the population of analysis to only those workers who consider that they should not have gone to work due to their health, and/or establishing low cut-off points to define someone as presenteeist, would more clearly delimit the study of SP to the exercise of a right to sick leave. In contrast, working with the entire population or using high cut-off points appears to relate the study of SP more with health status and less with the exercise of rights. On the other hand, taking the reasons for SP into account would probably help to improve interpretation of the phenomenon.

Specific psychosocial exposures for workers' mental health: A population-based study.

BACKGROUND: In order to plan interventions it is important to obtain evidence on the relation between a health outcome and specific exposures. However, there are few studies that identify the effect of specific psychosocial work exposures on poor mental health. This is the aim of this study. METHODS: Population-based cross-sectional study in Catalonia. We estimated prevalence ratios (PR) of minor psychiatric disorder associated with several psychosocial work exposures. RESULTS: The items with highest PR were "Are there times when you need to be at work and at home at the same time?" (PR = 1.81), "Are you worried about a variation in your salary?" (PR = 1.77), "Is your work emotionally demanding?" (PR = 1.65) and "Are you worried about it being difficult for you to find another job if you became unemployed?" (PR = 1.51). CONCLUSIONS: This study could be useful in order to begin planning interventions on specific psychosocial exposures to protect mental health in the workplace.

Inflammation, glucose, and vascular cell damage: the role of the pentose phosphate pathway.

BACKGROUND: Hyperglycemia is acknowledged as a pro-inflammatory condition and a major cause of vascular damage. Nevertheless, we have previously described that high glucose only promotes inflammation in human vascular cells previously primed with pro-inflammatory stimuli, such as the cytokine interleukin (IL)1ß. Here, we aimed to identify the cellular mechanisms by which high glucose exacerbates the vascular inflammation induced by IL1ß. METHODS: Cultured human aortic smooth muscle cells (HASMC) and isolated rat mesenteric microvessels were treated with IL1ß in medium containing 5.5-22 mmol/L glucose. Glucose uptake and consumption, lactate production, GLUT1 levels, NADPH oxidase activity and inflammatory signalling (nuclear factor-κB activation and inducible nitric oxide synthase expression) were measured in HASMC, while endothelium-dependent relaxations to acetylcholine were determined in rat microvessels. Pharmacological inhibition of IL1 receptors, NADPH oxidase and glucose-6-phosphate dehydrogenase (G6PD), as well as silencing of G6PD, were also performed. Moreover, the pentose phosphate pathway (PPP) activity and the levels of reduced glutathione were determined. RESULTS: We found that excess glucose uptake in HASMC cultured in 22 mM glucose only occurred following activation with IL1ß. However, the simple entry of glucose was not enough to be deleterious since over-expression of the glucose transporter GLUT1 or increased glucose uptake following inhibition of mitochondrial respiration by sodium azide was not sufficient to trigger inflammatory mechanisms. In fact, besides allowing glucose entry, IL1ß activated the PPP, thus permitting some of the excess glucose to be metabolized via this route. This in turn led to an over-activation NADPH oxidase, resulting in increased generation of free radicals and the subsequent downstream pro-inflammatory signalling. Moreover, in rat mesenteric microvessels high glucose incubation enhanced the endothelial dysfunction induced by IL1ß by a mechanism which was abrogated by the inhibition of the PPP. CONCLUSIONS: A pro-inflammatory stimulus like IL1ß transforms excess glucose into a vascular deleterious agent by causing an increase in glucose uptake and its subsequent diversion into the PPP, promoting the pro-oxidant conditions required for the exacerbation of pro-oxidant and pro-inflammatory pathways. We propose that over-activation of the PPP is a crucial mechanism for the vascular damage associated to hyperglycemia.

Is the worsening of psychosocial exposures associated with mental health? Comparing two population-based cross-sectional studies in Spain, 2005-2010.

AIMS: To analyze whether associations between workplace psychosocial exposures and the mental health of the working population in Spain changed between 2005 and 2010. METHODS: Two representative samples of the Spanish working population have been analyzed, 2005 (n = 5073) and 2010 (n = 3544). RESULTS: In 2010 there was a significant association between poor mental health and exposure to high Demands, low Social Support and high Insecurity over working conditions, and exposure to high Insecurity over losing the job only for men. In 2005 there was a significant association with exposure to high Demands and low Social Support. CONCLUSION: Changes in the associations between psychosocial risks and mental health may be related to the socioeconomic context marked by the rise in unemployment and the destruction of jobs as a result of the 2008 economic crisis.

Glycolysis: a bioenergetic or a survival pathway?

Following inhibition of mitochondrial respiration neurons die rapidly, whereas astrocytes utilize glycolytically-generated ATP to increase their mitochondrial membrane potential, thus becoming more resistant to pro-apoptotic stimuli. Neurons are unable to increase glycolysis due to the lack of activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase, isoform 3 (PFKFB3). In neurons, PFKFB3 is degraded constantly via the E3 ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)- CDH1. Glucose metabolism in neurons is directed mainly to the pentose phosphate pathway, leading to regeneration of reduced glutathione. In addition to their relevance to brain physiology and pathophysiology, these observations suggest that APC/C-CDH1 might link activation of glycolysis and cell proliferation as it is also involved in the regulation of cell cycle proteins.

Oncogenic transformation of mesenchymal stem cells decreases Nrf2 expression favoring in vivo tumor growth and poorer survival.

BACKGROUND: The transcription factor Nrf2 is a key regulator of the cellular antioxidant response, and its activation by chemoprotective agents has been proposed as a potential strategy to prevent cancer. However, activating mutations in the Nrf2 pathway have been found to promote tumorigenesis in certain models. Therefore, the role of Nrf2 in cancer remains contentious. METHODS: We employed a well-characterized model of stepwise human mesenchymal stem cell (MSC) transformation and breast cancer cell lines to investigate oxidative stress and the role of Nrf2 during tumorigenesis. The Nrf2 pathway was studied by microarray analyses, qRT-PCR, and western-blotting. To assess the contribution of Nrf2 to transformation, we established tumor xenografts with transformed MSC expressing Nrf2 (n = 6 mice per group). Expression and survival data for Nrf2 in different cancers were obtained from GEO and TCGA databases. All statistical tests were two-sided. RESULTS: We found an accumulation of reactive oxygen species during MSC transformation that correlated with the transcriptional down-regulation of antioxidants and Nrf2-downstream genes. Nrf2 was repressed in transformed MSC and in breast cancer cells via oncogene-induced activation of the RAS/RAF/ERK pathway. Furthermore, restoration of Nrf2 function in transformed cells decreased reactive oxygen species and impaired in vivo tumor growth (P = 0.001) by mechanisms that included sensitization to apoptosis, and a decreased hypoxic/angiogenic response through HIF-1α destabilization and VEGFA repression. Microarray analyses showed down-regulation of Nrf2 in a panel of human tumors and, strikingly, low Nrf2 expression correlated with poorer survival in patients with melanoma (P = 0.0341), kidney (P = 0.0203) and prostate (P = 0.00279) cancers. CONCLUSIONS: Our data indicate that oncogene-induced Nrf2 repression is an adaptive response for certain cancers to acquire a pro-oxidant state that favors cell survival and in vivo tumor growth.

The changing patterns of psychosocial exposures at work in the south of Europe: Spain as a labor market laboratory.

AIMS: To examine the pattern of psychosocial risk exposures at work among wage-earners in Spain in 2005 and 2010, and to analyze changes in exposure inequalities by gender and job category. METHODS: Psychosocial exposures were compared using the COPSOQ-ISTAS21 method, based on two surveys representative of the Spanish wage-earning population (2005 and 2010). Statistical analysis was conducted using correspondence analysis. RESULTS: There was an increase in exposure to high Double Presence, low Social Support, high Work Pace, and high Insecurity about finding a job; and reduction in exposure to high Insecurity about losing a job, and to high Insecurity over worsening of employment conditions. A gender- and occupation-related gradient was maintained. CONCLUSION: Although this study analyzes wage-earner "survivors" after the outbreak of the current economic crisis, it shows a worsening of harmful exposures to some psychosocial risks. In a context of job destruction, concerns about worsening working conditions appear to be subordinate to insecurity about job loss.

The copenhagen psychosocial questionnaire II (COPSOQ II) in Spain--a tool for psychosocial risk assessment at the workplace.

AIMS: To describe the second version of the Spanish Copenhagen Psychosocial Questionnaire and to present evidence of its validity and reliability. METHODS: The original Danish long COPSOQ II questionnaire was adapted to the labor market, cultural, and linguistic setting of Spain and included in the 2010 Spanish Psychosocial Risks Survey. Analysis involved the assessment of psychometric characteristics and associations among psychosocial scales and health scales. Medium and short versions were derived from the long one. RESULTS: The long questionnaire was configured with 24 dimensions (92 items); medium-length questionnaire with 20 dimensions (69 items); and short questionnaire with 14 dimensions (28 items). All scales showed acceptable reliability and concordance between versions. Most associations among psychosocial scales and Mental Health, Stress, and Burnout scales were in the expected direction, except the scale of Influence, that showed some incongruent associations. CONCLUSION: Results support the validity and reliability of Spanish COPSOQ II questionnaires as tools for psychosocial risk assessment at the workplace, however, better scales should be developed specially for the dimension of Influence.

Two ubiquitin ligases, APC/C-Cdh1 and SKP1-CUL1-F (SCF)-beta-TrCP, sequentially regulate glycolysis during the cell cycle.

During cell proliferation, the abundance of the glycolysis-promoting enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is controlled by the ubiquitin ligase APC/C-Cdh1 via a KEN box. We now demonstrate in synchronized HeLa cells that PFKFB3, which appears in mid-to-late G1, is essential for cell division because its silencing prevents progression into S phase. In cells arrested by glucose deprivation, progression into S phase after replacement of glucose occurs only when PFKFB3 is present or is substituted by the downstream glycolytic enzyme 6-phosphofructo-1-kinase. PFKFB3 ceases to be detectable during late G1/S despite the absence of Cdh1; this disappearance is prevented by proteasomal inhibition. PFKFB3 contains a DSG box and is therefore a potential substrate for SCF-ß-TrCP, a ubiquitin ligase active during S phase. In synchronized HeLa cells transfected with PFKFB3 mutated in the KEN box, the DSG box, or both, we established the breakdown routes of the enzyme at different stages of the cell cycle and the point at which glycolysis is enhanced. Thus, the presence of PFKFB3 is tightly controlled to ensure the up-regulation of glycolysis at a specific point in G1. We suggest that this up-regulation of glycolysis and its associated events represent the nutrient-sensitive restriction point in mammalian cells.

Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells.

During cell division, the activation of glycolysis is tightly regulated by the action of two ubiquitin ligases, anaphase-promoting complex/cyclosome-Cdh1 (APC/C-Cdh1) and SKP1/CUL-1/F-box protein-ß-transducin repeat-containing protein (SCF-ß-TrCP), which control the transient appearance and metabolic activity of the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3). We now demonstrate that the breakdown of PFKFB3 during S phase occurs specifically via a distinct residue (S(273)) within the conserved recognition site for SCF-ß-TrCP. Glutaminase 1 (GLS1), the first enzyme in glutaminolysis, is also targeted for destruction by APC/C-Cdh1 and, like PFKFB3, accumulates after the activity of this ubiquitin ligase decreases in mid-to-late G1. However, our results show that GLS1 differs from PFKFB3 in that its recognition by APC/C-Cdh1 requires the presence of both a Lys-Glu-Asn box (KEN box) and a destruction box (D box) rather than a KEN box alone. Furthermore, GLS1 is not a substrate for SCF-ß-TrCP and is not degraded until cells progress from S to G2/M. The presence of PFKFB3 and GLS1 coincides with increases in generation of lactate and in utilization of glutamine, respectively. The contrasting posttranslational regulation of PFKFB3 and GLS1, which we have verified by studies of ubiquitination and protein stability, suggests the different roles of glucose and glutamine at distinct stages in the cell cycle. Indeed, experiments in which synchronized cells were deprived of either of these substrates show that both glucose and glutamine are required for progression through the restriction point in mid-to-late G1, whereas glutamine is the only substrate essential for the progression through S phase into cell division.

Fulfilling the metabolic requirements for cell proliferation.

The activity of key metabolic enzymes is regulated by the ubiquitin ligases that control the function of the cyclins; therefore the activity of these ubiquitin ligases explains the coordination of cell-cycle progression with the supply of substrates necessary for cell duplication. APC/C (anaphase-promoting complex/cyclosome)-Cdh1, the ubiquitin ligase that controls G(1)- to S-phase transition by targeting specific degradation motifs in cell-cycle proteins, also regulates the glycolysis-promoting enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) and GLS1 (glutaminase 1), a critical enzyme in glutaminolysis. A decrease in the activity of APC/C-Cdh1 in mid-to-late G(1) releases both proteins, thus explaining the simultaneous increase in the utilization of glucose and glutamine during cell proliferation. This occurs at a time consistent with the point in G(1) that has been described as the nutrient-sensitive restriction point and is responsible for the transition from G(1) to S. PFKFB3 is also a substrate at the onset of S-phase for the ubiquitin ligase SCF (Skp1/cullin/F-box)-ß-TrCP (ß-transducin repeat-containing protein), so that the activity of PFKFB3 is short-lasting, coinciding with a peak in glycolysis in mid-to-late G(1), whereas the activity of GLS1 remains high throughout S-phase. The differential regulation of the activity of these proteins indicates that a finely-tuned set of mechanisms is activated to fulfil specific metabolic demands at different stages of the cell cycle. These findings have implications for the understanding of cell proliferation in general and, in particular, of cancer, its prevention and treatment.

E3 ubiquitin ligase APC/C-Cdh1 accounts for the Warburg effect by linking glycolysis to cell proliferation.

Cell proliferation is known to be accompanied by activation of glycolysis. We have recently discovered that the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, isoform 3 (PFKFB3), is degraded by the E3 ubiquitin ligase APC/C-Cdh1, which also degrades cell-cycle proteins. We now show in two different cell types (neoplastic and nonneoplastic) that both proliferation and aerobic glycolysis are prevented by overexpression of Cdh1 and enhanced by its silencing. Furthermore, we have coexpressed Cdh1 with PFKFB3--either wild-type or a mutant form resistant to ubiquitylation by APC/C-Cdh1--or with the glycolytic enzyme 6-phosphofructo-1-kinase and demonstrated that whereas glycolysis is essential for cell proliferation, its initiation in the presence of active Cdh1 does not result in proliferation. Our experiments indicate that the proliferative response, regardless of whether it occurs in normal or neoplastic cells, is dependent on a decrease in the activity of APC/C-Cdh1, which activates both proliferation and glycolysis. These observations have implications for cell proliferation, neoplastic transformation, and the prevention and treatment of cancer.

Anaphase-promoting complex/cyclosome-Cdh1 coordinates glycolysis and glutaminolysis with transition to S phase in human T lymphocytes.

Cell proliferation is accompanied by an increase in the utilization of glucose and glutamine. The proliferative response is dependent on a decrease in the activity of the ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C)-Cdh1 which controls G1-to-S-phase transition by targeting degradation motifs, notably the KEN box. This occurs not only in cell cycle proteins but also in the glycolysis-promoting enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3), as we have recently demonstrated in cells in culture. We now show that APC/C-Cdh1 controls the proliferative response of human T lymphocytes. Moreover, we have found that glutaminase 1 is a substrate for this ubiquitin ligase and appears at the same time as PFKFB3 in proliferating T lymphocytes. Glutaminase 1 is the first enzyme in glutaminolysis, which converts glutamine to lactate, yielding intermediates for cell proliferation. Thus APC/C-Cdh1 is responsible for the provision not only of glucose but also of glutamine and, as such, accounts for the critical step that links the cell cycle with the metabolic substrates essential for its progression.

The relationship between immigration and mental health: what is the role of workplace psychosocial factors.

BACKGROUND: To study the relationship between immigration and mental health considering the psychosocial factors in the workplace. METHODS: Multistage cluster sampling was used (final sample: 7,612 workers). Workers whose country of origin was unknown were excluded from the study (study population: 7,555). The information was collected between 2004 and 2005 using a standardized questionnaire, and interviews were conducted in respondents' homes. The risk of poor mental health according to psychosocial factor, using the native, non-exposed workers as a reference, was calculated using log-binomial models. The prevalence ratio (PR) and confidence intervals (CI 95%) were estimated from crude data and from data adjusted for sex, age, and occupational category. RESULTS: Immigrants who experienced high quantitative demands (PR = 1.46; CI 95%:1.34-1.59), high emotional demands (PR = 1.42; CI 95%:1.301.56), high demands for hiding emotions (PR = 1.35; CI 95%:1.21-1.50), low possibilities for development (PR = 1.21; CI 95%:1.09-1.33), low levels of support from coworkers (PR = 1.41; CI 95%:1.30-1.53), and low esteem (PR = 1.53; CI 95%:1.42-1.66) perceived worse mental health. Equally, the study found that the immigrants with a high influence (PR = 1.19; CI 95%:1.09-1.29) and high control over working times (PR = 1.25; CI 95%:1.14-1.36) also reported worse mental health. We also found that native workers exposed to these factors also perceived worse mental health than those who were not exposed and that even, at times, they were at greater risk than exposed immigrants. CONCLUSIONS: Differences in mental health between exposed and non-exposed wage earners, whether immigrant or native workers, indicate the importance of taking action to reduce psychosocial factors, as this would benefit both native and immigrant workers.