COVID-19 epidemic spread and green areas Italy and Spain between 2020 and 2021: An observational multi-country retrospective study.

Several studies have proposed that environmental factors influencing human wellbeing, such as chronic exposures to high levels of particulate matter, could indirectly or even directly affect also the severity of COVID-19 disease in case of infection by novel coronavirus SARS-COV2. This study has investigated the association between COVID-19 infections, hospitalizations or deaths and the extension of public green areas (km2 per 100,000 based on OECD data of 2014), an indicator that has been chosen as independent endpoint variable to test the research hypothesis in 10 Italian and 8 Spanish Provinces with more than 500.000 inhabitants, including capitals (Rome and Madrid) and bigger cities (Bologna, Catania, Florence, Genoa, Milan, Naples, Palermo, Turin and Venice for Italy; Barcelona, Valencia, Seville, Zaragoza, Malaga, Las Palmas and Bilbao for Spain). Two different methodologies have been applied: a bottom-up approach was applied to Spanish institutional data concerning contagions/hospitalizations/deaths and the extent of public green areas for each responder to an official questionnaire in the frame of a nationwide survey (with detailed data granularity per province) containing specific georeferenced information; a top-down approach was used for Italy, starting from the official figures of contagions/hospitalizations/deaths of each province and linking them to the OECD statistics about the extension of public green areas in the different areas. Linear and generalized models were used for statistical analyses including also PM2.5 in a multivariate approach (with annual average concentrations from official air quality monitoring stations) and were able to adjust for the different number inhabitants living in each province, in order to take into account the difference in contagion dynamics related to the different density of population. The results obtained for Spain are consistent with those observed for Italy, as for both countries, it has clearly emerged a statistically significant association between COVID-19 clinical features (contagions, hospitalizations, and deaths) and the extension of public green areas, as well as the annual average concentrations of PM2.5 (with this latter variable loosing statistical significance in some province). Therefore, the extension of public green areas and air pollution seem to have a high correlation with COVID-19 severity.

Activation of Kv7 Potassium Channels Inhibits Intracellular Ca2+ Increases Triggered By TRPV1-Mediated Pain-Inducing Stimuli in F11 Immortalized Sensory Neurons.

Kv7.2-Kv7.5 channels mediate the M-current (IKM), a K+-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively; however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i). The results obtained revealed the expression of transcripts of all Kv7 genes, leading to an IKM-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%); at least for BK-induced Ca2+ responses, the potency of retigabine (IC50~1 µM) was higher than that of ICA-27243 (IC50~5 µM) and (S)-1 (IC50~7 µM). Altogether, these results suggest that IKM activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.

Epileptic Encephalopathy In A Patient With A Novel Variant In The Kv7.2 S2 Transmembrane Segment: Clinical, Genetic, and Functional Features.

Kv7.2 subunits encoded by the KCNQ2 gene provide a major contribution to the M-current (IKM), a voltage-gated K+ current crucially involved in the regulation of neuronal excitability. Heterozygous missense variants in Kv7.2 are responsible for epileptic diseases characterized by highly heterogeneous genetic transmission and clinical severity, ranging from autosomal-dominant Benign Familial Neonatal Seizures (BFNS) to sporadic cases of severe epileptic and developmental encephalopathy (DEE). Here, we describe a patient with neonatal onset DEE, carrying a previously undescribed heterozygous KCNQ2 c.418G > C, p.Glu140Gln (E140Q) variant. Patch-clamp recordings in CHO cells expressing the E140Q mutation reveal dramatic loss of function (LoF) effects. Multistate structural modelling suggested that the E140Q substitution impeded an intrasubunit electrostatic interaction occurring between the E140 side chain in S2 and the arginine at position 210 in S4 (R210); this interaction is critically involved in stabilizing the activated configuration of the voltage-sensing domain (VSD) of Kv7.2. Functional results from coupled charge reversal or disulfide trapping experiments supported such a hypothesis. Finally, retigabine restored mutation-induced functional changes, reinforcing the rationale for the clinical use of Kv7 activators as personalized therapy for DEE-affected patients carrying Kv7.2 LoF mutations.

Critical role of large-conductance calcium- and voltage-activated potassium channels in leptin-induced neuroprotection of N-methyl-d-aspartate-exposed cortical neurons.

In the present study, the neuroprotective effects of the adipokine leptin, and the molecular mechanism involved, have been studied in rat and mice cortical neurons exposed to N-methyl-d-aspartate (NMDA) in vitro. In rat cortical neurons, leptin elicited neuroprotective effects against NMDA-induced cell death, which were concentration-dependent (10-100 ng/ml) and largest when the adipokine was preincubated for 2h before the neurotoxic stimulus. In both rat and mouse cortical neurons, leptin-induced neuroprotection was fully antagonized by paxilline (Pax, 0.01-1 µM) and iberiotoxin (Ibtx, 1-100 nM), with EC50s of 38 ± 10 nM and 5 ± 2 nM for Pax and Ibtx, respectively, close to those reported for Pax- and Ibtx-induced Ca(2+)- and voltage-activated K(+) channels (Slo1 BK channels) blockade; the BK channel opener NS1619 (1-30 µM) induced a concentration-dependent protection against NMDA-induced excitotoxicity. Moreover, cortical neurons from mice lacking one or both alleles coding for Slo1 BK channel pore-forming subunits were insensitive to leptin-induced neuroprotection. Finally, leptin exposure dose-dependently (10-100 ng/ml) increased intracellular Ca(2+) levels in rat cortical neurons. In conclusion, our results suggest that Slo1 BK channel activation following increases in intracellular Ca(2+) levels is a critical step for leptin-induced neuroprotection in NMDA-exposed cortical neurons in vitro, thus highlighting leptin-based intervention via BK channel activation as a potential strategy to counteract neurodegenerative diseases.

Downgraded dreams: Labor market outcomes and mental health in undocumented migration.

Undocumented immigrant workers are particularly exposed to mental health risk factors, including occupational downgrading - i.e. the loss in occupational status upon arrival. This study breaks new ground by examining the relationship between occupational downgrading and mental health among this hard-to-reach population, offering the first-ever investigation of its kind. Leveraging a unique dataset collected by a primary care outpatient clinic in Milan, Italy, which combines medical evaluations with detailed occupational information, we construct a direct measure of occupational downgrading, which adds to the literature. We employ logistic regression models to estimate odds ratios (ORs) for mental and behavioral disorders. The study also offers fresh evidence on the socioeconomic and health status of a sizable sample of undocumented migrants. The study sample consists of 1738 individuals that had their first medical examination in 2017-18. Prevalence of mental health conditions is 5.58%. Data also highlight poor labor market integration: one third of individuals in the sample is employed, mostly in elementary occupations; 66.63% of immigrant workers experienced occupational downgrading. Regression results show that undocumented immigrants who undergo occupational downgrading are at considerably higher risk of mental disorders. ORs range from 1.729 (95% CI 1.071-2.793), when the model only includes individual characteristics determined prior to migration, to 2.659 (CI 1.342-5.271), when it accounts for all the available controls. From a policy perspective, our study underscores the need to consider the broader impact of policies, including restrictive entry and integration policies, on migrant health. Additionally, ensuring access to primary care for all immigrants is crucial for early detection and treatment of mental health conditions.

Traditional Atlantic Diet and Its Effect on Health and the Environment: A Secondary Analysis of the GALIAT Cluster Randomized Clinical Trial.

Importance: The universal call to action for healthier and more sustainable dietary choices is the framework of the United Nations's Sustainable Development Goals. The Atlantic diet, originating from the northwest of the Iberian Peninsula, represents an example of a traditional diet that aligns with these principles. Objective: To explore a 6-month intervention based on the Atlantic diet's effects on metabolic and environmental health, assessing metabolic syndrome (MetS) incidence and the carbon footprint. Design, Setting, and Participants: The Galician Atlantic Diet study was a 6-month randomized clinical trial designed to assess the effects of this regional traditional diet on families' eating habits. The study was conducted from March 3, 2014, to May 29, 2015, at a local primary health care center in the rural town of A Estrada in northwestern Spain and involved a multisectoral collaboration. Families were randomly selected from National Health System records and randomized 1:1 to an intervention or control group. This secondary analysis of the trial findings was performed between March 24, 2021, and November 7, 2023. Interventions: Over 6 months, families in the intervention group received educational sessions, cooking classes, written supporting material, and foods characteristic of the Atlantic diet, whereas those randomized to the control group continued with their habitual lifestyle. Main Outcomes and Measures: The main outcomes were MetS incidence, defined per National Cholesterol Education Program Adult Treatment Panel III guidelines, and carbon footprint emissions as an environmental metric using life cycle assessment with daily dietary intake as the functional unit. Results: Initially, 250 families were randomized (574 participants; mean [SD] age, 46.8 [15.7] years; 231 males [40.2%] and 343 females [59.8%]). The intervention group included 126 families (287 participants) and the control group, 124 families (287 participants). Ultimately, 231 families completed the trial. The intervention significantly reduced the risk of incident cases of MetS (rate ratio, 0.32; 95% CI, 0.13-0.79) and had fewer MetS components (proportional odds ratio, 0.58; 95% CI, 0.42-0.82) compared with the control condition. The intervention group did not have a significantly reduced environmental impact in terms of carbon footprint emissions compared with the control group (-0.17 [95% CI, -0.46 to 0.12] kg CO2 equivalents/person/d). Conclusions and Relevance: These findings provide important evidence that a family-focused dietary intervention based on a traditional diet can reduce the risk of incident MetS. Further research is needed to understand the underlying mechanisms and determine the generalizability to other populations, taking into account regional cultural and dietary variations. Trial Registration: ClinicalTrials.gov Identifier: NCT02391701.

Zinc homeostasis and redox alterations in obesity.

Impairment of both cellular zinc and redox homeostasis is a feature of several chronic diseases, including obesity. A significant two-way interaction exists between redox metabolism and the relatively redox-inert zinc ion. Redox metabolism critically influences zinc homeostasis and controls its cellular availability for various cellular functions by regulating zinc exchange from/to zinc-binding proteins. Zinc can regulate redox metabolism and exhibits multiple pro-antioxidant properties. On the other hand, even minor disturbances in zinc status and zinc homeostasis affect systemic and cellular redox homeostasis. At the cellular level, zinc homeostasis is regulated by a multi-layered machinery consisting of zinc-binding molecules, zinc sensors, and two selective families of zinc transporters, the Zinc Transporter (ZnT) and Zrt, Irt-like protein (ZIP). In the present review, we summarize the current state of knowledge on the role of the mutual interaction between zinc and redox homeostasis in physiology and pathophysiology, pointing to the role of zinc in the alterations responsible for redox stress in obesity. Since zinc transporters primarily control zinc homeostasis, we describe how changes in the expression and activity of these zinc-regulating proteins are associated with obesity.

Chronic exposure to l-BMAA cyanotoxin induces cytoplasmic TDP-43 accumulation and glial activation, reproducing an amyotrophic lateral sclerosis-like phenotype in mice.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive and often fatal neurodegenerative disease characterized by the loss of Motor Neurons (MNs) in spinal cord, motor cortex and brainstem. Despite significant efforts in the field, the exact pathogenetic mechanisms underlying both familial and sporadic forms of ALS have not been fully elucidated, and the therapeutic possibilities are still very limited. Here we investigate the molecular mechanisms of neurodegeneration induced by chronic exposure to the environmental cyanotoxin L-BMAA, which causes a form of ALS/Parkinson's disease (PD) in several populations consuming food and/or water containing high amounts of this compound. METHODS: In this effort, mice were chronically exposed to L-BMAA and analyzed at different time points to evaluate cellular and molecular alterations and behavioral deficits, performing MTT assay, immunoblot, immunofluorescence and immunohistochemistry analysis, and behavioral tests. RESULTS: We found that cyanotoxin L-BMAA determines apoptotic cell death and a marked astrogliosis in spinal cord and motor cortex, and induces neurotoxicity by favoring TDP-43 cytoplasmic accumulation. CONCLUSIONS: Overall, our results characterize a new versatile neurotoxic animal model of ALS that may be useful for the identification of new druggable targets to develop innovative therapeutic strategies for this disease.

Co-benefits of the EAT-Lancet diet for environmental protection in the framework of the Spanish dietary pattern.

The immediate need to build resilient food systems with lower greenhouse gas (GHG) emissions and protection of water resources is a global challenge. To address this, the EAT-Lancet Commission described the global reference diet with principles of nutritional quality and environmental sustainability. With this in mind, the carbon and water footprints of the current Spanish dietary pattern have been compared with the EAT-Lancet global dietary recommendations, taking into account deviations in food intake. To provide additional context, differences between the average Spanish dietary pattern and dietary guidelines applied in other countries in Europe (Italy, the Netherlands and the Mediterranean region) and America have also been analyzed and discussed from a sustainability approach. We found that the EAT-Lancet diet requires less water resources (3056 L·person-1·day-1) and lower level of GHG emissions (2.13 kgCO2eq·person-1·day-1) in comparison with the Spanish dietary pattern (3732 L·person-1·day-1 and 3.62 kgCO2eq·person-1·day-1, respectively). Starch-based products and oils and fats were identified as largest contributors to both environmental indicators in the EAT-Lancet diet. On the other hand, meat and dairy were the environmental hotspots in the Spanish dietary pattern. Comparison with other food-based dietary patterns also raises environmental concerns about the high meat consumption in Spain. Overall, this analysis suggests that reducing the consumption of beef meat and dairy to a level in line with the global environmental targets set by the EAT-Lancet Commission would ensure a shift in Spanish dietary habits towards more environmentally sustainable food consumption patterns.

Driving commitment to sustainable food policies within the framework of American and European dietary guidelines.

Diet and nutrition are essential factors in promoting good health throughout life. Their role as determinants of chronic non-communicable diseases is widely recognized. Additionally, the demand for food involves relevant environmental burdens that have to be taken into account on the way to achieving the Sustainable Development Goals. As an important part of nutrition policy, food-based dietary guidelines (FBDGs) have been revised. The key question is: Are environmental considerations being incorporated into them? To address this issue, we modeled and compared both the main environmental indicators in terms of carbon footprint (CF) and water footprint (WF), and nutritional quality (according to the Nutrient Rich Diet index, NRD9.3 and a health gain score) of dietary guidelines from Northern and Southern Europe and America. Particularly, the FBDGs compared were Dutch Dietary Guidelines (DDG), New Nordic Diet (NND), Spanish Strategy for Nutrition, Physical Activity and Obesity Prevention (NAOS), Mediterranean Diet (MD), Italian Dietary Guidelines (IDG) and American Dietary Guidelines (DGA). The IDG and MD offered the best profiles from a climate change perspective (2.04 and 2.21 kgCO2eq·day-1). Overall, DGA had the highest CF (2.98 kgCO2eq·day-1). WF presented greater fluctuations, not only due to daily-recommended amounts, but also because of different climate conditions and production systems of the reference countries. Hence, WF ranged from 1760 L·person-1·day-1 in IDG to 3181 L·person-1·day-1 in NAOS. Finally, the nutritional value of MD, which had the highest NRD9.3 (477) and health gain score (178), has been demonstrated when the comparison was made with DDG, the one with the lowest health gain score (97) and DGA, the worst in terms of NRD9.3 (391). To go ahead of the FBDGs that bet on all dimensions of sustainability, multi-criteria analysis is needed. Nutrition and environmental performance are not the only aspects of the problem; economy and sociocultural variables should be considered.

Plasma Membrane and Organellar Targets of STIM1 for Intracellular Calcium Handling in Health and Neurodegenerative Diseases.

Located at the level of the endoplasmic reticulum (ER) membrane, stromal interacting molecule 1 (STIM1) undergoes a complex conformational rearrangement after depletion of ER luminal Ca2+. Then, STIM1 translocates into discrete ER-plasma membrane (PM) junctions where it directly interacts with and activates plasma membrane Orai1 channels to refill ER with Ca2+. Furthermore, Ca2+ entry due to Orai1/STIM1 interaction may induce canonical transient receptor potential channel 1 (TRPC1) translocation to the plasma membrane, where it is activated by STIM1. All these events give rise to store-operated calcium entry (SOCE). Besides the main pathway underlying SOCE, which mainly involves Orai1 and TRPC1 activation, STIM1 modulates many other plasma membrane proteins in order to potentiate the influxof Ca2+. Furthermore, it is now clear that STIM1 may inhibit Ca2+ currents mediated by L-type Ca2+ channels. Interestingly, STIM1 also interacts with some intracellular channels and transporters, including nuclear and lysosomal ionic proteins, thus orchestrating organellar Ca2+ homeostasis. STIM1 and its partners/effectors are significantly modulated in diverse acute and chronic neurodegenerative conditions. This highlights the importance of further disclosing their cellular functions as they might represent promising molecular targets for neuroprotection.

Effects of low-dose methylcyclopentadienyl manganese tricarbonyl-derived manganese on the development of diencephalic dopaminergic neurons in zebrafish.

Fuel additive methylcyclopentadienyl manganese tricarbonyl (MMT) is counted as an organic manganese (Mn)-derived compound. The toxic effects of Mn (alone and complexed) on dopaminergic (DA) neurotransmission have been investigated in both cellular and animal models. However, the impact of environmentally relevant Mn exposure on DA neurodevelopment is rather poorly understood. In the present study, the MMT dose of 100 µM (about 5 mg Mn/L) caused up-regulation of DA-related genes in association with cell body swelling and increase in the number of DA neurons of the ventral diencephalon subpopulation DC2. Furthermore, our analysis identified significant brain Mn bioaccumulation and enhancement of total dopamine levels in association with locomotor hyperactivity. Although DA levels were restored at adulthood, we observed a deficit in the acquisition and consolidation of memory. Collectively, these findings suggest that developmental exposure to low-level MMT-derived Mn is responsible for the selective alteration of diencephalic DA neurons and with long-lasting effects on fish explorative behaviour in adulthood.

Identification of Plasma Glycosphingolipids as Potential Biomarkers for Prostate Cancer (PCa) Status.

Prostate cancer (PCa) is the most common male cancer and the second leading cause of cancer death in United States men. Controversy continues over the effectiveness of prostate-specific antigen (PSA) for distinguishing aggressive from indolent PCa. There is a critical need for more specific and sensitive biomarkers to detect and distinguish low- versus high-risk PCa cases. Discovery metabolomics were performed utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) on plasma samples from 159 men with treatment naïve prostate cancer participating in the North Carolina-Louisiana PCa Project to determine if there were metabolites associated with aggressive PCa. Thirty-five identifiable plasma small molecules were associated with PCa aggressiveness, 15 of which were sphingolipids; nine common molecules were present in both African-American and European-American men. The molecules most associated with PCa aggressiveness were glycosphingolipids; levels of trihexosylceramide and tetrahexosylceramide were most closely associated with high-aggressive PCa. The Cancer Genome Atlas was queried to determine gene alterations within glycosphingolipid metabolism that are associated with PCa and other cancers. Genes that encode enzymes associated with the metabolism of glycosphingolipids were altered in 12% of PCa and >30% of lung, uterine, and ovarian cancers. These data suggest that the identified plasma (glyco)sphingolipids should be further validated for their association with aggressive PCa, suggesting that specific sphingolipids may be included in a diagnostic signature for PCa.

Amyloid ß-Induced Upregulation of Nav1.6 Underlies Neuronal Hyperactivity in Tg2576 Alzheimer's Disease Mouse Model.

Hyperexcitability and alterations in neuronal networks contribute to cognitive impairment in Alzheimer's Disease (AD). Voltage-gated sodium channels (NaV), which are crucial for regulating neuronal excitability, have been implicated in AD-related hippocampal hyperactivity and higher incidence of spontaneous non-convulsive seizures. Here, we show by using primary hippocampal neurons exposed to amyloid-ß1-42 (Aß1-42) oligomers and from Tg2576 mouse embryos, that the selective upregulation of NaV1.6 subtype contributes to membrane depolarization and to the increase of spike frequency, thereby resulting in neuronal hyperexcitability. Interestingly, we also found that NaV1.6 overexpression is responsible for the aberrant neuronal activity observed in hippocampal slices from 3-month-old Tg2576 mice. These findings identify the NaV1.6 channels as a determinant of the hippocampal neuronal hyperexcitability induced by Aß1-42 oligomers. The selective blockade of NaV1.6 overexpression and/or hyperactivity might therefore offer a new potential therapeutic approach to counteract early hippocampal hyperexcitability and subsequent cognitive deficits in the early stages of AD.

Ionic Homeostasis Maintenance in ALS: Focus on New Therapeutic Targets.

Amyotrophic lateral sclerosis (ALS) is one of the most threatening neurodegenerative disease since it causes muscular paralysis for the loss of Motor Neurons in the spinal cord, brainstem and motor cortex. Up until now, no effective pharmacological treatment is available. Two forms of ALS have been described so far: 90% of the cases presents the sporadic form (sALS) whereas the remaining 10% of the cases displays the familiar form (fALS). Approximately 20% of fALS is associated with inherited mutations in the Cu, Zn-superoxide dismutase 1 (SOD1) gene. In the last decade, ionic homeostasis dysregulation has been proposed as the main trigger of the pathological cascade that brings to motor-neurons loss. In the light of these premises, the present review will analyze the involvement in ALS pathophysiology of the most well studied metal ions, i.e., calcium, sodium, iron, copper and zinc, with particular focus to the role of ionic channels and transporters able to contribute in the regulation of ionic homeostasis, in order to propose new putative molecular targets for future therapeutic strategies to ameliorate the progression of this devastating neurodegenerative disease.

The expression and activity of KV3.4 channel subunits are precociously upregulated in astrocytes exposed to Aß oligomers and in astrocytes of Alzheimer's disease Tg2576 mice.

Astrocyte dysfunction emerges early in Alzheimer's disease (AD) and may contribute to its pathology and progression. Recently, the voltage gated potassium channel KV3.4 subunit, which underlies the fast-inactivating K+ currents, has been recognized to be relevant for AD pathogenesis and is emerging as a new target candidate for AD. In the present study, we investigated both in in vitro and in vivo models of AD the expression and functional activity of KV3.4 potassium channel subunits in astrocytes. In primary astrocytes our biochemical, immunohistochemical, and electrophysiological studies demonstrated a time-dependent upregulation of KV3.4 expression and functional activity after exposure to amyloid-ß (Aß) oligomers. Consistently, astrocytic KV3.4 expression was upregulated in the cerebral cortex, hippocampus, and cerebellum of 6-month-old Tg2576 mice. Further, confocal triple labeling studies revealed that in 6-month-old Tg2576 mice, KV3.4 was intensely coexpressed with Aß in nonplaque associated astrocytes. Interestingly, in the cortical and hippocampal regions of 12-month-old Tg2576 mice, plaque-associated astrocytes much more intensely expressed KV3.4 subunits, but not Aß. More important, we evidenced that the selective knockdown of KV3.4 expression significantly downregulated both glial fibrillary acidic protein levels and Aß trimers in the brain of 6-month-old Tg2576 mice. Collectively, our results demonstrate that the expression and function of KV3.4 channel subunits are precociously upregulated in cultured astrocytes exposed to Aß oligomers and in reactive astrocytes of AD Tg2576 mice.

Polychlorinated Biphenyls Induce Mitochondrial Dysfunction in SH-SY5Y Neuroblastoma Cells.

Chronic exposure to polychlorinated biphenyls (PCBs), ubiquitous environmental contaminants, can adversely affect the development and function of the nervous system. Here we evaluated the effect of PCB exposure on mitochondrial function using the PCB mixture Aroclor-1254 (A1254) in SH-SY5Y neuroblastoma cells. A 6-hour exposure to A1254 (5 µg/ml) reduced cellular ATP production by 45%±7, and mitochondrial membrane potential, detected by TMRE, by 49%±7. Consistently, A1254 significantly decreased oxidative phosphorylation and aerobic glycolysis measured by extracellular flux analyzer. Furthermore, the activity of mitochondrial protein complexes I, II, and IV, but not V (ATPase), measured by BN-PAGE technique, was significantly reduced after 6-hour exposure to A1254. The addition of pyruvic acid during exposure to A1254 significantly prevent A1254-induced cell injury, restoring resting mitochondrial membrane potential, ATP levels, oxidative phosphorylation and aerobic glycolysis. Furthermore, pyruvic acid significantly preserved the activity of mitochondrial complexes I, II and IV and increased basal activity of complex V. Collectively, the present results indicate that the neurotoxicity of A1254 depends on the impairment of oxidative phosphorylation, aerobic glycolysis, and mitochondrial complexes I, II, and IV activity and it was counteracted by pyruvic acid.

A bacterial engineered glycoprotein as a novel antigen for diagnosis of bovine brucellosis.

Brucellosis is a highly contagious zoonosis that affects livestock and human beings. Laboratory diagnosis of bovine brucellosis mainly relies on serological diagnosis using serum and/or milk samples. Although there are several serological tests with different diagnostic performance and capacity to differentiate vaccinated from infected animals, there is still no standardized reference antigen for the disease. Here we validate the first recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide-protein conjugate (OAg-AcrA), for diagnosis of bovine brucellosis. This antigen can be produced in homogeneous batches without the need of culturing pathogenic brucellae; all characteristics that make it appropriate for standardization. An indirect immunoassay based on the detection of anti O-polysaccharide IgG antibodies in bovine samples was developed coupling OAg-AcrA to magnetic beads or ELISA plates. As a proof of concept and to validate the antigen, we analyzed serum, whole blood and milk samples obtained from non-infected, experimentally infected and vaccinated animals included in a vaccination/infection trial performed in our laboratory as well as more than 1000 serum and milk samples obtained from naturally infected and S19-vaccinated animals from Argentina. Our results demonstrate that OAg-AcrA-based assays are highly accurate for diagnosis of bovine brucellosis, even in vaccinated herds, using different types of samples and in different platforms. We propose this novel recombinant glycoprotein as an antigen suitable for the development of new standard immunological tests for screening and confirmatory diagnosis of bovine brucellosis in regions or countries with brucellosis-control programs.