A membrane-associated phosphoswitch in Rad controls adrenergic regulation of cardiac calcium channels.

The ability to fight or flee from a threat relies on an acute adrenergic surge that augments cardiac output, which is dependent on increased cardiac contractility and heart rate. This cardiac response depends on ß-adrenergic-initiated reversal of the small RGK G protein Rad-mediated inhibition of voltage-gated calcium channels (CaV) acting through the Cavß subunit. Here, we investigate how Rad couples phosphorylation to augmented Ca2+ influx and increased cardiac contraction. We show that reversal required phosphorylation of Ser272 and Ser300 within Rad's polybasic, hydrophobic C-terminal domain (CTD). Phosphorylation of Ser25 and Ser38 in Rad's N-terminal domain (NTD) alone was ineffective. Phosphorylation of Ser272 and Ser300 or the addition of 4 Asp residues to the CTD reduced Rad's association with the negatively charged, cytoplasmic plasmalemmal surface and with CaVß, even in the absence of CaVα, measured here by FRET. Addition of a posttranslationally prenylated CAAX motif to Rad's C-terminus, which constitutively tethers Rad to the membrane, prevented the physiological and biochemical effects of both phosphorylation and Asp substitution. Thus, dissociation of Rad from the sarcolemma, and consequently from CaVß, is sufficient for sympathetic upregulation of Ca2+ currents.

Höffding step and beyond: The impact of visual sensory impairment on cognitive performance in neuropsychological testing of survivors of acute methanol poisoning.

BACKGROUND: Sensory deficits can result in limitations regarding how well neuropsychological test findings can be interpreted. Only a few studies have investigated the influence of vision alteration on neuropsychological tests. In 2012 the Czech Republic experienced mass methanol poisoning. Methanol metabolites cause histotoxic hypoxia to the optic nerve. OBJECTIVE: In the current study, the effect of the toxic damage on the parts of the visual pathway on visual and non-visual neuropsychological measures was investigated using electrophysiological methods (visual evoked potential (VEP) and optical coherence tomography (OCT) with retinal nerve fibre layer (RNFL) thickness measurement. METHODS: 53 individuals who experienced methanol poisoning participated in this research (76% men; ages 24 to 74 years, mean = 43.8±14.6 years; education 11.9±1.4 years). Each participant underwent comprehensive neurological, ophthalmological, and neuropsychological examinations. RESULTS: The results of mixed-effect models revealed significant small to a medium association between the Stroop test weak interference and Grooved Pegboard with the left eye global, nasal and temporal RNFL thickness. Also, medium associations between the Finger Tapping test and the Stroop test weak interference and left eye temporal RNFL, right eye temporal RNFL, and the latency P1 of VEP in the left eye were significant. CONCLUSION: The results of this study found a small to medium association (r = .15- .33; p = .010- .046) between RNFL thickness and cognitive visual test performance. Careful interpretation is suggested regarding results obtained from visual tests of the executive or motor functioning with participants with RNFL decrease or other types of early visual processing damage.

Engineered cardiac tissue model of restrictive cardiomyopathy for drug discovery.

Restrictive cardiomyopathy (RCM) is defined as increased myocardial stiffness and impaired diastolic relaxation leading to elevated ventricular filling pressures. Human variants in filamin C (FLNC) are linked to a variety of cardiomyopathies, and in this study, we investigate an in-frame deletion (c.7416_7418delGAA, p.Glu2472_Asn2473delinAsp) in a patient with RCM. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with this variant display impaired relaxation and reduced calcium kinetics in 2D culture when compared with a CRISPR-Cas9-corrected isogenic control line. Similarly, mutant engineered cardiac tissues (ECTs) demonstrate increased passive tension and impaired relaxation velocity compared with isogenic controls. High-throughput small-molecule screening identifies phosphodiesterase 3 (PDE3) inhibition by trequinsin as a potential therapy to improve cardiomyocyte relaxation in this genotype. Together, these data demonstrate an engineered cardiac tissue model of RCM and establish the translational potential of this precision medicine approach to identify therapeutics targeting myocardial relaxation.

Acute and chronic blood serum proteome changes in patients with methanol poisoning.

Twenty-four blood serum samples from patients with acute methanol poisoning (M) from the mass methanol poisoning outbreak in the Czech Republic in 2012 were compared with 46 patient samples taken four years after poisoning (S) (overlap of 10 people with group M) and with a control group (C) of 24 samples of patients with a similar proportion of chronic alcohol abuse. When comparing any two groups, tens to hundreds of proteins with a significant change in concentration were identified. Fifteen proteins showed significant changes when compared between any two groups. The group with acute methanol poisoning showed significant changes in protein concentrations for at least 64 proteins compared to the other groups. Among the most important identified proteins closely related to intoxication are mainly those involved in blood coagulation, metabolism of vitamin A (increased retinol-binding protein), immune response (e.g., increased complement factor I, complement factors C3 and C5), and lipid transport (increased apolipoprotein A I, apolipoprotein A II, adiponectin). For blood coagulation, the most affected proteins with significant changes in the methanol poisoning group were von Willebrand factor, carboxypeptidase N, alpha-2-antiplasmin (all increased), inter-alpha-trypsin inhibitor heavy chain H4, kininogen-1, plasma serine protease inhibitor, plasminogen (all decreased). However, heparin administration used for the methanol poisoning group could have interfered with some of the changes in their concentrations. Data are available via ProteomeXchange with the identifier PXD035726.

Rad regulation of CaV1.2 channels controls cardiac fight-or-flight response.

Fight-or-flight responses involve ß-adrenergic-induced increases in heart rate and contractile force. In the present study, we uncover the primary mechanism underlying the heart's innate contractile reserve. We show that four protein kinase A (PKA)-phosphorylated residues in Rad, a calcium channel inhibitor, are crucial for controlling basal calcium current and essential for ß-adrenergic augmentation of calcium influx in cardiomyocytes. Even with intact PKA signaling to other proteins modulating calcium handling, preventing adrenergic activation of calcium channels in Rad-phosphosite-mutant mice (4SA-Rad) has profound physiological effects: reduced heart rate with increased pauses, reduced basal contractility, near-complete attenuation of ß-adrenergic contractile response and diminished exercise capacity. Conversely, expression of mutant calcium-channel ß-subunits that cannot bind 4SA-Rad is sufficient to enhance basal calcium influx and contractility to adrenergically augmented levels of wild-type mice, rescuing the failing heart phenotype of 4SA-Rad mice. Hence, disruption of interactions between Rad and calcium channels constitutes the foundation toward next-generation therapeutics specifically enhancing cardiac contractility.

DPODv2: Dense Correspondence-Based 6 DoF Pose Estimation.

We propose a three-stage 6 DoF object detection method called DPODv2 (Dense Pose Object Detector) that relies on dense correspondences. We combine a 2D object detector with a dense correspondence estimation network and a multi-view pose refinement method to estimate a full 6 DoF pose. Unlike other deep learning methods that are typically restricted to monocular RGB images, we propose a unified deep learning network allowing different imaging modalities to be used (RGB or Depth). Moreover, we propose a novel pose refinement method, that is based on differentiable rendering. The main concept is to compare predicted and rendered correspondences in multiple views to obtain a pose which is consistent with predicted correspondences in all views. Our proposed method is evaluated rigorously on different data modalities and types of training data in a controlled setup. The main conclusions is that RGB excels in correspondence estimation, while depth contributes to the pose accuracy if good 3D-3D correspondences are available. Naturally, their combination achieves the overall best performance. We perform an extensive evaluation and an ablation study to analyze and validate the results on several challenging datasets. DPODv2 achieves excellent results on all of them while still remaining fast and scalable independent of the used data modality and the type of training data.

Cognitive changes after methanol exposure: Longitudinal perspective.

BACKGROUND: From 2012 to 2013, there was a mass methanol poisoning outbreak in the Czech Republic. Methanol metabolites can cause specific lesions in the basal ganglia, subcortical white matter, and optic nerve. However, long-term sequelae of methanol poisoning on cognitive functioning have not yet been explored. The current study aimed to delineate the cognitive changes observed in methanol poisoning survivors in the seven years since 2012. METHODS: We conducted longitudinal research with repeated measurements in 2013, 2015, 2017 and 2019 to evaluate the development of cognitive changes after acute methanol poisoning. A complex neuropsychological battery consisted of tests of global cognitive performance, auditory and visual attention, executive functioning, learning and memory, working memory and language. Motor performance measures and depression scale were also included. RESULTS: Repeated measures ANOVA of four measurements with post-hoc tests showed a significant decline in the Mini-Mental State Examination (p = 0.007); however, other parameters were not significantly decreasing. In comparison to normative values, the z-scores for each test measure, in the memory domain, in particular, ranged from 43 to 60 % of participants below 1.5 SD. Mild to severe depression levels from the onset of poisoning improved during the seven years, returning to normal in up to 27 % of participants. CONCLUSION: In the longitudinal perspective, methanol poisoning survivors manifest progressive global cognitive decline and overall persistent below-average cognitive performance with some improvements in the frequency of depressive symptoms.

Estimation of long-term costs of postacute care in survivors of the methanol poisoning outbreak.

OBJECTIVES: To fill the existing research gap related to long-term costs of postacute care in methanol poisoning survivors, healthcare cost for 6 years after the outbreak has been modelled and estimated. DESIGN: In a prospective longitudinal cohort study, data collected from 55 survivors of the Czech methanol mass poisoning outbreak in 2012 were collected in four rounds (5 months, then 2, 4 and 6 years after the discharge) in the General University Hospital in Prague according to the same predefined study protocol. The collected data were used to inform the cost model. SETTING AND PARTICIPANTS: All 83 patients discharged from a hospital poisoning treatment after the 2012 methanol outbreak were informed about the study and invited to participate. Fifty-five patients (66%) gave their written informed consent and were followed until their death or the last follow-up 6 years later. The costs were modelled from the Czech healthcare service (general health insurance) perspective. MAIN OUTCOME MEASURES: Long-term national budget impact of the methanol poisoning outbreak, frequencies of sequelae and their average costs. RESULTS: The postacute cost analysis concentrated on visual and neurological sequelae that were shown to be dominant. Collected data were used to create process maps portraying gradual changes in long-term sequelae over time. Individual process maps were created for the central nervous system, peripheral nervous system, sequelae detected during eye examinations and sequelae concerning the visual evoked potentials. Based on the process maps the costs of the postacute outpatient care were estimated. CONCLUSIONS: In 2013-2019 the highest costs per patient related to postacute care were found in the first year; the average costs decreased afterwards, and remained almost constant for the rest of the studied period of time. These costs per patient ranged from CZK4142 in 2013 to CZK1845 in 2018, when they raised to CZK2519 in 2019 again.

Adrenergic CaV1.2 Activation via Rad Phosphorylation Converges at α1C I-II Loop.

RATIONALE: Changing activity of cardiac CaV1.2 channels under basal conditions, during sympathetic activation, and in heart failure is a major determinant of cardiac physiology and pathophysiology. Although cardiac CaV1.2 channels are prominently upregulated via activation of PKA (protein kinase A), essential molecular details remained stubbornly enigmatic. OBJECTIVE: The primary goal of this study was to determine how various factors converging at the CaV1.2 I-II loop interact to regulate channel activity under basal conditions, during ß-adrenergic stimulation, and in heart failure. METHODS AND RESULTS: We generated transgenic mice with expression of CaV1.2 α1C subunits with (1) mutations ablating interaction between α1C and ß-subunits, (2) flexibility-inducing polyglycine substitutions in the I-II loop (GGG-α1C), or (3) introduction of the alternatively spliced 25-amino acid exon 9* mimicking a splice variant of α1C upregulated in the hypertrophied heart. Introducing 3 glycine residues that disrupt a rigid IS6-α-interaction domain helix markedly reduced basal open probability despite intact binding of CaVß to α1C I-II loop and eliminated ß-adrenergic agonist stimulation of CaV1.2 current. In contrast, introduction of the exon 9* splice variant in the α1C I-II loop, which is increased in ventricles of patients with end-stage heart failure, increased basal open probability but did not attenuate stimulatory response to ß-adrenergic agonists when reconstituted heterologously with ß2B and Rad or transgenically expressed in cardiomyocytes. CONCLUSIONS: Ca2+ channel activity is dynamically modulated under basal conditions, during ß-adrenergic stimulation, and in heart failure by mechanisms converging at the α1C I-II loop. CaVß binding to α1C stabilizes an increased channel open probability gating mode by a mechanism that requires an intact rigid linker between the ß-subunit binding site in the I-II loop and the channel pore. Release of Rad-mediated inhibition of Ca2+ channel activity by ß-adrenergic agonists/PKA also requires this rigid linker and ß-binding to α1C.

Efficiency of 123I-ioflupane SPECT as the marker of basal ganglia damage in acute methanol poisoning: 6-year prospective study.

CONTEXT: Investigate whether 123I-ioflupane SPECT (DaT SPECT) has the potential as a marker of basal ganglia damage in acute methanol poisoning. METHODS: Prospective, single-centre, cohort study of patients with confirmed methanol poisoning was conducted. DaT SPECT was performed twice with semi-quantification using DaTQUANTTM and MRI-based volumetry was calculated. Specific binding ratios (SBR) of striatum, caudate nucleus, and putamen were correlated with laboratory parameters of outcome, volumetric data, and retinal nerve fibres layer (RNFL) thickness measurements. RESULTS: Forty-two patients (mean age 46.3 ± 4.2 years; 8 females), including 15 with MRI-detected putamen lesions (group I) and 27 patients with intact putamen (group II), underwent DaT SPECT. Volumetry was calculated in 35 of the patients assessed. SBR values for the left putamen correlated with putamen volume (r = 0.665; p < 0.001). Decreased bilateral SBR values were determined for the striatum and the putamen, but not for the nucleus caudate, in group I (p < 0.05). Significant correlation was observed between the SBR of the posterior putamen and arterial blood pH (r = 0.574; p < 0.001) and other toxicological parameters of severity of poisoning/outcome including serum lactate, glucose, and creatinine concentrations (p < 0.05). The SBR of the posterior putamen positively correlated with the global RNFL thickness (p < 0.05). ROC analysis demonstrated a significant discriminatory ability of SBR of the posterior putamen with AUC = 0.753 (95%CI 0.604-0.902; p = 0.007). The multivariate regression model demonstrated that arterial blood pH, age, and gender were the most significant factors associated with SBR of the posterior putamen. CONCLUSION: DaT SPECT demonstrates significant potential for the diagnosis of methanol-induced basal ganglia damage.

Fibroblast growth factor homologous factors tune arrhythmogenic late NaV1.5 current in calmodulin binding-deficient channels.

The Ca2+-binding protein calmodulin has emerged as a pivotal player in tuning Na+ channel function, although its impact in vivo remains to be resolved. Here, we identify the role of calmodulin and the NaV1.5 interactome in regulating late Na+ current in cardiomyocytes. We created transgenic mice with cardiac-specific expression of human NaV1.5 channels with alanine substitutions for the IQ motif (IQ/AA). The mutations rendered the channels incapable of binding calmodulin to the C-terminus. The IQ/AA transgenic mice exhibited normal ventricular repolarization without arrhythmias and an absence of increased late Na+ current. In comparison, transgenic mice expressing a lidocaine-resistant (F1759A) human NaV1.5 demonstrated increased late Na+ current and prolonged repolarization in cardiomyocytes, with spontaneous arrhythmias. To determine regulatory factors that prevent late Na+ current for the IQ/AA mutant channel, we considered fibroblast growth factor homologous factors (FHFs), which are within the NaV1.5 proteomic subdomain shown by proximity labeling in transgenic mice expressing NaV1.5 conjugated to ascorbate peroxidase. We found that FGF13 diminished late current of the IQ/AA but not F1759A mutant cardiomyocytes, suggesting that endogenous FHFs may serve to prevent late Na+ current in mouse cardiomyocytes. Leveraging endogenous mechanisms may furnish an alternative avenue for developing novel pharmacology that selectively blunts late Na+ current.

MRI-based brain volumetry and retinal optical coherence tomography as the biomarkers of outcome in acute methanol poisoning.

BACKGROUND: Basal ganglia lesions are typical findings on magnetic resonance imaging (MRI) of the brain in survivors of acute methanol poisoning. However, no data are available on the association between the magnitude of damaged brain regions, serum concentrations of markers of acute methanol toxicity, oxidative stress, neuroinflammation, and the rate of retinal nerve ganglion cell loss. OBJECTIVES: To investigate the association between MRI-based volumetry of the basal ganglia, retinal nerve fibre layer (RNFL) thickness and prognostic laboratory markers of outcomes in acute methanol poisoning. METHODS: MRI-based volumetry of putamen, nucleus caudatus and globus pallidus was performed and compared with laboratory parameters of severity of poisoning and acute serum markers of oxidative damage of lipids (8-isoprostan, MDA, HHE, HNE), nucleic acids (8-OHdG, 8-OHG, 5-OHMU), proteins (o-Thyr, NO-Thyr, Cl-Thyr) and leukotrienes (LTC4, LTD4, LTE4, LTB4), as well as with the results of RNFL measurements by optic coherence tomography (OCT) in 16 patients with acute methanol poisoning (Group I) and in 28 survivors of poisoning two years after discharge with the same markers measured within the follow-up examination (Group II). The control group consisted of 28 healthy subjects without methanol poisoning. RESULTS: The survivors of acute methanol poisoning had significantly lower volumes of basal ganglia than the controls. The patients with MRI signs of methanol-induced toxic brain damage had significantly lower volumes of basal ganglia than those without these signs. A positive correlation was found between the volume of putamen and arterial blood pH on admission (r = 0.45; p = 0.02 and r = 0.44; p = 0.02 for left and right putamen, correspondingly). A negative correlation was present between the volumes of putamen and acute serum lactate (r = -0.63; p < 0.001 and r = -0.59; p = 0.01), creatinine (r = -0.53; p = 0.01 and r = -0.47; p = 0.01) and glucose (r = -0.55; p < 0.001 and r = -0.50; p = 0.01) concentrations. The volume of basal ganglia positively correlated with acute concentrations of markers of lipoperoxidation (8-isoprostan: r = 0.61; p < 0.05 and r = 0.59; p < 0.05 for left and right putamen, correspondingly) and inflammation (leukotriene LTB4: r = 0.61; p < 0.05 and r = 0.61; p < 0.05 for left and right putamen, correspondingly). The higher the volume of the basal ganglia, the higher the thickness of the RNFL, with the strongest positive association between global RNFL and the volume of putamen bilaterally (all p < 0.01). In the follow-up markers of oxidative stress and inflammation, only o-Thyr concentration negatively correlated with the volume of putamen bilaterally (r = -0.39; p < 0.05 and r = -0.37; p < 0.05 for left and right putamen, correspondingly). CONCLUSION: In survivors of acute methanol poisoning with signs of toxic brain damage, the magnitude of affected areas correlated with acute parameters of severity of poisoning, markers of oxidative stress and neuroinflammation. There was a positive association between the basal ganglia volume and the thickness of RNFL, making OCT an important screening test and MRI-based volumetry the confirmative diagnostic method for the detection of CNS sequelae of methanol poisoning.

Peripheral polyneuropathy after acute methanol poisoning: Six-year prospective cohort study.

BACKGROUND: Methanol is a widely used industrial short-chain aliphatic alcohol with known neurotoxic properties. Mass poisoning outbreaks due to the consumption of methanol-adulterated alcoholic drinks present a challenge to healthcare providers due to the high mortality and serious central nervous system (CNS) damage in survivors. However, the impact of methanol exposure on the peripheral nervous system is unknown. OBJECTIVES: To investigate the role of acute methanol exposure in the development of peripheral polyneuropathy (PNP) during the years following discharge from the hospital. METHODS: A total of 55 patients with confirmed methanol poisoning (mean age of 47.9 ± 3.6 years; 9 females) were examined 4 times within a 6-year prospective longitudinal cohort study. The program included neurological and electromyographic examinations, visual evoked potentials, ocular examinations with retinal nerve fibre layer thickness measurements, brain magnetic resonance imaging, and a series of biochemical and toxicological tests. RESULTS: PNP was observed in 20/55 (36 %) patients, which, in most of the cases, was mild axonal sensorimotor neuropathy. In 8/55 (15 %) patients, worsening of electromyographic findings was registered during the follow-up period, including 5 cases with newly diagnosed PNP and 3 cases of PNP progression. In one subject, complete reversal of PNP was registered after cessation of alcohol intake. The patients with PNP were significantly older (57.3 ± 5.3 versus 42.5 ± 3.9 years; p < 0.001), with higher blood glucose (5.93 ± 0.97 versus 4.81 ± 0.32 mmol/L; p = 0.035) and lower vitamin B1 (45.5 ± 7.4 versus 57.5 ± 5.2 ug/L; p = 0.015) concentrations. The number of chronic alcohol abusers was significantly higher in the PNP group (17/20 versus 20/35; p = 0.034). No associations between PNP prevalence/ dynamics and acute parameters of poisoning severity, arterial blood pH (7.26 ± 0.07 with PNP versus 7.18 ± 0.09 without PNP; p = 0.150), or serum methanol (1320.0 ± 700.0 with PNP versus 1430.0 ± 510.0 mg/L without PNP; p = 0.813) and ethanol (460.0 ± 560.0 with PNP versus 340.0 ± 230.0 mg/L without PNP; p = 0.675) concentrations at admission were found. No difference in the number of patients with visual (9/20 with PNP versus 12/35 patients without PNP; p = 0.431) and CNS sequelae (9/20 with PNP versus 15/35 patients without PNP; p = 0.877) of poisoning was present. DISCUSSION: Despite the relatively high number of PNP cases, no association was found between the severity of acute methanol poisoning and the prevalence of PNP and its dynamics during six years of observation. We did not find an association between methanol-induced visual/ brain damage and the prevalence of PNP in survivors of poisoning. A high prevalence of PNP and its progression might be attributed to other causes, mainly a history of chronic alcohol abuse and insufficiently treated diabetes mellitus. Our results highlight the importance of complete cessation of alcohol consumption and better control of glycaemia in diabetic patients in the prevention and treatment of peripheral PNP.

Formaldehyde Reacts with Amino Acids and Peptides with a Potential Role in Acute Methanol Intoxication.

Methanol, an aliphatic alcohol widely used in the industry, causes acute and chronic intoxications associated with severe long-term health damage, including permanent visual impairment, brain damage, mainly necrosis of the basal ganglia and high mortality due to cancer. However, the role of formaldehyde, an intermediate metabolite of methanol oxidation, in methanol toxicity remains unclear. Thus, we studied the reactivity of several amino acids and peptides in the presence of formaldehyde by identifying products by direct infusion electrospray high-resolution mass spectrometry (MS) and matrix-assisted laser desorption-ionization MS. Cysteine, homocysteine and two peptides, CG and CGAG, provided cyclic products with a +12 amu mass shift with respect to the original compounds. The proposed structures of the products were confirmed by high-resolution tandem MS. Moreover, the formation of the products with +12 amu mass shift was also shown for two biologically relevant peptides, fragments of ipilimumab, which is a human IgG1 monoclonal antibody against cytotoxic T-lymphocyte-associated protein 4. Overall, our experimental results indicate that formaldehyde reacts with some amino acids and peptides, yielding covalently modified structures. Such chemical modifications may induce undesirable changes in the properties and function of vital biomolecules (e.g., hormones, enzymes) and consequently pathogenesis.

Acute exposures to e-cigarettes and heat-not-burn products reported to the Czech Toxicological Information Centre over a 7-year period (2012-2018).

E-cigarettes and heat-not-burn cigarettes (HNBC) present new health risks due to their rising popularity, high content of nicotine and serious adverse effects. The objective of the study was to analyse the cases of acute exposure to e-cigarettes, e-liquids and HNBC products containing nicotine that led to toxicological consultations at our poisons control centre during a 7-year period (2012-2018) and identify the categories of special concern that require further investigation and intervention. The demographic, toxicological and clinical data were analysed by descriptive statistics. Poisoning severity score (PSS) was estimated. From 119 229 consultations, 148 cases concerned acute exposure to e-cigarettes. Children and adolescents were exposed in 91 (61%) cases, including exposure of neonates and infants in 54 (36%) cases. The main route of exposure was ingestion in 129 (87%), inhalation in nine (6%), ocular in six (4%) and intravenous administration in three (2%) cases. The source of exposure was the cartridge with e-liquid (107; 72%), refillable tank in 29 (20%) and HNBC refill in nine (6%) cases. The reason for exposure was accidental in 110 (74%), incorrect application of the device in 10 (7%), abuse in six (4%), suicide attempt in six (4%) and other/unknown in 16 (11%) cases. The dose estimation was severe/lethal in 6 (4%), toxic in 53 (36%), low-to-moderate in 35 (24%) and unknown in 54 (36%) cases. Vomiting was observed in 38 (26%) patients; 72% of patients were hospitalised. In symptomatic cases, 41 patient had PSS 1, 12 patients had PSS 2, and one patient had PSS 3. Activated charcoal was applied in 57 (39%) patients, and symptomatic treatment was recommended in 75 (51%) patients. Cases of unintentional exposure of children demonstrate the need for preventive risk reduction measures.

Mechanism of adrenergic CaV1.2 stimulation revealed by proximity proteomics.

Increased cardiac contractility during the fight-or-flight response is caused by ß-adrenergic augmentation of CaV1.2 voltage-gated calcium channels1-4. However, this augmentation persists in transgenic murine hearts expressing mutant CaV1.2 α1C and ß subunits that can no longer be phosphorylated by protein kinase A-an essential downstream mediator of ß-adrenergic signalling-suggesting that non-channel factors are also required. Here we identify the mechanism by which ß-adrenergic agonists stimulate voltage-gated calcium channels. We express α1C or ß2B subunits conjugated to ascorbate peroxidase5 in mouse hearts, and use multiplexed quantitative proteomics6,7 to track hundreds of proteins in the proximity of CaV1.2. We observe that the calcium-channel inhibitor Rad8,9, a monomeric G protein, is enriched in the CaV1.2 microenvironment but is depleted during ß-adrenergic stimulation. Phosphorylation by protein kinase A of specific serine residues on Rad decreases its affinity for ß subunits and relieves constitutive inhibition of CaV1.2, observed as an increase in channel open probability. Expression of Rad or its homologue Rem in HEK293T cells also imparts stimulation of CaV1.3 and CaV2.2 by protein kinase A, revealing an evolutionarily conserved mechanism that confers adrenergic modulation upon voltage-gated calcium channels.

Health-related quality of life determinants in survivors of a mass methanol poisoning outbreak: six-year prospective cohort study.

Purpose: The effect of acute methanol poisoning on the follow-up quality of life of survivors in mass poisoning outbreaks is not known. The objective of this is to study the impact of visual and central nervous system (CNS) sequelae of methanol poisoning on long-term health-related quality of life (QoL) of survivors, its clinical determinants, and dynamics.Materials and methods: A total of 54 patients with confirmed methanol poisoning (mean age 46.7 ± 13.4 years, 9 females) were examined consequently three times within six-year prospective cohort study and compared to 23 controls with the history of chronic alcohol abuse. The following tests were performed: SF-36 QoL questionnaire, visual evoked potentials (VEP) of optic nerve, ocular examination with retinal nerve fiber layer (RNFL) thickness measurement, brain magnetic resonance imaging (MRI), and biochemical and toxicological tests.Results: Acute methanol poisoning led to significant decrease in physical component summary (PCS) compared to PCS of age-adjusted controls (mean score with SD 46.8 ± 11.0 versus 52.3 ± 9.4 points; p = .003). In 17/40 (42.5%) patients with three rounds of examination, signs of severe disability (≤30 points in at least one score) were present six years after discharge, with negative dynamics of PCS score during the observation period. The patients with abnormal RNFL thickness had lower PCS (mean difference 10.5 points; 95%CI 3.5-17.5, p = .004) and mental component summary score (9.5 points; 95%CI 1.9-17.1, p = .015) compared to the patients with normal RNFL. Signs of physical and mental adaptation to long-term visual sequelae were registered with gradual reduction of difference in most of physical and mental components scores compared to the patients with normal RNFL during six years of observation. Signs of hemorrhagic brain lesions were associated with permanent decrease of PCS score (mean difference 7.4 points; 95%CI 0.6-14.0; p = .033), bodily pain (8.7 points; 95%CI 1.6-17.6; p = .018), and social functioning (8.2 points; 95%CI 3.0-17.4; p = .005) six years after discharge. No effect of type of antidote (fomepizole versus ethanol) and extracorporeal enhanced elimination modality (intermittent hemodialysis versus continuous renal replacement therapy) applied in hospital on long-term QoL was found (all p > .05).Conclusion: Acute methanol poisoning was associated with a significant decrease of health-related quality of life of survivors persisting for at least six years after discharge. The more pronounced decrease in QoL scores was observed in the patients with hemorrhagic brain lesions and visual sequelae of poisoning with abnormal RNFL thickness.

The impact of co-morbidities on a 6-year survival after methanol mass poisoning outbreak: possible role of metabolic formaldehyde.

Context: The influence of co-morbid conditions on the outcome of acute methanol poisoning in mass poisoning outbreaks is not known.Objective: The objective of this is to study the impact of burden of co-morbidities, complications, and methanol-induced brain lesions on hospital, follow-up, and total mortality.Methods: All patients hospitalized with methanol poisoning during a mass poisoning outbreak were followed in a prospective cohort study until death or final follow-up after 6 years. The age-adjusted Charlson co-morbidity index (ACCI) score was calculated for each patient. A multivariate Cox regression model was used to calculate the adjusted hazards ratio (HR) for death. The survival was modeled using the Kaplan-Meier method.Results: Of 108 patients (mean age with SD 50.9 ± 2.6 years), 24 (54.4 ± 5.9 years) died during hospitalization (mean survival with SD 8 ± 4 days) and 84 (49.9 ± 3.0 years; p = .159) were discharged, including 27 with methanol-induced brain lesions. Of the discharged patients, 15 (56.3 ± 6.8 years) died during the follow-up (mean survival 37 ± 11 months) and 69 (48.5 ± 3.3 years; p = .044) survived. The hospital mortality was 22%, the follow-up mortality was 18%; the total mortality was 36%. Cardiac/respiratory arrest, acute respiratory failure, multiorgan failure syndrome, and arterial hypotension increased the HR for hospital and total (but not follow-up) mortality after adjustment for age, sex, and arterial pH (all p < .05). All patients who died in the hospital had at least one complication. A higher ACCI score was associated with greater total mortality (HR 1.22; 1.00-1.48 95% CI; p = .046). Of those who died, 35 (90%) had a moderate-to-high ACCI. The Kaplan-Meier curve demonstrated that patients with a high ACCI had greater follow-up mortality compared to ones with low (p = .027) or moderate (p = .020) scores. For the patients who died during follow-up, cancers of different localizations were responsible for 7/15 (47%) of the deaths.Conclusions: The character and number of complications affected hospital but not follow-up mortality, while the burden of co-morbidities affected follow-up mortality. Methanol-induced brain lesions did not affect follow-up mortality. Relatively high cancer mortality rate may be associated with acute exposure to metabolic formaldehyde produced by methanol oxidation.

Consensus statements on the approach to patients in a methanol poisoning outbreak.

Background: Methanol poisoning is an important cause of mortality and morbidity worldwide. Although it often occurs as smaller sporadic events, epidemic outbreaks are not uncommon due to the illicit manufacture and sale of alcoholic beverages.Objective: We aimed to define methanol poisoning outbreak (MPO), outline an approach to triaging an MPO, and define criteria for prioritizing antidotes, extracorporeal elimination treatments (i.e., dialysis), and indications for transferring patients in the context of an MPO.Methods: We convened a group of experts from across the world to explore geographical, socio-cultural and clinical considerations in the management of an MPO. The experts answered specific open-ended questions based on themes aligned to the goals of this project. This project used a modified Delphi process. The discussion continued until there was condensation of themes.Results: We defined MPO as a sudden increase in the number of cases of methanol poisoning during a short period of time above what is normally expected in the population in that specific geographic area. Prompt initiation of an antidote is necessary in MPOs. Scarce hemodialysis resources require triage to identify patients most likely to benefit from this treatment. The sickest patients should not be transferred unless the time for transfer is very short. Transporting extracorporeal treatment equipment and antidotes may be more efficient.Conclusion: We have developed consensus statements on the response to a methanol poisoning outbreak. These can be used in any country and will be most effective when they are discussed by health authorities and clinicians prior to an outbreak.