Alcohol Exposure and Disease Associations: A Mendelian Randomization and Meta-Analysis on Weekly Consumption and Problematic Drinking.

Alcohol consumption significantly impacts disease burden and has been linked to various diseases in observational studies. However, comprehensive meta-analyses using Mendelian randomization (MR) to examine drinking patterns are limited. We aimed to evaluate the health risks of alcohol use by integrating findings from MR studies. A thorough search was conducted for MR studies focused on alcohol exposure. We utilized two sets of instrumental variables-alcohol consumption and problematic alcohol use-and summary statistics from the FinnGen consortium R9 release to perform de novo MR analyses. Our meta-analysis encompassed 64 published and 151 de novo MR analyses across 76 distinct primary outcomes. Results show that a genetic predisposition to alcohol consumption, independent of smoking, significantly correlates with a decreased risk of Parkinson's disease, prostate hyperplasia, and rheumatoid arthritis. It was also associated with an increased risk of chronic pancreatitis, colorectal cancer, and head and neck cancers. Additionally, a genetic predisposition to problematic alcohol use is strongly associated with increased risks of alcoholic liver disease, cirrhosis, both acute and chronic pancreatitis, and pneumonia. Evidence from our MR study supports the notion that alcohol consumption and problematic alcohol use are causally associated with a range of diseases, predominantly by increasing the risk.

Bulk and mosaic deletions of Egfr reveal regionally defined gliogenesis in the developing mouse forebrain.

The epidermal growth factor receptor (EGFR) plays a role in cell proliferation and differentiation during healthy development and tumor growth; however, its requirement for brain development remains unclear. Here we used a conditional mouse allele for Egfr to examine its contributions to perinatal forebrain development at the tissue level. Subtractive bulk ventral and dorsal forebrain deletions of Egfr uncovered significant and permanent decreases in oligodendrogenesis and myelination in the cortex and corpus callosum. Additionally, an increase in astrogenesis or reactive astrocytes in effected regions was evident in response to cortical scarring. Sparse deletion using mosaic analysis with double markers (MADM) surprisingly revealed a regional requirement for EGFR in rostrodorsal, but not ventrocaudal glial lineages including both astrocytes and oligodendrocytes. The EGFR-independent ventral glial progenitors may compensate for the missing EGFR-dependent dorsal glia in the bulk Egfr-deleted forebrain, potentially exposing a regenerative population of gliogenic progenitors in the mouse forebrain.

Tracking footprints of artificial and natural selection signatures in breeding and non-breeding cats.

Stray non-breeding cats (stray) represent the largest heterogeneous cat population subject to natural selection, while populations of the Siamese (SIAM) and Oriental Shorthair (OSH) breeds developed through intensive artificial selection for aesthetic traits. Runs of homozygosity (ROH) and demographic measures are useful tools to discover chromosomal regions of recent selection and to characterize genetic diversity in domestic cat populations. To achieve this, we genotyped 150 stray and 26 household non-breeding cats (household) on the Illumina feline 63 K SNP BeadChip and compared them to SIAM and OSH. The 50% decay value of squared correlation coefficients (r2) in stray (0.23), household (0.25), OSH (0.24) and SIAM (0.25) corresponded to a mean marker distance of 1.12 Kb, 4.55 Kb, 62.50 Kb and 175.07 Kb, respectively. The effective population size (Ne) decreased in the current generation to 55 in stray, 11 in household, 9 in OSH and 7 in SIAM. In the recent generation, the increase in inbreeding per generation (ΔF) reached its maximum values of 0.0090, 0.0443, 0.0561 and 0.0710 in stray, household, OSH and SIAM, respectively. The genomic inbreeding coefficient (FROH) based on ROH was calculated for three length categories. The FROH was between 0.014 (FROH60) and 0.020 (FROH5) for stray, between 0.018 (FROH60) and 0.024 (FROH5) for household, between 0.048 (FROH60) and 0.069 (FROH5) for OSH and between 0.053 (FROH60) and 0.073 (FROH5) for SIAM. We identified nine unique selective regions for stray through genome-wide analyses for regions with reduced heterozygosity based on FST statistics. Genes in these regions have previously been associated with reproduction (BUB1B), motor/neurological behavior (GPHN, GABRB3), cold-induced thermogenesis (DIO2, TSHR), immune system development (TSHR), viral carcinogenesis (GTF2A1), host immune response against bacteria, viruses, chemoattractant and cancer cells (PLCB2, BAHD1, TIGAR), and lifespan and aging (BUB1B, FGF23). In addition, we identified twelve unique selective regions for OSH containing candidate genes for a wide range of coat colors and patterns (ADAMTS20, KITLG, TYR, TYRO3-a MITF regulator, GPNMB, FGF7, RAB38) as well as congenital heart defects (PDE4D, PKP2) and gastrointestinal disorders (NLGN1, ALDH1B1). Genes in stray that represent unique selective events indicate, at least in part, natural selection for environmental adaptation and resistance to infectious disease, and should be the subject of future research. Stray cats represent an important genetic resource and have the potential to become a research model for disease resistance and longevity, which is why we recommend preserving semen before neutering.

Safety and immunogenicity of COVID-19 vaccination in patients with hepatocellular carcinoma (CHESS-NMCID 2101): A multicenter prospective study.

Data on safety and immunogenicity of coronavirus disease 2019 (COVID-19) vaccinations in hepatocellular carcinoma (HCC) patients are limited. In this multicenter prospective study, HCC patients received two doses of inactivated whole-virion COVID-19 vaccines. The safety and neutralizing antibody were monitored. Totally, 74 patients were enrolled from 10 centers in China, and 37 (50.0%), 25 (33.8%), and 12 (16.2%) received the CoronaVac, BBIBP-CorV, and WIBP-CorV, respectively. The vaccines were well tolerated, where pain at the injection site (6.8% [5/74]) and anorexia (2.7% [2/74]) were the most frequent local and systemic adverse events. The median level of neutralizing antibody was 13.5 (interquartile range [IQR]: 6.9-23.2) AU/ml at 45 (IQR: 19-72) days after the second dose of vaccinations, and 60.8% (45/74) of patients had positive neutralizing antibody. Additionally, lower γ-glutamyl transpeptidase level was related to positive neutralizing antibody (odds ratio = 1.022 [1.003-1.049], p = 0.049). In conclusion, this study found that inactivated COVID-19 vaccinations are safe and the immunogenicity is acceptable or hyporesponsive in patients with HCC. Given that the potential benefits may outweigh the risks and the continuing emergences of novel severe acute respiratory syndrome coronavirus 2 variants, we suggest HCC patients to be vaccinated against COVID-19. Future validation studies are warranted.

Health-adjusted life expectancy (HALE) in Chongqing, China, 2017: An artificial intelligence and big data method estimating the burden of disease at city level.

BACKGROUND: A universally applicable approach that provides standard HALE measurements for different regions has yet to be developed because of the difficulties of health information collection. In this study, we developed a natural language processing (NLP) based HALE estimation approach by using individual-level electronic medical records (EMRs), which made it possible to calculate HALE timely in different temporal or spatial granularities. METHODS: We performed diagnostic concept extraction and normalisation on 13•99 million EMRs with NLP to estimate the prevalence of 254 diseases in WHO Global Burden of Disease Study (GBD). Then, we calculated HALE in Chongqing, 2017, by using the life table technique and Sullivan's method, and analysed the contribution of diseases to the expected years "lost" due to disability (DLE). FINDINGS: Our method identified a life expectancy at birth (LE0) of 77•9 years and health-adjusted life expectancy at birth (HALE0) of 71•7 years for the general Chongqing population of 2017. In particular, the male LE0 and HALE0 were 76•3 years and 68•9 years, respectively, while the female LE0 and HALE0 were 80•0 years and 74•4 years, respectively. Cerebrovascular diseases, cancers, and injuries were the top three deterioration factors, which reduced HALE by 2•67, 2•15, and 1•19 years, respectively. INTERPRETATION: The results demonstrated the feasibility and effectiveness of EMRs-based HALE estimation. Moreover, the method allowed for a potentially transferable framework that facilitated a more convenient comparison of cross-sectional and longitudinal studies on HALE between regions. In summary, this study provided insightful solutions to the global ageing and health problems that the world is facing. FUNDING: National Key R and D Program of China (2018YFC2000400).

Clonal Analysis of Gliogenesis in the Cerebral Cortex Reveals Stochastic Expansion of Glia and Cell Autonomous Responses to Egfr Dosage.

Development of the nervous system undergoes important transitions, including one from neurogenesis to gliogenesis which occurs late during embryonic gestation. Here we report on clonal analysis of gliogenesis in mice using Mosaic Analysis with Double Markers (MADM) with quantitative and computational methods. Results reveal that developmental gliogenesis in the cerebral cortex occurs in a fraction of earlier neurogenic clones, accelerating around E16.5, and giving rise to both astrocytes and oligodendrocytes. Moreover, MADM-based genetic deletion of the epidermal growth factor receptor (Egfr) in gliogenic clones revealed that Egfr is cell autonomously required for gliogenesis in the mouse dorsolateral cortices. A broad range in the proliferation capacity, symmetry of clones, and competitive advantage of MADM cells was evident in clones that contained one cellular lineage with double dosage of Egfr relative to their environment, while their sibling Egfr-null cells failed to generate glia. Remarkably, the total numbers of glia in MADM clones balance out regardless of significant alterations in clonal symmetries. The variability in glial clones shows stochastic patterns that we define mathematically, which are different from the deterministic patterns in neuronal clones. This study sets a foundation for studying the biological significance of stochastic and deterministic clonal principles underlying tissue development, and identifying mechanisms that differentiate between neurogenesis and gliogenesis.

Biochemical parameters, dynamic tensiometry and circulating nucleic acids for cattle blood analysis: a review.

The animal's blood is the most complicated and important biological liquid for veterinary medicine. In addition to standard methods that are always in use, recent technologies such as dynamic tensiometry (DT) of blood serum and PCR analysis of particular markers are in progress. The standard and modern biochemical tests are commonly used for general screening and, finally, complete diagnosis of animal health. Interpretation of major biochemical parameters is similar across animal species, but there are a few peculiarities in each case, especially well-known for cattle. The following directions are discussed here: hematological indicators; "total protein" and its fractions; some enzymes; major low-molecular metabolites (glucose, lipids, bilirubin, etc.); cations and anions. As example, the numerous correlations between DT data and biochemical parameters of cattle serum have been obtained and discussed. Changes in the cell-free nucleic acids (cfDNA) circulating in the blood have been studied and analyzed in a variety of conditions; for example, pregnancy, infectious and chronic diseases, and cancer. CfDNA can easily be detected using standard molecular biological techniques like DNA amplification and next-generation sequencing. The application of digital PCR even allows exact quantification of copy number variations which are for example important in prenatal diagnosis of chromosomal aberrations.

Interdigital Hyperplasia in Holstein Cattle Is Associated With a Missense Mutation in the Signal Peptide Region of the Tyrosine-Protein Kinase Transmembrane Receptor Gene.

Bovine interdigital hyperplasia (IH) is a typical disease of the foot with varying prevalence depending on age, breed, and environmental factors resulting in different degrees of lameness. In studies based on assessments of claw health status at time of hoof trimming and applying genetic-statistical models to analyze this data, IH consistently exhibits high estimates of heritability in the range of 0.30-0.40. Although some studies have identified chromosomal regions that could possibly harbor causative genes, a clear identification of molecular causes for IH is lacking. While analyzing the large database of claw health status as documented at time of hoof trimming, we identified one herd with extreme prevalence of IH of > 50% of affected Holstein dairy cows. This herd subsequently was chosen as the object of a detailed study. A total of n = 91 cows was assessed and revealed a prevalence of 59.3% and 38.5% for IH cases, documented as "one-sided" or "two-sided", respectively. Cows were genotyped using the BovineSNP50 BeadChip. A genome wide association study revealed two significantly associated chromosomal positions (-log10P = 5.57) on bovine chromosome 8 (BTA8) located in intron 5 and downstream of the receptor tyrosine kinase-like orphan receptor 2 (ROR2) gene. As ROR2 plays a key role in ossification of the distal limbs and is associated with brachydactylies in humans, it was a reasonable candidate for IH. A comparative sequencing of the ROR2 gene between cases and controls revealed two missense variants in exon 1 (NC_037335.1:g.85,905,534T > A, ARS-UCD1.2) and exon 9 (NC_037335.1:g.86,140,379A > G, ARS-UCD1.2), respectively. Genotyping of both variants in the cohort of 91 cattle showed that the exon 1 variant (rs377953295) remained significantly associated with IH (p < 0.0001) as a risk factor of the disease. This variant resulted in an amino acid exchange (ENSBTAP00000053765.2:p.Trp9Arg) in the N-terminal region of the ROR2 signal peptide which is necessary for proper topology of the polypeptide during translocation. Quantification of ROR2 mRNA and ROR2 protein showed that the variant resulted in a significant suppression of ROR2 expression in homozygous affected compared to wild type and carrier cows.

Transplantation of GABAergic Interneurons into the Neonatal Primary Visual Cortex Reduces Absence Seizures in Stargazer Mice.

Epilepsies are debilitating neurological disorders characterized by repeated episodes of pathological seizure activity. Absence epilepsy (AE) is a poorly understood type of seizure with an estimated 30% of affected patients failing to respond to antiepileptic drugs. Thus, novel therapies are needed for the treatment of AE. A promising cell-based therapeutic strategy is centered on transplantation of embryonic neural stem cells from the medial ganglionic eminence (MGE), which give rise to gamma-aminobutyric acidergic (GABAergic) interneurons during embyronic development. Here, we used the Stargazer (Stg) mouse model of AE to map affected loci using c-Fos immunohistochemistry, which revealed intense seizure-induce activity in visual and somatosensory cortices. We report that transplantation of MGE cells into the primary visual cortex (V1) of Stg mice significantly reduces AE episodes and lowers mortality. Electrophysiological analysis in acute cortical slices of visual cortex demonstrated that Stg V1 neurons exhibit more pronounced increases in activity in response to a potassium-mediated excitability challenge than wildtypes (WT). The defective network activity in V1 was significantly altered following WT MGE transplantation, associating it with behavioral rescue of seizures in Stgs. Taken together, these findings present MGE grafting in the V1 as a possible clinical approach in the treatment of AE.

Identification of substituted 3-hydroxy-2-mercaptocyclohex-2-enones as potent inhibitors of human lactate dehydrogenase.

A novel class of 3-hydroxy-2-mercaptocyclohex-2-enone-containing inhibitors of human lactate dehydrogenase (LDH) was identified through a high-throughput screening approach. Biochemical and surface plasmon resonance experiments performed with a screening hit (LDHA IC50=1.7 µM) indicated that the compound specifically associated with human LDHA in a manner that required simultaneous binding of the NADH co-factor. Structural variation of this screening hit resulted in significant improvements in LDHA biochemical inhibition activity (best IC50=0.18 µM). Two crystal structures of optimized compounds bound to human LDHA were obtained and explained many of the observed structure-activity relationships. In addition, an optimized inhibitor exhibited good pharmacokinetic properties after oral administration to rats (F=45%).

Transcription factor C/EBPß and 17ß-estradiol promote transcription of the porcine p53 gene.

The tumor protein 53 (p53) gene played a crucial role in maternal reproduction except its classic roles in maintaining genomic stability and preventing tumorigenesis. However, little is known concerning the regulatory elements which control the expression of p53 gene. In this study, we predicted two binding sites (-490/-477 and -405/-392) of transcription factor CCAAT/enhancer binding protein beta (C/EBPß) within the core promoter (-985/-273) determined by promoter deletion analysis, and discovered that the second site (-405/-392) was important for p53 promoter activity by site-directed mutagenesis. Then the binding of C/EBPß to the p53 promoter was identified by electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP). Moreover, evidence from C/EBPß overexpression and RNAi studies showed C/EBPß regulated p53 promoter activity and endogenous p53 expression. Meanwhile, we observed p53 mRNA at the peak in 10(-6)mol/L 17ß-estradiol treated cells for 24h via enhancing its core promoter activity. Taken together, our study indicates that C/EBPß and 17ß-estradiol are the essential regulatory factors for p53 transcription.

Polysialylated NCAM and ephrinA/EphA regulate synaptic development of GABAergic interneurons in prefrontal cortex.

A novel function for the neural cell adhesion molecule (NCAM) was identified in ephrinA/EphA-mediated repulsion as an important regulatory mechanism for development of GABAergic inhibitory synaptic connections in mouse prefrontal cortex. Deletion of NCAM, EphA3, or ephrinA2/3/5 in null mutant mice increased the numbers and size of perisomatic synapses between GABAergic basket interneurons and pyramidal cells in the developing cingulate cortex (layers II/III). A functional consequence of NCAM loss was increased amplitudes and faster kinetics of miniature inhibitory postsynaptic currents in NCAM null cingulate cortex. NCAM and EphA3 formed a molecular complex and colocalized with the inhibitory presynaptic marker vesicular GABA transporter (VGAT) in perisomatic puncta and neuropil in the cingulate cortex. EphrinA5 treatment promoted axon remodeling of enhanced green fluorescent protein-labeled basket interneurons in cortical slice cultures and induced growth cone collapse in wild-type but not NCAM null mutant neurons. NCAM modified with polysialic acid (PSA) was required to promote ephrinA5-induced axon remodeling of basket interneurons in cortical slices, likely by providing a permissive environment for ephrinA5/EphA3 signaling. These results reveal a new mechanism in which NCAM and ephrinAs/EphA3 coordinate to constrain GABAergic interneuronal arborization and perisomatic innervation, potentially contributing to excitatory/inhibitory balance in prefrontal cortical circuitry.

Anticonvulsant activity of BmK AS, a sodium channel site 4-specific modulator.

The anticonvulsant activity of BmK AS, a sodium channel site 4-selective modulator purified from scorpion venom (Buthus martensi Karsch), was investigated in unanesthetized rats with acute pentylenetetrazole (PTZ)- and pilocarpine-induced seizures. Rats were microinjected in the CA1 region with either saline or BmK AS, followed by epileptogenic doses of PTZ or pilocarpine 30 minutes later. The anticonvulsant efficacy of BmK AS in PTZ- or pilocarpine-evoked seizure-like behavior and cortical epileptiform EEG activity was assessed. Intrahippocampal injections of BmK AS (0.05-1 µg in 1 µL) produced dose-dependent anticonvulsant activity in the PTZ model, suppressing seizure-associated behavior and reducing both the number and duration of high-amplitude, high-frequency discharges (HAFDs) on the EEG. In contrast, BmK AS did not affect the epileptiform EEG in the pilocarpine model over the same dose range, although it did increase the latency to status epilepticus onset and slightly, but significantly, reduced the seizure score. In summary, our results demonstrate that the sodium channel site 4-selective modulator BmK AS is an effective inhibitor of PTZ- but not pilocarpine-induced acute seizures. These results indicate that BmK AS may serve as a novel probe in exploring the role of different sodium channel subtypes in an epileptogenic setting and as a potential lead in developing antiepileptic drugs specifically for the therapy of sodium channel site 4-related epilepsy.

The epileptic seizures induced by BmK I, a modulator of sodium channels.

In the present study, the susceptibility to rat epileptic seizures induced by the intrahippocampal administration of BmK I, a modulator of sodium channels purified from the venom of Chinese scorpion, has been investigated. The results showed that the strong epileptic behaviors and discharges in the hippocampus were evoked by BmK I dose-dependently. The hippocampal c-Fos expression displayed two peak waves in a specific spatio-temporal pattern elicited by BmK I. The whole cell patch clamp recordings showed that the inactivation of sodium currents in rat cultured hippocampal neurons was prolonged significantly by BmK I, and restored partially after washing. These results indicated that the rat hippocampus is a susceptible target for the proconvulsant effects of BmK I, and the induction of epileptic seizures may be ascribed to the modulation of BmK I on the inactivation of voltage-gated sodium channels distributing in the rat hippocampal neurons.

Decrease of morphine-induced reward effects and withdrawal symptoms in mice overexpressing gamma-aminobutyric acid transporter I.

Morphine addiction has been shown to result from neural adaptations produced by repeated drug exposure, but the mechanism is still unclear. In the present study, we found that gamma-aminobutyric acid (GABA) uptake was increased in mouse brain 120 min after, but not 20 min after, morphine (10 mg/kg, s.c.) injection. We generated GABA transporter I (GAT1)-overexpressing mice to investigate whether the GABAergic system and GABA transporter are involved in morphine-induced reward effects and withdrawal symptoms. Our results revealed that the rewarding effects induced by morphine were significantly decreased in GAT1-overexpressing mice as measured by the conditioned place preference (CPP) paradigm. Moreover, both somatic and vegetative signs of naloxone-induced morphine withdrawal symptoms were substantially reduced in GAT1-overexpressing mice. In addition, the decreased morphine rewarding in transgenic mice could be recovered when mice were coinjected with NO-711 (a GAT1 selective inhibitor) in the CPP paradigm. These findings suggest that the GABAergic system plays an important role in morphine addiction and point to the possibility of developing drugs that target GAT1 and extend the clinical application of opiates.

Fos expression in rat spinal cord induced by peripheral injection of BmK I, an alpha-like scorpion neurotoxin.

In this paper, the central neuronal activities elicited by BmK I, a specific voltage-gated Na+ channel modulator, were examined by monitoring the c-Fos expression pattern of rat spinal cord. c-Fos protein in laminae I-II, V-VI, and VII-X could be detected at 0.5 h, increased steadily at 1 h, reached a peak at 2 h, and then decreased rapidly from 4 to 24 h after Bmk I was subcutaneously injected into the rat hind paw. However, c-Fos expression in laminae III-IV was activated to a peak at 0.5 h and then declined gradually from 0.5 to 24 h. Furthermore, c-Fos expression could be induced by BmK I in a dose-dependent manner. In addition, the increase of c-Fos expression in laminae I-II, V-VI, and VII-X induced by BmK I, and not in laminae III-IV, could be partially inhibited by systemic morphine in a dose-dependent manner. The results suggested that peripheral administration of BmK I could evoke a profound change of spinal neuronal activities manifested as specific patterns of c-Fos expression, which may be partially attributed to the selective modulation of BmK I on voltage-gated Na+ channels located in peripheral nociceptors.

Suppressive effects of BmK IT2 on nociceptive behavior and c-Fos expression in spinal cord induced by formalin.

In this study, the suppressive effects of BmK IT2, a kind of Na+ channel-specific modulator from the venom of the scorpion Buthus martensi Karsch, on biphasic nociceptive behavior in rats and c-Fos expression in rat spinal cord induced by formalin were investigated. Fifty microliters of 2.5% formalin were subcutaneously injected into the rat hind paw; 0.1 and 1 microg doses of BmK IT2 were subcutaneously administered into the rat ipsilateral hind paw 1 min before or 10 min after formalin injection individually, and the number of flinches per 5 min was counted. The detection of c-Fos expression induced by formalin in either the absence or the presence of BmK IT2 was carried out with the ABC method. Biphasic nociceptive behavior in rats was significantly suppressed by pretreatment with BmK IT2. No. of flinches/5 min in the second phase was also decreased by posttreatment with BmK IT2. In addition, c-Fos expression induced by formalin was significantly inhibited in all laminae of L4-5 spinal cord by pre- or posttreatment with BmK IT2. The suppression of BmK IT2 in the first- and second-phase behaviors may be attributed to the anesthesia of the toxin toward nociceptors and primary afferents and its selective modulation of tetrodotoxin-resistant Na+ currents of dorsal root ganglion neurons, respectively. In addition, the nonparallel suppression of BmK IT2 on flinch behavior and c-Fos expression induced by formalin may be ascribed to the different activity patterns of afferent fibers and central neurons.

c-Fos expression in rat spinal cord induced by scorpion BmK venom via plantar subcutaneous injection.

The aim of this study was to assess the cell-type and distribution of highly activated neurons in rat spinal cord underlying nociceptive responses induced by scorpion BmK venom using Fos immunohistochemistry. BmK venom was intraplantarly injected into one hind paw of a conscious rat. Fos-like immunoreactive neurons were found to predominantly distribute at L4-5 segments in the rat spinal cord after BmK venom application. c-Fos labeling was most dense in the medial half portion of laminae I-II, moderately dense in laminae V-VI and less dense in laminae III-IV, VII-X. c-Fos labeling could be detected at 0.5 h, reached the peak at 2 h, decreased steeply from 4 h and then almost disappeared at 24 h. Ten to fifty micrograms of BmK venom was deemed to be a sufficient dosage to evoke c-Fos expression. On the other hand, c-Fos expression induced by BmK venom could be suppressed partially by systemic morphine in a dose-dependent manner. The results suggest that the different extent of activities of neuronal subpopulation in the spinal cord involved in nociceptive transmission manifesting as c-Fos expression, were mainly correlated with mechanisms underlying the generation, maintenance and/or modulation of spontaneous pain and hyperalgesia evoked by BmK venom.