Strategies for genomic predictions of an indicine multi-breed population using single-step GBLUP.

Brazilian livestock breeding programmes strive to enhance the genetics of beef cattle, with a strong emphasis on the Nellore breed, which has an extensive database and has achieved significant genetic progress in the last years. There are other indicine breeds that are economically important in Brazil; however, these breeds have more modest sets of phenotypes, pedigree and genotypes, slowing down their genetic progress as their predictions are less accurate. Combining several breeds in a multi-breed evaluation could help enhance predictions for those breeds with less information available. This study aimed to evaluate the feasibility of multi-breed, single-step genomic best linear unbiased predictor genomic evaluations for Nellore, Brahman, Guzerat and Tabapua. Multi-breed evaluations were contrasted to the single-breed ones. Data were sourced from the National Association of Breeders and Researchers of Brazil and included pedigree (4,207,516), phenotypic (328,748), and genomic (63,492) information across all breeds. Phenotypes were available for adjusted weight at 210 and 450 days of age, and scrotal circumference at 365 days of age. Various scenarios were evaluated to ensure pedigree and genomic information compatibility when combining different breeds, including metafounders (MF) or building the genomic relationship matrix with breed-specific allele frequencies. Scenarios were compared using the linear regression method for bias, dispersion and accuracy. The results showed that using multi-breed evaluations significantly improved accuracy, especially for smaller breeds like Guzerat and Tabapua. The validation statistics indicated that the MF approach provided accurate predictions, albeit with some bias. While single-breed evaluations tended to have lower accuracy, merging all breeds in multi-breed evaluations increased accuracy and reduced dispersion. This study demonstrates that multi-breed genomic evaluations are proper for indicine beef cattle breeds. The MF approach may be particularly beneficial for less-represented breeds, addressing limitations related to small reference populations and incompatibilities between G and A22. By leveraging genomic information across breeds, breeders and producers can make more informed selection decisions, ultimately improving genetic gain in these cattle populations.

In-Hospital Violence and Its Impact on Critical Care Practitioners.

OBJECTIVES: To provide a narrative review of hospital violence (HV) and its impact on critical care clinicians. DATA SOURCES: Detailed search strategy using PubMed and OVID Medline for English language articles describing HV, risk factors, precipitating events, consequences, and mitigation strategies. STUDY SELECTION: Studies that specifically addressed HV involving critical care medicine clinicians or their practice settings were selected. The time frame was limited to the last 15 years to enhance relevance to current practice. DATA EXTRACTION: Relevant descriptions or studies were reviewed, and abstracted data were parsed by setting, clinician type, location, social media events, impact, outcomes, and responses (agency, facility, health system, individual). DATA SYNTHESIS: HV is globally prevalent, especially in complex care environments, and correlates with a variety of factors including ICU stay duration, conflict, and has recently expanded to out-of-hospital occurrences; online violence as well as stalking is increasingly prevalent. An overlap with violent extremism and terrorism that impacts healthcare facilities and clinicians is similarly relevant. A number of approaches can reduce HV occurrence including, most notably, conflict management training, communication initiatives, and visitor flow and access management practices. Rescue training for HV occurrences seems prudent. CONCLUSIONS: HV is a global problem that impacts clinicians and imperils patient care. Specific initiatives to reduce HV drivers include individual training and system-wide adaptations. Future methods to identify potential perpetrators may leverage machine learning/augmented intelligence approaches.

Assessment of Genetic Diversity, Runs of Homozygosity, and Signatures of Selection in Tropical Milking Criollo Cattle Using Pedigree and Genomic Data.

The objective of this study was to evaluate the genetic diversity of the Tropical Milking Criollo cattle (TMC) breed in Mexico through parameters derived from pedigree and genomic information assessment. The pedigree file consisted of 3780 animals. Seventy-nine bovines were genotyped with the medium-density single nucleotide polymorphism chip and considered a reference population for pedigree analysis. The effective population size and the probability of gene origin used to assess the evolution of genetic diversity were calculated with pedigree information. Inbreeding coefficients were evaluated based on pedigree (FPed), the genomic relationship matrix (FGRM), and runs of homozygosity (FROH) of different length classes. The average inbreeding was 2.82 ± 2.66%, −0.7 ± 3.8%, and 10.9 ± 3.0% for FPED, FGRM, and FROH, respectively. Correlation between FPED and FROH was significant only for runs of homozygosity > 4 Mb, indicating the FPED of a population with an average equivalent complete generation of five only recovers the most recent inbreeding. The parameters of the probability of gene origin indicated the existence of genetic bottlenecks and the loss of genetic diversity in the history of the TMC cattle population; however, pedigree and genomic information revealed the existence of current sufficient genetic diversity to design a sustainable breeding program.

Impact of the Discordance Between Scales of Memorial Sloan-Kettering Cancer Center and International Metastatic Renal Cell Carcinoma Database Consortium in Patients' Prognosis With Metastatic Renal Cancer.

Background: In Mexico, about 30% of renal cancer patients are diagnosed in a metastatic state. Despite the recent advances in the treatment of cancer, metastatic renal cancer is still an incurable illness. Thus, identifying prognostic factors helps improve prognosis accuracy and survival prediction for patients. Methods: In this study, we retrospectively analyzed 26 patients with histological diagnosis of renal cell carcinoma, including clear cell and other subtypes in stage IV (metastatic), recurrent or unresectable disease. We performed a multivariate analysis of overall survival regarding the congruity between prognostic scales. Results: Our results showed a significant difference in favor of patients with congruity between scales for progression-free survival (18.9 vs. 3.1 months; P = 0.048) and a tendency towards better overall survival in patients with the congruity of both scales compared to the discordant patients (112 vs. 32 months; P = 0.99). Conclusion: This study highlights the discordance between Memorial Sloan-Kettering Cancer Center and International Metastatic Renal Cell Carcinoma Database Consortium scales, which was associated with worse prognosis with a significant difference in progression-free survival but not in overall survival.

Surnames and population structure in the Doctrine of Belén, Altos de Arica, Viceroyalty of Peru (1750-1813).

The analysis of multiple population structures (biodemographic, genetic and socio-cultural) and their inter-relations contribute to a deeper understanding of population structure and population dynamics. Genetically, the population structure corresponds to the deviation of random mating conditioned by a limited number of ancestors, by restricted migration in the social or geographic space, or by preference for certain consanguineous unions. Through the isonymic method, surname frequency and distribution across the population can supply quantitative information on the structure of a human population, as they constitute universal socio-cultural variables. Using documentary sources to undertake the Doctrine of Belén's (Altos de Arica, Chile) historical demography reconstruction between 1763 and 1820, this study identified an indigenous population with stable patronymics. The availability of complete marriage, baptism and death records, low rates of migration and the significant percentage of individuals registered and constantly present in this population favoured the application of the isonymic method. The aim of this work was to use given names and surnames recorded in these documentary sources to reconstruct the population structure and migration pattern of the Doctrine of Belén between 1750 and 1813 through the isonymic method. The results of the study were consistent with the ethno-historical data of this ethnic space, where social cohesion was, in multiple ways, related to the regulation of daily life in colonial Andean societies.

Prevalence and Molecular Profile of Breast Carcinoma Using Immunohistochemistry Markers in Mexican Women.

BACKGROUND: In Mexico, breast cancer is the leading cause of death by malignant tumors in women aged 20 and older. The World Health Organization estimates that 69% of deaths caused by breast cancer occur in developing countries. Little is known about the prevalence of breast carcinoma in Mexico and its molecular subclassification. METHODS: This retrospective cross-sectional study included patients who underwent a mastectomy (single, radical or lumpectomy) or a breast tumor biopsy (core-needle or excisional) from January 2002 to December 2018. The primary purpose of the study was to determine the prevalence and molecular profile of breast in comprehensive cancer center in Mexico and compare our results with those published in the US. This study was approved by our scientific and bioethical committee. RESULTS: The final analysis included 379 patients. The youngest patient was 23 years old and the oldest patient was 89; the mean age at diagnosis was 54.63 years. Patients of 40 years old or younger accounted for 48 of the cases (12.66%) and those older than 40 accounted for 331 of the cases (87.33%). The molecular subclassification showed luminal A subtype in 139 cases (36.67%), luminal B subtype in 143 cases (37.73%), human epidermal growth factor receptor 2-positive carcinomas in 32 cases (8.44%) and triple-negative carcinomas in 65 cases (17.15%). Diabetes mellitus was present in 43 patients (11.34%), hypertension in 78 patients (20.58%), obesity in 82 patients (21.63%) and 66 patients reported being treated with exogenous hormone therapy (17.41%). CONCLUSIONS: Breast carcinoma occurs at an earlier age in Mexican women compared to women in the US. Hormone-positive tumors were found to be more prevalent in older patients, while high-grade tumors were more frequently identified in younger patients.

Genetic Background and Inbreeding Depression in Romosinuano Cattle Breed in Mexico.

The ultimate goal of genetic selection is to improve genetic progress by increasing favorable alleles in the population. However, with selection, homozygosity, and potentially harmful recessive alleles can accumulate, deteriorating genetic variability and hampering continued genetic progress. Such potential adverse side effects of selection are of particular interest in populations with a small effective population size like the Romosinuano beef cattle in Mexico. The objective of this study was to evaluate the genetic background and inbreeding depression in Mexican Romosinuano cattle using pedigree and genomic information. Inbreeding was estimated using pedigree (FPED) and genomic information based on the genomic relationship matrix (FGRM) and runs of homozygosity (FROH) of different length classes. Linkage disequilibrium (LD) was evaluated using the correlation between pairs of loci, and the effective population size (Ne) was calculated based on LD and pedigree information. The pedigree file consisted of 4875 animals born between 1950 and 2019, of which 71 had genotypes. LD decreased with the increase in distance between markers, and Ne estimated using genomic information decreased from 610 to 72 animals (from 109 to 1 generation ago), the Ne estimated using pedigree information was 86.44. The reduction in effective population size implies the existence of genetic bottlenecks and the decline of genetic diversity due to the intensive use of few individuals as parents of the next generations. The number of runs of homozygosity per animal ranged between 18 and 102 segments with an average of 55. The shortest and longest segments were 1.0 and 36.0 Mb long, respectively, reflecting ancient and recent inbreeding. The average inbreeding was 2.98 ± 2.81, 2.98 ± 4.01, and 7.28 ± 3.68% for FPED, FGRM, and FROH, respectively. The correlation between FPED and FGRM was -0.25, and the correlations among FPED and FROH of different length classes were low (from 0.16 to 0.31). The correlations between FGRM and FROH of different length classes were moderate (from 0.44 to 0.58), indicating better agreement. A 1% increase in population inbreeding decreased birth weight by 0.103 kg and weaning weight by 0.685 kg. A strategy such as optimum genetic contributions to maximize selection response and manage the long-term genetic variability and inbreeding could lead to more sustainable breeding programs for the Mexican Romosinuano beef cattle breed.

Synaptotagmin-1 overexpression under inflammatory conditions affects secretion in salivary glands from Sjögren's syndrome patients.

Sjögren's syndrome (SS) is an autoimmune exocrinopathy associated with severe secretory alterations by disruption of the glandular architecture integrity, which is fundamental for a correct function and localization of the secretory machinery. Syt-1, PI(4,5)P2 and Ca2+ are significant factors controlling exocytosis in different secretory cells, the Ca2+ role being the most studied. Salivary acinar cells from SS-patients show a defective agonist-regulated intracellular Ca2+ release together with a decreased IP3R expression level, and this condition may explain a reduced water release. However, there are not reports where Syt-1, PI(4,5)P2 and Ca2+ in acinar cells of SS patients had been studied. In the present study, we analyzed the expression and/or localization of Syt-1 and PI(4,5)P2 in acinar cells of labial salivary gland biopsies from SS-patients and control individuals. Also, we evaluated whether the overexpression of Syt-1 and the loss of cell polarity induced by TNF-α or loss of interaction between acinar cell and basal lamina, alters directionality of the exocytosis process, Ca2+ signaling and α-amylase secretion in a 3D-acini model stimulated with cholinergic or ß-adrenergic agonists. In addition, the correlation between Syt-1 protein levels and clinical parameters was evaluated. The results showed an increase of Syt-1 mRNA and protein levels, and a high number of co-localization points of Syt-1/STX4 and PI(4,5)P2/Ezrin in the acinar basolateral region of LSG from SS-patients. With regard to 3D-acini, Syt-1 overexpression increased exocytosis in the apical pole compared to control acini. TNF-α stimulation increased exocytic events in the basal pole, which was further enhanced by Syt-1 overexpression. Additionally, altered acinar cell polarity affected Ca2+ signaling and amylase secretion. Overexpression of Syt-1 was associated with salivary gland alterations revealing that the secretory dysfunction in SS-patients is linked to altered expression and/or localization of secretory machinery components together with impaired epithelial cell polarity. These findings provide a novel insight on the pathological mechanism implicated in ectopic secretory products to the extracellular matrix of LSG from SS-patients, which might initiate inflammation.

Triclosan Impairs Hippocampal Synaptic Plasticity and Spatial Memory in Male Rats.

Triclosan, a widely used industrial and household agent, is present as an antiseptic ingredient in numerous products of everyday use, such as toothpaste, cosmetics, kitchenware, and toys. Previous studies have shown that human brain and animal tissues contain triclosan, which has been found also as a contaminant of water and soil. Triclosan disrupts heart and skeletal muscle Ca2+ signaling, damages liver function, alters gut microbiota, causes colonic inflammation, and promotes apoptosis in cultured neocortical neurons and neural stem cells. Information, however, on the possible effects of triclosan on the function of the hippocampus, a key brain region for spatial learning and memory, is lacking. Here, we report that triclosan addition at low concentrations to hippocampal slices from male rats inhibited long-term potentiation but did not affect basal synaptic transmission or paired-pulse facilitation and modified the content or phosphorylation levels of synaptic plasticity-related proteins. Additionally, incubation of primary hippocampal cultures with triclosan prevented both the dendritic spine remodeling induced by brain-derived neurotrophic factor and the emergence of spontaneous oscillatory Ca2+ signals. Furthermore, intra-hippocampal injection of triclosan significantly disrupted rat navigation in the Oasis maze spatial memory task, an indication that triclosan impairs hippocampus-dependent spatial memory performance. Based on these combined results, we conclude that triclosan exerts highly damaging effects on hippocampal neuronal function in vitro and impairs spatial memory processes in vivo.

Coastal and river flood risk analyses for guiding economically optimal flood adaptation policies: a country-scale study for Mexico.

Many countries around the world face increasing impacts from flooding due to socio-economic development in flood-prone areas, which may be enhanced in intensity and frequency as a result of climate change. With increasing flood risk, it is becoming more important to be able to assess the costs and benefits of adaptation strategies. To guide the design of such strategies, policy makers need tools to prioritize where adaptation is needed and how much adaptation funds are required. In this country-scale study, we show how flood risk analyses can be used in cost-benefit analyses to prioritize investments in flood adaptation strategies in Mexico under future climate scenarios. Moreover, given the often limited availability of detailed local data for such analyses, we show how state-of-the-art global data and flood risk assessment models can be applied for a detailed assessment of optimal flood-protection strategies. Our results show that especially states along the Gulf of Mexico have considerable economic benefits from investments in adaptation that limit risks from both river and coastal floods, and that increased flood-protection standards are economically beneficial for many Mexican states. We discuss the sensitivity of our results to modelling uncertainties, the transferability of our modelling approach and policy implications.This article is part of the theme issue 'Advances in risk assessment for climate change adaptation policy'.

Larval dispersal in three coral reef decapod species: Influence of larval duration on the metapopulation structure.

Most coral-associated decapod species have non-migratory adult populations and depend on their planktonic larvae for dispersal. This study examined the metapopulation structure of three decapod species with different pelagic larval duration (PLD) from twelve coral reef complexes of the Gulf of Mexico. The dispersion of larvae was analyzed through the use of a realistic numerical simulation of the Gulf of Mexico with the Hybrid Coordinate Ocean Model. To study the transport and dispersion of particles in near-surface waters, a particle-tracking subroutine was run using as input the currents from the model. The simulation consisted of the launch of 100 passive particles (virtual larvae) every 24 hours from each reef throughout five years, and tracked for as long as 210 days. Results indicated that species with a short PLD, Mithraculus sculptus (PLD 8‒13 days), had a weak connection among the reefs, but higher self-recruitment, especially on the narrow western shelf. The species with a longer PLD, Dromia erythropus (28‒30 days), had a stronger connection among neighboring reefs (< 300 km). Finally, the species with an even longer PLD, Stenopus hispidus (123‒210 days), had a wider potential distribution than the other species. Circulation on synoptic, seasonal and interannual scales had differential effects on the larval dispersal of each species. The metapopulation structure of M. sculptus and D. erythropus seemed to combine features of the non-equilibrium and the patchy models, whereas that of S. hispidus presumably fit to a patchy model. These findings support previous observations that indicate that species with longer PLD tend to occupy larger areas than species with short PLD, although recruitment of juveniles to the adult populations will also depend on other factors, such as the availability of suitable habitats and the ability to colonize them.

Delphinidin Reduces Glucose Uptake in Mice Jejunal Tissue and Human Intestinal Cells Lines through FFA1/GPR40.

Anthocyanins are pigments with antihyperglycemic properties, and they are potential candidates for developing functional foods for the therapy or prevention of Diabetes mellitus type 2 (DM2). The mechanism of these beneficial effects of anthocyanins are, however, hard to explain, given their very low bioavailability due to poor intestinal absorption. We propose that free fatty acid receptor 1 (FFA1, also named GPR40), is involved in an inhibitory effect of the anthocyanidin delphinidin over intestinal glucose absorption. We show the direct effects of delphinidin on the intestine using jejunum samples from RF/J mice, and the human intestinal cell lines HT-29, Caco-2, and NCM460. By the use of specific pharmacological antagonists, we determined that delphinidin inhibits glucose absorption in both mouse jejunum and a human enterocytic cell line in a FFA1-dependent manner. Delphinidin also affects the function of sodium-glucose cotransporter 1 (SGLT1). Intracellular signaling after FFA1 activation involved cAMP increase and cytosolic Ca2+ oscillations originated from intracellular Ca2+ stores and were followed by store-operated Ca2+ entry. Taken together, our results suggest a new GPR-40 mediated local mechanism of action for delphinidin over intestinal cells that may in part explain its antidiabetic effect. These findings are promising for the search for new prevention and pharmacological treatment strategies for DM2 management.

Cholesterol removal from adult skeletal muscle impairs excitation-contraction coupling and aging reduces caveolin-3 and alters the expression of other triadic proteins.

Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction (E-C) coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-ß-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca(2+) transients without affecting membrane integrity or causing sarcoplasmic reticulum (SR) calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not SR membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na(+)/K(+)-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX) activity and protein content of NOX2 subunits (p47(phox) and gp91(phox)), implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs E-C coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged animals.

Seroprevalencia de hepatitis B, C y VIH en indigentes en Costa Rica / Seroprevalence of hepatitis B, C and VIH en homeless people in Costa Rica

Establecer la seroprevalencia de los infectados con hepatitis B, C y del Virus de Inmunodeficiencia Humana en población indigente. Determinar los factores de riesgo para la presencia de estas infecciones en la población de estudio. Métodos: Se realizó un estudio exploratorio descriptivo, transversal y de prevalencia, por medio de toma de muestra sanguínea en una población de indigentes que acudieron por alimentación a un centro de ayuda social ubicado en el distrito de la Merced del cantón central de San José, durante el 2011. Aquellos casos de tamizaje por hepatitis B que fueron positivos se les realizó el perfil inmunológico para establecer el tipo de infección actual.Resultados: Se encontró una alta prevalencia de hepatitis B(16 por ciento), de enfermedad por VIH (7 por ciento) y hepatitis (4 por ciento). Los casos de hepatitis B en su mayoría correponden con infecciones previas por este virus. Solo hubo un caso de hepatitis B activa debido a la presencia de antígeno de superficie y dos casos catalogados como posibles co-infecciones VHB/VIH. Conclusiones: La población de estudio mostró una alta prevalencia de infecciones previas por hepatitis B y VIH sin embargo, la presencia de hepatitis C no es prevalente...

High-frequency field stimulation of primary neurons enhances ryanodine receptor-mediated Ca2+ release and generates hydrogen peroxide, which jointly stimulate NF-κB activity.

Neuronal electrical activity increases intracellular Ca(2+) concentration and generates reactive oxygen species. Here, we show that high frequency field stimulation of primary hippocampal neurons generated Ca(2+) signals with an early and a late component, and promoted hydrogen peroxide generation via a neuronal NADPH oxidase. Hydrogen peroxide generation required both Ca(2+) entry through N-methyl-D-aspartate receptors and Ca(2+) release mediated by ryanodine receptors (RyR). Field stimulation also enhanced nuclear translocation of the NF-κB p65 protein and NF-κB -dependent transcription, and increased c-fos mRNA and type-2 RyR protein content. Preincubation with inhibitory ryanodine or with the antioxidant N-acetyl L-cysteine abolished the increase in hydrogen peroxide generation and the late Ca(2+) signal component induced by electrical stimulation. Primary cortical cells behaved similarly as primary hippocampal cells. Exogenous hydrogen peroxide also activated NF-κB-dependent transcription in hippocampal neurons; inhibitory ryanodine prevented this effect. Selective inhibition of the NADPH oxidase or N-acetyl L-cysteine also prevented the enhanced translocation of p65 in hippocampal cells, while N-acetyl L-cysteine abolished the increase in RyR2 protein content induced by high frequency stimulation. In conclusion, the present results show that electrical stimulation induced reciprocal activation of ryanodine receptor-mediated Ca(2+) signals and hydrogen peroxide generation, which stimulated jointly NF-κB activity.

Cetuximab Plus irinotecan in pretreated metastatic colorectal cancer progressing on irinotecan: the LABEL study.

BACKGROUND: The aim of this multicenter phase II study was to demonstrate the activity of the epidermal growth factor receptor (EGFR)-targeting monoclonal antibody cetuximab combined with irinotecan in the treatment of Latin American patients with EGFR-expressing metastatic colorectal cancer (mCRC) in whom previous treatment with an irinotecan-containing regimen had failed. PATIENTS AND METHODS: Patients received cetuximab, as a 400 mg/m2 initial infusion followed by 250 mg/m2 weekly, plus the same irinotecan regimen that had previously failed, until the occurrence of disease progression or unacceptable toxicity. The primary endpoint was response. Secondary endpoints included progression-free survival (PFS), duration of response, overall survival (OS), and safety. RESULTS: Seventy-nine patients received treatment. One patient had a complete response, 20 had partial responses, and disease was stabilized in 23 patients, giving an overall response rate of 27% and a disease control rate of 56%. The median duration of response was 23.9 weeks. Median PFS was 17.4 weeks, median OS was 9.2 months, and the 12-month OS rate was 38%. The most common adverse events according to System Organ Class were skin and subcutaneous tissue disorders (91% of the patients). Grade 3/4 adverse events occurred in 45 patients (57%), with the most common being diarrhea (20%), neutropenia (11%), and rash (6%). Seven patients (9%) had grade 3/4 acne-like skin rash. No grade 3/4 infusion-related reactions were reported. CONCLUSION: Cetuximab in combination with irinotecan is active and tolerable in Latin American patients with mCRC progressing on irinotecan, with a safety profile similar to that described in European studies.

Hypoxia stimulates via separate pathways ERK phosphorylation and NF-kappaB activation in skeletal muscle cells in primary culture.

Mammalian cells sense oxygen levels and respond to hypoxic conditions through the regulation of multiple signaling pathways and transcription factors. Here, we investigated the effects of hypoxia on the activity of two transcriptional regulators, ERK1/2 and NF-kappaB, in skeletal muscle cells in primary culture. We found that hypoxia significantly enhanced ERK1/2 phosphorylation and that it stimulated NF-kappaB-dependent gene transcription as well as nuclear translocation of a green fluorescent protein-labeled p65 NF-kappaB isoform. Phosphorylation of ERK1/2- and NF-kappaB-dependent transcription by hypoxia required calcium entry through L-type calcium channels. Calcium release from ryanodine-sensitive stores was also necessary for ERK1/2 activation but not for NF-kappaB-dependent-transcription. N-acetylcysteine, a general scavenger of reactive oxygen species, blocked hypoxia-induced ROS generation but did not affect the stimulation of ERK1/2 phosphorylation induced by hypoxia. In contrast, NF-kappaB activation was significantly inhibited by N-acetylcysteine and did not depend on ERK1/2 stimulation, as shown by the lack of effect of the upstream ERK inhibitor U-0126. These separate pathways of activation of ERK1/2 and NF-kappaB by hypoxia may contribute to muscle adaptation in response to hypoxic conditions.

Sodium-dependent action potentials induced by brevetoxin-3 trigger both IP3 increase and intracellular Ca2+ release in rat skeletal myotubes.

Brevetoxin-3 (PbTx-3), described to increase the open probability of voltage-dependent sodium channels, caused trains of action potentials and fast oscillatory changes in fluorescence intensity of fluo-3-loaded rat skeletal muscle cells in primary culture, indicating that the toxin increased intracellular Ca2+ levels. PbTx-3 did not elicit calcium transients in dysgenic myotubes (GLT cell line), lacking the alpha1 subunit of the dihydropyridine receptor (DHPR), but after transfection of the alpha1DHPR cDNA to GLT cells, PbTx-3 induced slow calcium transients that were similar to those of normal cells. Ca2+ signals evoked by PbTx-3 were inhibited by blocking either IP3 receptors, with 2-aminoethoxydiphenyl borate, or phospholipase C with U73122. PbTx-3 caused a tetrodotoxin-sensitive increase in intracellular IP3 mass levels, dependent on extra-cellular Na+. A similar increase in IP3 mass was induced by high K+ depolarization but no action potential trains (nor calcium signals) were elicited by prolonged depolarization under current clamp conditions. The increase in IP3 mass induced by either PbTx-3 or K+ was also detected in Ca2+-free medium. These results establish that the effect of the toxin on both intracellular Ca2+ and IP3 levels occurs via a membrane potential sensor instead of directly by Na+ flux and supports the notion of a train of action potentials being more efficient as a stimulus than sustained depolarization, suggesting that tetanus is the physiological stimulus for the IP3-dependent calcium signal involved in regulation of gene expression.

NFAT activation by membrane potential follows a calcium pathway distinct from other activity-related transcription factors in skeletal muscle cells.

Depolarization of skeletal muscle cells triggers intracellular Ca2+ signals mediated by ryanodine and inositol 1,4,5-trisphosphate (IP3) receptors. Previously, we have reported that K+-induced depolarization activates transcriptional regulators ERK, cAMP response element-binding protein, c-fos, c-jun, and egr-1 through IP3-dependent Ca2+ release, whereas NF-kappa B activation is elicited by both ryanodine and IP3 receptor-mediated Ca2+ signals. We have further shown that field stimulation with electrical pulses results in an NF-kappa B activation increase dependent of the amount of pulses and independent of their frequency. In this work, we report the results obtained for nuclear factor of activated T cells (NFAT)-mediated transcription and translocation generated by both K+ and electrical stimulation protocols in primary skeletal muscle cells and C2C12 cells. The Ca2+ source for NFAT activation is through release by ryanodine receptors and extracellular Ca2+ entry. We found this activation to be independent of the number of pulses within a physiological range of stimulus frequency and enhanced by long-lasting low-frequency stimulation. Therefore, activation of the NFAT signaling pathway differs from that of NF-kappa B and other transcription factors. Calcineurin enzyme activity correlated well with the relative activation of NFAT translocation and transcription using different stimulation protocols. Furthermore, both K+-induced depolarization and electrical stimulation increased mRNA levels of the type 1 IP3 receptor mediated by calcineurin activity, which suggests that depolarization may regulate IP3 receptor transcription. These results confirm the presence of at least two independent pathways for excitation-transcription coupling in skeletal muscle cells, both dependent on Ca2+ release and triggered by the same voltage sensor but activating different intracellular release channels.

NF-kappaB activation by depolarization of skeletal muscle cells depends on ryanodine and IP3 receptor-mediated calcium signals.

Depolarization of skeletal muscle cells by either high external K(+) or repetitive extracellular field potential pulses induces calcium release from internal stores. The two components of this release are mediated by either ryanodine receptors or inositol 1,4,5-trisphosphate (IP(3)) receptors and show differences in kinetics, amplitude, and subcellular localization. We have reported that the transcriptional regulators including ERKs, cAMP/Ca(2+)-response element binding protein, c-fos, c-jun, and egr-1 are activated by K(+)-induced depolarization and that their activation requires IP(3)-dependent calcium release. We presently describe the activation of the nuclear transcription factor NF-kappaB in response to depolarization by either high K(+) (chronic) or electrical pulses (fluctuating). Calcium transients of relative short duration activate an NF-kappaB reporter gene to an intermediate level, whereas long-lasting calcium increases obtained by prolonged electrical stimulation protocols of various frequencies induce maximal activation of NF-kappaB. This activation is independent of extracellular calcium, whereas calcium release mediated by either ryanodine or IP(3) receptors contribute in all conditions tested. NF-kappaB activation is mediated by IkappaBalpha degradation and p65 translocation to the nucleus. Partial blockade by N-acetyl-l-cysteine, a general antioxidant, suggests the participation of reactive oxygen species. Calcium-dependent signaling pathways such as those linked to calcineurin and PKC also contribute to NF-kappaB activation by depolarization, as assessed by blockade through pharmacological agents. These results suggest that NF-kappaB activation in skeletal muscle cells is linked to membrane depolarization and depends on the duration of elevated intracellular calcium. It can be regulated by sequential activation of calcium release mediated by the ryanodine and by IP(3) receptors.