Non-canonical endogenous expression of voltage-gated sodium channel NaV 1.7 subtype by the TE671 rhabdomyosarcoma cell line.

The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription-PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250 kDa. Elicited sodium currents had a mean amplitude of 2.6 ± 0.7 nA with activation and fast inactivation V50 values of -31.9 ± 1.1 and -69.6 ± 1.0 mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100 nm (IC50  = 22.3 nm). They were also very susceptible to the NaV 1.7 specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6 nm and 0.8 nm, respectively, characteristic of those previously determined for NaV 1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV 1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV 1.7, a major target for the development of analgesic drugs and more generally for the study of pain. KEY POINTS: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV 1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV 1.7-targeting drugs

Nutritional status and disease severity in children acutely presenting to a primary health clinic in rural Gambia

BACKGROUND: Accurate and timely data on the health of a population are key for evidence-based decision making at both the policy and programmatic level. In many low-income settings, such data are unavailable or outdated. Using an electronic medical records system, we determined the association between nutritional status and severe illness and mortality among young children presenting to a rural primary health care facility in the Gambia. METHODS: Clinical data collected over five years (2010-2014) on children aged under 60 months making acute visits to a primary health care clinic in the rural Gambian district of Kiang West were retrospectively extracted from the medical records system. Generalised estimating equation models were used to investigate associations between nutritional status and illness severity, accounting for repeat visits, gender, age and access to transport to the clinic. The Population Attributable Fraction (PAF) was used to determine the proportion of severe illness likely attributable to different grades of malnutrition. RESULTS: 3839/5021 (77%) children under 60 months of age living in Kiang West presented acutely to the clinic at least once, yielding 21,278 visits (47% girls, median age 20.2 months (Interquartile Range (IQR) 23.92 months)) and 26,001 diagnoses, 86% being infectious diseases. Severe illness was seen in 4.5% of visits (961/21,278). Wasting was associated with an increased risk of severe illness in a dose-dependent manner, ('WHZ < -1' adjusted Odds Ratio (aOR) 1.68, 95% CI:1.43-1.98, p < 0.001, 'WHZ <-2 and ≥-3' aOR 2.78, 95% CI:2.31-3.36, p < 0.001 and 'WHZ < -3' aOR 7.82, 95% CI:6.40-9.55, p < 0.001) the PAF for wasting (WHZ < -2) was 0.21 (95% CI: 0.18-0.24). Stunting, even in the most severe form (HAZ < -3), was not significantly associated with severe illness (aOR 1.19 95% CI:0.94-1.51) but was associated with a significantly increased risk of death (aOR 6.04 95% CI:1.94-18.78). CONCLUSION: In this population-based cohort of young children in rural Gambia, wasting was associated with disease severity in a dose-dependent manner. Further research is needed into strategies to identify and reach these children with effective interventions to improve their nutritional status

In rural Gambia, do adolescents have increased nutritional vulnerability compared with adults?

Adolescents may be particularly susceptible to malnutrition owing to the energy and nutrient costs of the pubertal growth spurt. Here, our aim is to compare differences in selected markers of nutritional status between adolescents and adults in rural Gambia. The Keneba Biobank collects cross-sectional data and samples for all consenting individuals resident in the West Kiang region of the Gambia. For this study, participants between the ages of 10 and 40 years were selected (n = 4201, females 2447). Height, body mass index, body composition, hemoglobin concentration, fasting glucose concentration, and blood pressure were compared using linear regression models adjusting for age, parity, season of measurement, and residence, across three age groups: early adolescent (10–14.9 years), late adolescent (15–19.9 years), and adult (20–39.9 years). Adolescents, particularly early-adolescent girls and boys, were shorter, lighter, and leaner than adults. By late adolescence, differences were smaller, particularly in girls where, notably, the prevalence of overweight, hypertension, and impaired fasting glucose was low. Given the importance of maternal health for reproductive outcomes and intergenerational health, the results of the study, albeit with limited biomarkers available, indicate that adolescent girls are no more compromised than adult women or males from the same population

Non‐canonical endogenous expression of voltage‐gated sodium channel NaV1.7 subtype by the TE671 rhabdomyosarcoma cell line

Abstract: The human TE671 cell line was originally used as a model of medulloblastoma but has since been reassigned as rhabdomyosarcoma. Despite the characterised endogenous expression of voltage-sensitive sodium currents in these cells, the specific voltage-gated sodium channel (VGSC) subtype underlying these currents remains unknown. To profile the VGSC subtype in undifferentiated TE671 cells, endpoint and quantitative reverse transcription–PCR (qRT-PCR), western blot and whole-cell patch clamp electrophysiology were performed. qRT-PCR profiling revealed that expression of the SCN9A gene was ∼215-fold greater than the SCN4A gene and over 400-fold greater than any of the other VGSC genes, while western blot confirmed that the dominant SCN9A RNA was translated to a protein with a molecular mass of ∼250kDa. Elicited sodium currents had a mean amplitude of 2.6±0.7nA with activation and fast inactivation V50 values of −31.9±1.1 and −69.6±1.0mV, respectively. The currents were completely and reversibly blocked by tetrodotoxin at concentrations greater than 100nm (IC50=22.3nm). They were also very susceptible to the NaV1.7specific blockers Huwentoxin-IV and Protoxin-II with IC50 values of 14.6nm and 0.8nm, respectively, characteristic of those previously determined for NaV1.7. Combined, the results revealed the non-canonical and highly dominant expression of NaV1.7 in the human TE671 rhabdomyosarcoma cell line. We show that the TE671 cell line is an easy to maintain and cost-effective model for the study of NaV1.7, a major target for the development of analgesic drugs and more generally for the study of pain. Key points: Undifferentiated TE671 cells produce a voltage-sensitive sodium current when depolarised. The voltage-gated sodium channel isoform expressed in undifferentiated TE671 cells was previously unknown. Through qRT-PCR, western blot and toxin pharmacology, it is shown that undifferentiated TE671 cells dominantly (>99.5%) express the NaV1.7 isoform that is strongly associated with pain. The TE671 cell line is, therefore, a very easy to maintain and cost-effective model to study NaV1.7-targeting drugs

In rural Gambia, do adolescents have increased nutritional vulnerability compared to adults?

Adolescents may be particularly susceptible to malnutrition due to the energy and nutrient costs of the pubertal growth spurt. The aim of this study was to compare differences in selected markers of nutritional status between adolescents and adults in rural Gambia.The Keneba Biobank collects cross-sectional data and samples for all consenting individuals resident in the West Kiang region of The Gambia. For this study, participants between the ages of 10 and 40 years (y) were selected (n = 4201, female 2447). Height, body mass index, body composition, haemoglobin concentration, fasting glucose concentration and blood pressure were compared using linear regression models adjusting for age, parity, season of measurement and residence, across three age groups: early adolescent (10-14.9y), late adolescent (15-19.9y) and adult (20-39.9y).Adolescents, particularly early adolescent girls and boys, were shorter, lighter and leaner than adults. By late adolescence differences were smaller, particularly in girls where, notably, the prevalence of overweight, hypertension and impaired fasting glucose was low. Given the importance of maternal health for reproductive outcomes and intergenerational health, the results of the study, albeit with limited biomarkers available, indicate adolescent girls are no more compromised than adult women or males from the same population

Protocol for the EMPHASIS study; epigenetic mechanisms linking maternal pre-conceptional nutrition and children’s health in India and Sub-Saharan Africa

Abstract Background Animal studies have shown that nutritional exposures during pregnancy can modify epigenetic marks regulating fetal development and susceptibility to later disease, providing a plausible mechanism to explain the developmental origins of health and disease. Human observational studies have shown that maternal peri-conceptional diet predicts DNA methylation in offspring. However, a causal pathway from maternal diet, through changes in DNA methylation, to later health outcomes has yet to be established. The EMPHASIS study (Epigenetic Mechanisms linking Pre-conceptional nutrition and Health Assessed in India and Sub-Saharan Africa, ISRCTN14266771) will investigate epigenetically mediated links between peri-conceptional nutrition and health-related outcomes in children whose mothers participated in two randomized controlled trials of micronutrient supplementation before and during pregnancy. Methods The original trials were the Mumbai Maternal Nutrition Project (MMNP, ISRCTN62811278) in which Indian women were offered a daily snack made from micronutrient-rich foods or low-micronutrient foods (controls), and the Peri-conceptional Multiple Micronutrient Supplementation Trial (PMMST, ISRCTN13687662) in rural Gambia, in which women were offered a daily multiple micronutrient (UNIMMAP) tablet or placebo. In the EMPHASIS study, DNA methylation will be analysed in the children of these women (~1100 children aged 5–7 y in MMNP and 298 children aged 7–9 y in PMMST). Cohort-specific and cross-cohort effects will be explored. Differences in DNA methylation between allocation groups will be identified using the Illumina Infinium MethylationEPIC array, and by pyrosequencing top hits and selected candidate loci. Associations will be analysed between DNA methylation and health-related phenotypic outcomes, including size at birth, and children’s post-natal growth, body composition, skeletal development, cardio-metabolic risk markers (blood pressure, serum lipids, plasma glucose and insulin) and cognitive function. Pathways analysis will be used to test for enrichment of nutrition-sensitive loci in biological pathways. Causal mechanisms for nutrition-methylation-phenotype associations will be explored using Mendelian Randomization. Associations between methylation unrelated to supplementation and phenotypes will also be analysed. Conclusion The study will increase understanding of the epigenetic mechanisms underpinning the long-term impact of maternal nutrition on offspring health. It will potentially lead to better nutritional interventions for mothers preparing for pregnancy, and to identification of early life biomarkers of later disease risk