Identification of valid reference genes during the differentiation of human myoblasts

<p>Abstract</p> <p>Background</p> <p>Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input.</p> <p>Results</p> <p>Using the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked.</p> <p>Conclusion</p> <p>RNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.</p

Microbiome dynamics during the HI-SEAS IV mission, and implications for future crewed missions beyond Earth.

Background: Human health is closely interconnected with its microbiome. Resilient microbiomes in, on, and around the human body will be key for safe and successful long-term space travel. However, longitudinal dynamics of microbiomes inside confined built environments are still poorly understood. Herein, we used the Hawaii Space Exploration Analog and Simulation IV (HI-SEAS IV) mission, a 1 year-long isolation study, to investigate microbial transfer between crew and habitat, in order to understand adverse developments which may occur in a future outpost on the Moon or Mars. Results: Longitudinal 16S rRNA gene profiles, as well as quantitative observations, revealed significant differences in microbial diversity, abundance, and composition between samples of the built environment and its crew. The microbiome composition and diversity associated with abiotic surfaces was found to be rather stable, whereas the microbial skin profiles of individual crew members were highly dynamic, resulting in an increased microbiome diversity at the end of the isolation period. The skin microbiome dynamics were especially pronounced by a regular transfer of the indicator species Methanobrevibacter between crew members within the first 200 days. Quantitative information was used to track the propagation of antimicrobial resistance in the habitat. Together with functional and phenotypic predictions, quantitative and qualitative data supported the observation of a delayed longitudinal microbial homogenization between crew and habitat surfaces which was mainly caused by a malfunctioning sanitary facility. Conclusions: This study highlights main routes of microbial transfer, interaction of the crew, and origins of microbial dynamics in an isolated environment. We identify key targets of microbial monitoring, and emphasize the need for defined baselines of microbiome diversity and abundance on surfaces and crew skin. Targeted manipulation to counteract adverse developments of the microbiome could be a highly important strategy to ensure safety during future space endeavors

Association of Cardiometabolic Genes with Arsenic Metabolism Biomarkers in American Indian Communities: The Strong Heart Family Study (SHFS)

Background: Metabolism of inorganic arsenic (iAs) is subject to inter-individual variability, which is explained partly by genetic determinants. Objectives: We investigated the association of genetic variants with arsenic species and principal components of arsenic species in the Strong Heart Family Study (SHFS). Methods: We examined variants previously associated with cardiometabolic traits (~ 200,000 from Illumina Cardio MetaboChip) or arsenic metabolism and toxicity (670) among 2,428 American Indian participants in the SHFS. Urine arsenic species were measured by high performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC-ICP-MS), and percent arsenic species [iAs, monomethylarsonate (MMA), and dimethylarsinate (DMA), divided by their sum × 100] were logit transformed. We created two orthogonal principal components that summarized iAs, MMA, and DMA and were also phenotypes for genetic analyses. Linear regression was performed for each phenotype, dependent on allele dosage of the variant. Models accounted for familial relatedness and were adjusted for age, sex, total arsenic levels, and population stratification. Single nucleotide polymorphism (SNP) associations were stratified by study site and were meta-analyzed. Bonferroni correction was used to account for multiple testing. Results: Variants at 10q24 were statistically significant for all percent arsenic species and principal components of arsenic species. The index SNP for iAs%, MMA%, and DMA% (rs12768205) and for the principal components (rs3740394, rs3740393) were located near AS3MT, whose gene product catalyzes methylation of iAs to MMA and DMA. Among the candidate arsenic variant associations, functional SNPs in AS3MT and 10q24 were most significant (p \u3c 9.33 × 10–5). Conclusions: This hypothesis-driven association study supports the role of common variants in arsenic metabolism, particularly AS3MT and 10q24

Dietary determinants of cadmium exposure in the Strong Heart Family Study

Urinary cadmium (Cd) concentrations in the Strong Heart Family Study (SHFS) participants are higher than in the general US population. This difference is unlikely to be related to tobacco smoking. We evaluated the association of consumption of processed meats and other dietary products with urinary Cd concentrations in the SHFS, a family-based study conducted in American Indian communities. We included 1725 participants with urine Cd concentrations (standardized to urine creatinine) and food frequency questionnaire data grouped in 24 categories, including processed meat. Median (IQR) urinary Cd concentrations were 0.42 (0.20–0.85) μg/g creatinine. The age, sex, smoking, education, center, body mass index, and total kcal adjusted geometric mean ratio (GMR) (95%CI) of urinary cadmium concentrations per IQR increase in each dietary category was 1.16 (1.04–1.29) for processed meat, 1.10 (1.00–1.21) for fries and chips, 0.87 (0.80–0.95) for dairy products, and 0.89 (0.82–0.97) for fruit juices. The results remained similar after further adjustment for the dietary categories associated with urinary Cd in the previous model except for fries and chips, which was no longer statistically significant. These findings revealed the potential importance of processed meat products as a dietary source of cadmium

Associations of maternal arsenic exposure with adult fasting glucose and insulin resistance in the Strong Heart Study and Strong Heart Family Study

Experimental and prospective epidemiologic evidence suggest that arsenic exposure has diabetogenic effects. However, little is known about how family exposure to arsenic may affect risk for type 2 diabetes (T2D)-related outcomes in adulthood. We evaluated the association of both maternal and offspring arsenic exposure with fasting glucose and incident T2D in 466 participants of the Strong Heart Family Study. Total arsenic (ΣAs) exposure was calculated as the sum of inorganic arsenic (iAs) and methylated (MMA, DMA) arsenic species in maternal and offspring baseline urine. Median maternal ΣAs at baseline (1989-91) was 7.6 µg/g creatinine, while median offspring ΣAs at baseline (2001-03) was 4.5 µg/g creatinine. Median offspring glucose in 2006-2009 was 94 mg/dL, and 79 participants developed T2D. The fully adjusted mean difference (95% CI) for offspring glucose was 4.40 (-3.46, 12.26) mg/dL per IQR increase in maternal ΣAs vs. 2.72 (-4.91 to 10.34) mg/dL per IQR increase in offspring ΣAs. The fully adjusted odds ratio (95%CI) of incident T2D was 1.35 (1.07, 1.69) for an IQR increase in maternal ΣAs and 1.15 (0.92, 1.43) for offspring ΣAs. The association of maternal ΣAs with T2D outcomes were attenuated with adjustment for offspring adiposity markers. Familial exposure to arsenic, as measured in mothers 15-20 years before offspring follow-up, is associated with increased odds of offspring T2D. More research is needed to confirm findings and better understand the importance of family exposure to arsenic in adult-onset diabetes.This study was supported by the National Institute of EnvironmentalHealth Sciences, Unites States (P42ES010349, P30ES009089,R01ES028758, R01ES025216).N.T., P.F.-L., and A.N.-A. contributed to the preparation of researchdata and writing of the manuscript. N.T, M.J.S, A.D.-R., M.T.-P., M.G.-P., and A.N.-A. contributed to the statistical analysis. B.V.H., J.M., K.N.,J.G.U., and S.C. contributed as the primary investigators of the SHS andSHFS, and to the preparation of the research data. K.A.F. and W.G.contributed to the arsenic measurements in the SHS and SHFS partici-pants. A.N.-A. is the guarantor of this work and, as such, had full accessto all the data in the study and takes responsibility for the integrity ofthe data and the accuracy of the data analysis.S

Association of Cardiometabolic Genes with Arsenic Metabolism Biomarkers in American Indian Communities: The Strong Heart Family Study (SHFS)

BACKGROUND: Metabolism of inorganic arsenic (iAs) is subject to inter-individual variability, which is explained partly by genetic determinants. OBJECTIVES: We investigated the association of genetic variants with arsenic species and principal components of arsenic species in the Strong Heart Family Study (SHFS). METHODS: We examined variants previously associated with cardiometabolic traits (~ 200,000 from Illumina Cardio MetaboChip) or arsenic metabolism and toxicity (670) among 2,428 American Indian participants in the SHFS. Urine arsenic species were measured by high performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS), and percent arsenic species [iAs, monomethylarsonate (MMA), and dimethylarsinate (DMA), divided by their sum × 100] were logit transformed. We created two orthogonal principal components that summarized iAs, MMA, and DMA and were also phenotypes for genetic analyses. Linear regression was performed for each phenotype, dependent on allele dosage of the variant. Models accounted for familial relatedness and were adjusted for age, sex, total arsenic levels, and population stratification. Single nucleotide polymorphism (SNP) associations were stratified by study site and were meta-analyzed. Bonferroni correction was used to account for multiple testing. RESULTS: Variants at 10q24 were statistically significant for all percent arsenic species and principal components of arsenic species. The index SNP for iAs%, MMA%, and DMA% (rs12768205) and for the principal components (rs3740394, rs3740393) were located near AS3MT, whose gene product catalyzes methylation of iAs to MMA and DMA. Among the candidate arsenic variant associations, functional SNPs in AS3MT and 10q24 were most significant (p < 9.33 × 10-5). CONCLUSIONS: This hypothesis-driven association study supports the role of common variants in arsenic metabolism, particularly AS3MT and 10q24. Citation: Balakrishnan P, Vaidya D, Franceschini N, Voruganti VS, Gribble MO, Haack K, Laston S, Umans JG, Francesconi KA, Goessler W, North KE, Lee E, Yracheta J, Best LG, MacCluer JW, Kent J Jr., Cole SA, Navas-Acien A. 2017. Association of cardiometabolic genes with arsenic metabolism biomarkers in American Indian communities: the Strong Heart Family Study (SHFS). Environ Health Perspect 125:15-22; http://dx.doi.org/10.1289/EHP251

Chitosan/polyester-based scaffolds for cartilage tissue engineering: assessment of extracellular matrix formation

Naturally derived polymers have been extensively used in scaffold production for cartilage tissue engineering. The present work aims to evaluate and characterize extracellular matrix (ECM) formation in two types of chitosan-based scaffolds, using bovine articular chondrocytes (BACs). The influence of these scaffolds’ porosity, as well as pore size and geometry, on the formation of cartilagineous tissue was studied. The effect of stirred conditions on ECM formation was also assessed. Chitosan-poly(butylene succinate) (CPBS) scaffolds were produced by compression moulding and salt leaching, using a blend of 50% of each material. Different porosities and pore size structures were obtained. BACs were seeded onto CPBS scaffolds using spinner flasks. Constructs were then transferred to the incubator, where half were cultured under stirred conditions, and the other half under static conditions for 4 weeks. Constructs were characterized by scanning electron microscopy, histology procedures, immunolocalization of collagen type I and collagen type II, and dimethylmethylene blue assay for glycosaminoglycan (GAG) quantification. Both materials showed good affinity for cell attachment. Cells colonized the entire scaffolds and were able to produce ECM. Large pores with random geometry improved proteoglycans and collagen type II production. However, that structure has the opposite effect on GAG production. Stirred culture conditions indicate enhancement of GAG production in both types of scaffold.M.L. Alves da Silva would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for her grant (SFRH/BD/28708/2006), Marie Curie Actions-ALEA JACTA EST (MEST-CT-2004-008104), European NoE EXPERTISSUES (NMP3-CT-2004-500283), IP GENOSTEM (LSHB-CT-2003-503161) and CARTISCAFF (POCTI/SAUIBMA/58982

Plasmapheresis reverses all side-effects of a cisplatin overdose – a case report and treatment recommendation

BACKGROUND: Cisplatin is widely used as an antineoplastic agent since it is effective against a broad spectrum of different tumours. Nevertheless, it has several potential side effects affecting different organ systems and an overdose may lead to life-threatening complications and even death. CASE PRESENTATION: We report on a 46-year old woman with non-small cell lung cancer who accidentally received 225 mg/m(2 )of cisplatin, which was threefold the dose as scheduled, within a 3-day period. Two days later, the patient presented with hearing loss, severe nausea and vomiting, acute renal failure as well as elevated liver enzymes. In addition, she developed a severe myelodepression. After plasmapheresis on two consecutive days and vigorous supportive treatment, the toxicity-related symptoms improved and the patient recovered without any sequelae. CONCLUSION: To date, no general accepted guidelines for the treatment of cisplatin overdoses are available. Along with the experience from other published cases, our report shows that plasmapheresis is capable of lowering cisplatin plasma and serum levels efficiently. Therefore, plasma exchange performed as soon as possible can ameliorate all side effects of a cisplatin overdose and be a potential tool for clinicians for treatment. However, additional intensive supportive treatment-modalities are necessary to control all occurring side effects

Low-moderate arsenic exposure and respiratory in American Indian communities in the Strong Heart Study

Background Arsenic exposure through drinking water is an established lung carcinogen. Evidence on non-malignant lung outcomes is less conclusive and suggests arsenic is associated with lower lung function. Studies examining low-moderate arsenic (< 50 μg/L), the level relevant for most populations, are limited. We evaluated the association of arsenic exposure with respiratory health in American Indians from the Northern Plains, the Southern Plains and the Southwest United States, communities with environmental exposure to inorganic arsenic through drinking water. Methods The Strong Heart Study is a prospective study of American Indian adults. This analysis used urinary arsenic measurements at baseline (1989–1991) and spirometry at Visit 2 (1993–1995) from 2132 participants to evaluate associations of arsenic exposure with airflow obstruction, restrictive pattern, self-reported respiratory disease, and symptoms. Results Airflow obstruction was present in 21.5% and restrictive pattern was present in 14.4%. The odds ratio (95% confidence interval) for obstruction and restrictive patterns, based on the fixed ratio definition, comparing the 75th to 25th percentile of arsenic, was 1.17 (0.99, 1.38) and 1.27 (1.01, 1.60), respectively, after adjustments, and 1.28 (1.02, 1.60) and 1.33 (0.90, 1.50), respectively, based on the lower limit of normal definition. Arsenic was associated with lower percent predicted FEV1 and FVC, self-reported emphysema and stopping for breath. Conclusion Low-moderate arsenic exposure was positively associated with restrictive pattern, airflow obstruction, lower lung function, self-reported emphysema and stopping for breath, independent of smoking and other lung disease risk factors. Findings suggest that low-moderate arsenic exposure may contribute to restrictive lung disease

The effect of an external magnetic force on cell adhesion and proliferation of magnetically labeled mesenchymal stem cells

<p>Abstract</p> <p>Background</p> <p>As the strategy for tissue regeneration using mesenchymal stem cells (MSCs) for transplantation, it is necessary that MSCs be accumulated and kept in the target area. To accumulate MSCs effectively, we developed a novel technique for a magnetic targeting system with magnetically labeled MSCs and an external magnetic force. In this study, we examined the effect of an external magnetic force on magnetically labeled MSCs in terms of cell adhesion and proliferation.</p> <p>Methods</p> <p>Magnetically labeled MSCs were plated at the bottom of an insert under the influence of an external magnetic force for 1 hour. Then the inserts were turned upside down for between 1 and 24 hours, and the number of MSCs which had fallen from the membrane was counted. The gene expression of MSCs affected magnetic force was analyzed with microarray. In the control group, the same procedure was done without the external magnetic force.</p> <p>Results</p> <p>At 1 hour after the inserts were turned upside down, the average number of fallen MSCs in the magnetic group was significantly smaller than that in the control group, indicating enhanced cell adhesion. At 24 hours, the average number of fallen MSCs in the magnetic group was also significantly smaller than that in control group. In the magnetic group, integrin alpha2, alpha6, beta3 BP, intercellular adhesion molecule-2 (ICAM-2), platelet/endothelial cell adhesion molecule-1 (PECAM-1) were upregulated. At 1, 2 and 3 weeks after incubation, there was no statistical significant difference in the numbers of MSCs in the magnetic group and control group.</p> <p>Conclusions</p> <p>The results indicate that an external magnetic force for 1 hour enhances cell adhesion of MSCs. Moreover, there is no difference in cell proliferation after using an external magnetic force on magnetically labeled MSCs.</p