Microsatellite primers for red drum (Sciaenops ocellatus)

In this note, we document polymerase-chain-reaction (PCR) primer pairs for 101 nuclear-encoded microsatellites designed and developed from a genomic library for red drum (Sciaenops ocellatus). Details of the genomic library construction, the sequencing of positive clones, primer design, and PCR protocols may be found in Karlsson et al. (2008). The 101 microsatellites (GENBA NK Accession Numbers EU015882-EU015982) were amplified successfully and used to genotype 24 red drum obtained from Galveston Bay, Texas (Table 1). A total of 69 of the microsatellites had an uninterrupted (perfect) dinucleotide motif, and 30 had an imperfect dinucleotide motif; one microsatellite had an imperfect tetranucleotide motif, and one had an imperfect and compound motif (Table 1 ). Sizes of the cloned alleles ranged from 84 to 252 base pairs. A ‘blast’ search of the GENBANK database indicated that all of the primers and the cloned alleles were unique (i.e., not duplicated)

Population structure, long-term connectivity, and effective size of mutton snapper (Lutjanus analis) in the Caribbean Sea and Florida Keys

Genetic structure and average long-term connectivity and effective size of mutton snapper (Lutjanus analis) sampled from offshore localities in the U.S. Caribbean and the Florida Keys were assessed by using nuclear-encoded microsatellites and a fragment of mitochondrial DNA. No significant differences in allele, genotype (microsatellites), or haplotype (mtDNA) distributions were detected; tests of selective neutrality (mtDNA) were nonsignificant after Bonferroni correction. Heuristic estimates of average long-term rate of migration (proportion of migrant individuals/generation) between geographically adjacent localities varied from 0.0033 to 0.0054, indicating that local subpopulations could respond independently of environmental perturbations. Estimates of average longterm effective population sizes varied from 341 to 1066 and differed significantly among several of the localities. These results indicate that over time larval drift and interregional adult movement may not be sufficient to maintain population sustainability across the region and that there may be different demographic stocks at some of the localities studied. The estimate of long-term effective population size at the locality offshore of St. Croix was below the minimum threshold size considered necessary to maintain the equilibrium between the loss of adaptive genetic variance from genetic drift and its replacement by mutation. Genetic variability in mutton snapper likely is maintained at the intraregional level by aggregate spawning and random mating of local populations. This feature is perhaps ironic in that aggregate spawning also renders mutton snapper especially vulnerable to overexploitation

Investigating the effects of 5.5 mmoL vs 25 mmoL glucose concentration in culture media on LHCN-M2 cell viability, proliferation, metabolism and differentiation

Introduction: In vitro skeletal muscle cell models are vitally important for investigating the molecular mechanisms of skeletal muscle in metabolic and endocrine diseases, such as obesity and type 2 diabetes. Culture media for skeletal muscle cells can often contain glucose concentrations (GC) five times higher than what’s considered normal in fasting human plasma, thus is not representative of the in vivo environment. Hyperglycaemia in culture media may negatively impact metabolic function, by creating a model of cell toxicity that’s representative of diseases such as diabetes mellitus. The aim of these experiments was to determine the impact of media containing GC of 5.5 mmol (physiological) vs 25 mmol (supraphysiological) on cell viability, proliferation, ATP production and differentiation in human LHCN-m2 myoblasts.Methods: LHCN-m2 myoblasts were cultured in 5.5 mmol or 25 mmol glucose growth media and cell viability, ATP production, and proliferation were determined. Differentiation of LHCN-m2 myoblasts into multinucleated myotubes was induced by reducing levels of human serum within the culture media and analysed by immunofluorescence following 10 days of differentiation.Results: We observed no differences in the viability, proliferation or basal ATP production rates of LHCN-m2 cells grown in 5.5 mmol compared to 25 mmol glucose (P> 0.05 for all). However cells had a trend of higher ATP production rates and faster proliferation in 5.5 mmol compared to 25 mmol. Fluorescence microscopy revealed the formation of multinucleated myotubes differentiated in 5.5 mmol glucose media containing various concentrations of human serum (0.5%, 1% and 2%).Conclusions: Our data demonstrates the ability to differentiate LHCN-m2 cells in 5.5 mmol GC, which allows our in vitro model to be more physiologically-relevant and more comparable to what is observed in vivo in humans. Further work is required to determine the implications of GC on the wider metabolic function in LHCN-m2 myoblasts

Characterization of hepcidin response to holotransferrin in novel recombinant TfR1 HepG2 cells

Hepcidin is the key regulator of systemic iron homeostasis. The iron-sensing mechanisms and the role of intracellular iron in modulating hepatic hepcidin secretion are unclear. Therefore, we created a novel cell line, recombinant-TfR1 HepG2,expressing iron-response-element-independent TFRC mRNA to promote cellular iron overload and examined the effect of excess holotransferrin (5 g/L) on cell-surface TfR1, iron content, hepcidin secretion and mRNA expressions of TFRC, HAMP, SLC40A1,HFE and TFR2. Results showed that the recombinant cells exceeded levels of cell surface TfR1 in wild-type cells under basal (2.8-fold; p<0.03) and holotransferrin supplemented conditions for 24 h and 48 h (4.4- and 7.5-fold, respectively; p<0.01). Also, these cells showed higher intracellular iron content than wild-type cells under basal (3-fold; p<0.03) and holotransferrin-supplemented conditions (6.6-fold at 4 h; p<0.01). However, hepcidin secretion was not higher than wild-type cells. Moreover, holotransferrin treatment to recombinant cells did not elevate HAMP responses compared to untreated or wild-type cells. In conclusion, increased intracellular iron content in recombinant cells did not increase hepcidin responses compared to wild-type cells, resembling hemochromatosis. Furthermore, TFR2 expression altered within 4 h of treatment, while HFE expression altered later at 24 h and 48 h, suggesting that TFR2 may function prior to HFE in HAMP regulation

The role of informal, unstructured practice in developing football expertise: the case of Brazilian Pelada

The aim of this paper is to provide explanation and discussion on how unconventional socio-cultural constraints influence the development of skill and expertise of Brazilian football players. On this basis, the central question of this research is this: What are the influential environmental constraints on the development of perceptual-motor skills and expertise of Brazilian football players? The epistemological and methodological assumptions of the “contextualised skill acquisition research” (CSAR) (see Uehara et al., 2014) are used as an underpinning framework for data collection and organization of material. Drawing upon the notion of ethnographic strategies of inquiry for generating and analyzing data, we used qualitative methods such as contextual analysis, participant-observation, and open-ended interviews. At the micro-level of Brazilian society “pelada” emerges as one of the socio-cultural constraints that shapes the talent of Brazilian football players by influencing the development of their perceptual-motor expertise

Serum concentration impacts myosin heavy chain expression but not cellular respiration in human LHCN‐M2 myoblasts undergoing differentiation

Abstract The human LHCN‐M2 myoblast cell line has the potential to be used to investigate skeletal muscle development and metabolism. Experiments were performed to determine how different concentrations of human serum affect myogenic differentiation and mitochondrial function of LHCN‐M2 cells. LHCN‐M2 myoblasts were differentiated in serum‐free medium, 0.5% or 2% human serum for 5 and 10 days. Myotube formation was assessed by immunofluorescence staining of myosin heavy chain (MHC) and molecularly by mRNA expression of Myogenic differentiation 1 (MYOD1) and Myoregulatory factor 5 (MYF5). Following differentiation, mitochondrial function was assessed to establish the impact of serum concentration on mitochondrial function. Time in differentiation increased mRNA expression of MYOD1 (day 5, 6.58 ± 1.33‐fold; and day 10, 4.28 ± 1.71‐fold) (P = 0.012), while suppressing the expression of MYF5 (day 5, 0.21 ± 0.11‐fold; and day 10, 0.06 ± 0.03‐fold) (P = 0.001), regardless of the serum concentration. Higher serum concentrations increased MHC area (serum free, 11.92 ± 0.85%; 0.5%, 23.10 ± 5.82%; 2%, 43.94 ± 8.92%) (P = 0.001). Absolute basal respiration approached significance (P = 0.06) with significant differences in baseline oxygen consumption rate (P = 0.025) and proton leak (P = 0.006) when differentiated in 2% human serum, but these were not different between conditions when normalised to total protein. Our findings show that increasing concentrations of serum of LHCN‐M2 skeletal muscle cells into multinucleated myotubes, but this does not affect relative mitochondrial function

Suppression of Integrin Activation: A Novel Function of a Ras/Raf-Initiated MAP Kinase Pathway

AbstractRapid modulation of ligand binding affinity (“activation”) is a central property of the integrin cell adhesion receptors. Using a screen for suppressors of integrin activation, we identified the small GTP-binding protein, H-Ras, and its effector kinase, Raf-1, as negative regulators of integrin activation. H-Ras inhibited the activation of integrins with three distinct α and β subunit cytoplasmic domains. Suppression was not associated with integrin phosphorylation and was independent of both mRNA transcription and protein synthesis. Furthermore, suppression correlated with activation of the ERK MAP kinase pathway. Thus, regulation of integrin affinity state is a novel, transcription-independent function of a Ras-linked MAP kinase pathway that may mediate a negative feedback loop in integrin function