Tissue proteomics of the low‐molecular weight proteome using an integrated cLC‐ESI‐QTOFMS approach

Analysis of the protein/peptide composition of tissue has provided meaningful insights into tissue biology and even disease mechanisms. However, little has been published regarding top down methods to investigate lower molecular weight (MW) (500–5000 Da) species in tissue. Here, we evaluate a tissue proteomics approach involving tissue homogenization followed by depletion of large proteins and then cLC‐MS (where c stands for capillary) analysis to interrogate the low MW/low abundance tissue proteome. In the development of this method, sheep heart, lung, liver, kidney, and spleen were surveyed to test our ability to observe tissue differences. After categorical tissue differences were demonstrated, a detailed study of this method's reproducibility was undertaken to determine whether or not it is suitable for analyzing more subtle differences in the abundance of small proteins and peptides. Our results suggest that this method should be useful in exploring the low MW proteome of tissues.     

Insulin-Receptor Substrate-2 (IRS-2) Is Required for Maintaining Glucokinase and Glucokinase Regulatory Protein Expression in Mouse Liver

Insulin receptor substrate (IRS) proteins play important roles in hepatic nutrient homeostasis. Since glucokinase (GK) and glucokinase regulatory protein (GKRP) function as key glucose sensors, we have investigated the expression of GK and GKRP in liver of Irs-2 deficient mice and Irs2(−/−) mice where Irs2 was reintroduced specifically into pancreatic β-cells [RIP-Irs-2/IRS-2(−/−)]. We observed that liver GK activity was significantly lower (p<0.0001) in IRS-2(−/−) mice. However, in RIP-Irs-2/IRS-2(−/−) mice, GK activity was similar to the values observed in wild-type animals. GK activity in hypothalamus was not altered in IRS-2(−/−) mice. GK and GKRP mRNA levels in liver of IRS-2(−/−) were significantly lower, whereas in RIP-Irs-2/IRS-2(−/−) mice, both GK and GKRP mRNAs levels were comparable to wild-type animals. At the protein level, the liver content of GK was reduced in IRS-2(−/−) mice as compared with controls, although GKRP levels were similar between these experimental models. Both GK and GKRP levels were lower in RIP-Irs-2/IRS-2(−/−) mice. These results suggest that IRS-2 signalling is important for maintaining the activity of liver GK. Moreover, the differences between liver and brain GK may be explained by the fact that expression of hepatic, but not brain, GK is controlled by insulin. GK activity was restored by the β-cell compensation in the RIP-Irs-2/IRS-2 mice. Interestingly, GK and GKRP protein expression remained low in RIP-Irs-2/IRS-2(−/−) mice, perhaps reflecting different mRNA half-lives or alterations in the process of translation and post-translational regulation.     

Effect of resistance training to muscle failure vs non-failure on strength,hypertrophy and muscle architecture in trained individuals

The aim of this study was to compare the effects of resistance training to muscle failure (RT-F) and non-failure (RT-NF) on muscle mass, strength and activation of trained individuals. We also compared the effects of these protocols on muscle architecture parameters. A within-subjects design was used in which 14 participants had one leg randomly assigned to RT-F and the other to RT-NF. Each leg was trained 2 days per week for 10 weeks. Vastus lateralis (VL) muscle cross-sectional area (CSA), pennation angle (PA), fascicle length (FL) and 1-repetition maximum (1-RM) were assessed at baseline (Pre) and after 20 sessions (Post). The electromyographic signal (EMG) was assessed after the training period. RT-F and RT-NF protocols showed significant and similar increases in CSA (RT-F: 13.5% and RT-NF: 18.1%; P < 0.0001), PA (RT-F: 13.7% and RT-NF: 14.4%; P < 0.0001) and FL (RT-F: 11.8% and RT-NF: 8.6%; P < 0.0001). All protocols showed significant and similar increases in leg press (RT-F: 22.3% and RT-NF: 26.7%; P < 0.0001) and leg extension (RT-F: 33.3%, P < 0.0001 and RT-NF: 33.7%; P < 0.0001) 1-RM loads. No significant differences in EMG amplitude were detected between protocols (P > 0.05). In conclusion, RT-F and RT-NF are similarly effective in promoting increases in muscle mass, PA, FL, strength and activation.     

Genotypic variation in the persistence of transgenerational responses to seasonal cues*

Phenotypes respond to environments experienced directly by an individual, via phenotypic plasticity, or to the environment experienced by ancestors, via transgenerational environmental effects. The adaptive value of environmental effects depends not only on the strength and direction of the induced response but also on how long the response persists within and across generations, and how stably it is expressed across environments that are encountered subsequently. Little is known about the genetic basis of those distinct components, or even whether they exhibit genetic variation. We tested for genetic differences in the inducibility, temporal persistence, and environmental stability of transgenerational environmental effects in Arabidopsis thaliana. Genetic variation existed in the inducibility of transgenerational effects on traits expressed across the life cycle. Surprisingly, the persistence of transgenerational effects into the third generation was uncorrelated with their induction in the second generation. Although environmental effects for some traits in some genotypes weakened over successive generations, others were stronger or even in the opposite direction in more distant generations. Therefore, transgenerational effects in more distant generations are not merely caused by the retention or dissipation of those expressed in prior generations, but they may be genetically independent traits with the potential to evolve independently.     

Preparation of submicrometer monodispersed magnetic silica particles using a novel water in oil microemulsion: properties and application for enzyme immobilization

The synthesis of monodispersed magnetic silica nanoparticles (MSN) is described using a water-in-oil reverse microemulsion system that does not require the use of co-surfactants. Sodium silicate, Tween 20 as a neutral surfactant and 1-butanol as the organic phase were used. There are several advantages of the proposed method including a saturation magnetization value of 10 emu/g for the particles obtained, uniformity of size and that they are easily functionalized to bind urease covalently. Moreover, the intra-day, inter-day and long-term stability results confirm that the procedure was successful and the enzyme-linked MSNs were stable over repeated uses and storage retaining more than 75 % activity after 4 months.     

The influence of moss on grazers in high‐altitude streams: food,refuge or both?

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Molecular dynamics of water in the neighborhood of aquaporins

Present knowledge obtained by molecular dynamics (MD) simulation studies regarding the dynamics of water, both in the vicinity of biological membranes and within the proteinaceous water channels, also known as aquaporins (AQPs), is reviewed. A brief general summary of the water models most extensively employed in MD simulations (SPC, SPC/E, TIP3P, TIP4P), indicating their most relevant pros and cons, is likewise provided. Structural considerations of water are also discussed, based on different order parameters, which can be extracted from MD simulations as well as from experiments. Secondly, the behaviour of water in the neighbourhood of membranes by means of molecular dynamics simulations is addressed. Consequently, the comparison with previous experimental evidence is pointed out. In living cells, water is transported across the plasma membrane through the lipid bilayer and the aforementioned AQPs, which motivates this review to focus mostly on MD simulation studies of water within AQPs. Relevant contributions explaining peculiar properties of these channels are discussed, such as selectivity and gating. Water models used in these studies are also summarised. Finally, based on the information presented here, further MD studies are encouraged.     

On the origins,rapid expansion and genetic diversity of native Americans from hunting‐gatherers to agriculturalists

Given the importance of Y‐chromosome haplogroup Q to better understand the source populations of contemporary Native Americans, we studied 8 biallelic and 17 microsatellite polymorphisms on the background of 128 Q Y‐chromosomes from geographically targeted populations. The populations examined in this study include three from the Tuva Republic in Central Asia (Bai‐Tai, Kungurtug, and Toora‐Hem, n = 146), two from the northeastern tip of Siberia (New Chaplino and Chukchi, n = 32), and two from Mesoamerica (Mayans from Yucatan, Mexico n = 72, and Mayans from the Guatemalan Highlands, n = 43). We also see evidence of a dramatic Mesoamerican post‐migration population growth in the ubiquitous and diverse Y‐STR profiles of the Mayan and other Mesoamerican populations. In the case of the Mayans, this demographic growth was most likely fueled by the agricultural‐ and trade‐based subsistence adopted during the Pre‐Classic, Classic and Post‐Classic periods of their empire. The limited diversity levels observed in the Altaian and Tuvinian regions of Central Asia, the lowest of all populations examined, may be the consequence of bottleneck events fostered by the spatial isolation and low effective population size characteristic of a nomadic lifestyle. Furthermore, our data illustrate how a sociocultural characteristic such as mode of subsistence may be of impact on the genetic structure of populations. We analyzed our genetic data using Multidimensional Scaling Analysis of populations, Principal Component Analysis of individuals, Median‐joining networks of M242, M346, L54, and M3 individuals, age estimations based on microsatellite variation utilizing genealogical and evolutionary mutation rates/generation times and estimation of Y‐ STR average gene diversity indices. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

27-Nor-Δ4-dafachronic acid is a synthetic ligand of Caenorhabditis elegans DAF-12 receptor

27-Nor-Δ⁴-dafachronic acid was prepared in nine steps and 14% overall yield by two sequential 2-carbon homologations from 20β-carboxyaldehyde-4-pregnen-3-one. Its activity was evaluated in vivo, where it rescued the Mig phenotype of daf-9(rh50) Caenorhabditis elegans mutants and restored their normal resistance to oxidative stress. 27-Nor-Δ⁴-dafachronic acid was also able to directly bind and activate DAF-12 in a transactivation cell-based luciferase reporter assay, although it was less active than the corresponding 25R-and 25S dafachronic acids. The binding mode of the 27-Nor steroid was studied by molecular dynamics using a homology model of the CeDAF-12 receptor.