Core–shell silver nanoparticles for optical labeling of cells

Silver nanoparticles have been modified with self-assembled monolayers of hydroxyl-terminated long chain thiols and encapsulated with a silica shell. The resulting core–shell nanoparticles were used as optical labels for cell analysis using flow cytometry and microscopy. The excitation of plasmon resonances in nanoparticles results in strong depolarized scattering of visible light, permitting detection at the single nanoparticle level. The nanoparticles were modified with neutravidin via epoxide–azide coupling chemistry, to which biotinylated antibodies targeting cell surface receptors were bound. The nanoparticle labels exhibited long-term stability in solutions with high salt concentrations without aggregation or silver etching. Labeled cells exhibited two orders of magnitude enhancement of the scattering intensity compared with unlabeled cells.     

IRS1 phosphorylation does not mediate mTORC1-induced insulin resistance

Increased mammalian target of rapamycin complex 1 (mTORC1) activity has been suggested to play important roles in development of insulin resistance in obesity. mTORC1 hyperactivity also increases endoplasmic reticulum (ER) stress, which in turn contributes to development of insulin resistance and glucose intolerance. Increased IRS1 phosphorylation at Ser307 in vitro is correlated with mTORC1- and ER stress-induced insulin resistance. This phosphorylation site correlates strongly with impaired insulin receptor signaling in diabetic mice and humans. In contrast, evidence from knock-in mice suggests that phosphorylation of IRS1 at Ser307 is actually required to maintain insulin sensitivity. To study the involvement of IRS1^Ser307 phosphorylation in mTORC1-mediated glucose intolerance and insulin sensitivity in vivo, we investigated the effects of liver specific TSC1 depletion in IRS1^Ser307Ala mice and controls. Our results demonstrate that blockade of IRS1^Ser307 phosphorylation in vivo does not prevent mTORC1-mediated glucose intolerance and insulin resistance.     

Streptococcus mutans Extracellular DNA Is Upregulated during Growth in Biofilms,Actively Released via Membrane Vesicles,and Influenced by Components of the Protein Secretion Machinery

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.     

Improved secretion of the cancer-testis antigen SSX2 in Pichia pastoris by deletion of its nuclear localization signal

The cancer-testis (CT) antigen synovial sarcoma X break point 2 (SSX2) was expressed in Pichia pastoris as a means to produce a delayed-type hypersensitivity skin test reagent for monitoring SSX2-specific anti-cancer immune responses. SSX2 was detected intracellularly in P. pastoris despite the addition of the Saccharomyces cerevisiae alpha-mating factor secretion signal. Increasing the SSX2 gene copy number did not improve its secretion but did enhance intracellular SSX2 levels. SSX2 with its C-terminal nuclear localization signal (NLS) deleted (SSX2NORD), however, was secreted. Indirect immunofluorescence indicated that SSX2 containing the NLS did not translocate to the nucleus but accumulated in the endoplasmic reticulum (ER). Experimental results further suggested that SSX2 containing the NLS was misfolded in the ER, while deletion of the NLS facilitated correct folding of SSX2 inside the ER and improved its secretion. Production of SSX2NORD was scaled-up to a 2-L fermentor using a fed-batch protocol to maintain methanol at a concentration of 1 g L⁻¹. Decreasing the cultivation temperature from 25 °C to 16 °C improved protein stability in the culture supernatant. In this process, after 120 h cultivation, the wet cell weight of P. pastoris reached 280 mg mL⁻¹, and the yield of SSX2NORD was 21.6 mg L⁻¹.     

Effects of a 4-week youth baseball conditioning program on throwing velocity

Effects of a 4-week youth baseball conditioning program on throwing velocity. This study examined the effects of a 4-week youth baseball conditioning program on maximum throwing velocity. Thirty-four youth baseball players (11-15 years of age) were randomly and equally divided into control and training groups. The training group performed 3 sessions (each 75 minutes) weekly for 4 weeks, which comprised a sport specific warm-up, resistance training with elastic tubing, a throwing program, and stretching. Throwing velocity was assessed initially and at the end of the 4-week conditioning program for both control and training groups. The level of significance used was p < 0.05. After the 4-week conditioning program, throwing velocity increased significantly (from 25.1 ± 2.8 to 26.1 ± 2.8 m·s) in the training group but did not significantly increase in the control group (from 24.2 ± 3.6 to 24.0 ± 3.9 m·s). These results demonstrate that the short-term 4-week baseball conditioning program was effective in increasing throwing velocity in youth baseball players. Increased throwing velocity may be helpful for pitchers (less time for hitters to swing) and position players (decreased time for a runner to advance to the next base).     

A comparison of high-speed power training and traditional slow-speed resistance training in older men and women

Muscle power, the product of force × velocity, is a critical determinant of function in older adults. Resistance training (RT) at high speed has been shown to improve peak muscle power in this population; however, different functional tasks may benefit from the improvement of power at values other than "peak" values, for example, tasks that require a greater velocity component or a greater force component. This study compared the effect of high-speed RT on muscle performance (peak power [PP] and its components [PP force and PP velocity] and overall peak velocity [VEL]) across a broad range of external resistances. Thirty-eight older men and women were randomized to high-speed power training at 40% of the 1-repetition maximum (1RM) (n = 13 [74.1 ± 6.4 years]); traditional RT at 80% 1RM (n = 13 [70.1 ± 7.0 years]); or control (n = 12 [72.8 ± 4.1 years]). Measures of muscle performance were obtained at baseline and after the 12-week training intervention. Muscle power and 1RM strength improved similarly with both high-speed and traditional slow-speed RT. However, speed-related muscle performance characteristics, PP velocity and overall VEL, were most positively impacted by high-speed power training, especially at lower external resistances. Because gains in speed-related measures with high-speed training compared to traditional RT do not come at the expense of other muscle performance outcomes, we recommend using an RT protocol in older adults that emphasizes high-speed movements at low external resistances.     

Comparative gene expression profiling analysis of urothelial carcinoma of the renal pelvis and bladder

Background

Tumor therapy mainly attacks the metabolism to interfere the tumor's anabolism and signaling of proliferative second messengers. However, the metabolic demands of different cancers are very heterogeneous and depend on their origin of tissue, age, gender and other clinical parameters. We investigated tumor specific regulation in the metabolism of breast cancer.

Methods

For this, we mapped gene expression data from microarrays onto the corresponding enzymes and their metabolic reaction network. We used Haar Wavelet transforms on optimally arranged grid representations of metabolic pathways as a pattern recognition method to detect orchestrated regulation of neighboring enzymes in the network. Significant combined expression patterns were used to select metabolic pathways showing shifted regulation of the aggressive tumors.

Results

Besides up-regulation for energy production and nucleotide anabolism, we found an interesting cellular switch in the interplay of biosynthesis of steroids and bile acids. The biosynthesis of steroids was up-regulated for estrogen synthesis which is needed for proliferative signaling in breast cancer. In turn, the decomposition of steroid precursors was blocked by down-regulation of the bile acid pathway.

Conclusion

We applied an intelligent pattern recognition method for analyzing the regulation of metabolism and elucidated substantial regulation of human breast cancer at the interplay of cholesterol biosynthesis and bile acid metabolism pointing to specific breast cancer treatment.     

Mixed model approaches for the identification of QTLs within a maize hybrid breeding program

Two outlines for mixed model based approaches to quantitative trait locus (QTL) mapping in existing maize hybrid selection programs are presented: a restricted maximum likelihood (REML) and a Bayesian Markov Chain Monte Carlo (MCMC) approach. The methods use the in-silico-mapping procedure developed by Parisseaux and Bernardo (2004) as a starting point. The original single-point approach is extended to a multi-point approach that facilitates interval mapping procedures. For computational and conceptual reasons, we partition the full set of relationships from founders to parents of hybrids into two types of relations by defining so-called intermediate founders. QTL effects are defined in terms of those intermediate founders. Marker based identity by descent relationships between intermediate founders define structuring matrices for the QTL effects that change along the genome. The dimension of the vector of QTL effects is reduced by the fact that there are fewer intermediate founders than parents. Furthermore, additional reduction in the number of QTL effects follows from the identification of founder groups by various algorithms. As a result, we obtain a powerful mixed model based statistical framework to identify QTLs in genetic backgrounds relevant to the elite germplasm of a commercial breeding program. The identification of such QTLs will provide the foundation for effective marker assisted and genome wide selection strategies. Analyses of an example data set show that QTLs are primarily identified in different heterotic groups and point to complementation of additive QTL effects as an important factor in hybrid performance.