Novel microstructures and technologies applied in chemical analysis techniques

Novel glass and silicon microstructures and their application in chemical analysis are presented. The micro technologies comprise (deep) dry etching, thin layer growth and anodic bonding. With this combination it is possible to create high resolution electrically isolating silicon dioxide structures with aspect ratio's similar to those possible in silicon. Main applications are chemical separation methods such as high performance liquid chromatography (HPLC) or electrophoresis (HPCE). Beside these channel structures, a capillary connector with very low dead and mixing volume has been designed and fabricated for use in (correlation) electrophoresis, and tested by means of precision of consecutive single injection

A microsensor array for biochemical sensing

A microsensor array to measure chemical properties of biological liquids is presented. A hybrid integration technique is used to mount four sensor chips on a micro flow channel: a pressure, temperature, pH, combined pO2 and pCO2 sensor chip. This results in a microsensor array which is developed to meet the technical requirements for space applications. The integration method allows to integrate other types of sensor chips. This multi-purpose and multi-user approach makes the microsensor array suitable for various biochemical applications

Collaborative coupling between polymerase and helicase for leading-strand synthesis

Rapid and processive leading-strand DNA synthesis in the bacteriophage T4 system requires functional coupling between the helicase and the holoenzyme, consisting of the polymerase and trimeric clamp loaded by the clamp loader. We investigated the mechanism of this coupling on a DNA hairpin substrate manipulated by a magnetic trap. In stark contrast to the isolated enzymes, the coupled system synthesized DNA at the maximum rate without exhibiting fork regression or pauses. DNA synthesis and unwinding activities were coupled at low forces, but became uncoupled displaying separate activities at high forces or low dNTP concentration. We propose a collaborative model in which the helicase releases the fork regression pressure on the holoenzyme allowing it to adopt a processive polymerization conformation and the holoenzyme destabilizes the first few base pairs of the fork thereby increasing the efficiency of helicase unwinding. The model implies that both enzymes are localized at the fork, but does not require a specific interaction between them. The model quantitatively reproduces homologous and heterologous coupling results under various experimental conditions

Mechanism of strand displacement synthesis by DNA replicative polymerases

Replicative holoenzymes exhibit rapid and processive primer extension DNA synthesis, but inefficient strand displacement DNA synthesis. We investigated the bacteriophage T4 and T7 holoenzymes primer extension activity and strand displacement activity on a DNA hairpin substrate manipulated by a magnetic trap. Holoenzyme primer extension activity is moderately hindered by the applied force. In contrast, the strand displacement activity is strongly stimulated by the applied force; DNA polymerization is favoured at high force, while a processive exonuclease activity is triggered at low force. We propose that the DNA fork upstream of the holoenzyme generates a regression pressure which inhibits the polymerization-driven forward motion of the holoenzyme. The inhibition is generated by the distortion of the template strand within the polymerization active site thereby shifting the equilibrium to a DNA-protein exonuclease conformation. We conclude that stalling of the holoenzyme induced by the fork regression pressure is the basis for the inefficient strand displacement synthesis characteristic of replicative polymerases. The resulting processive exonuclease activity may be relevant in replisome disassembly to reset a stalled replication fork to a symmetrical situation. Our findings offer interesting applications for single-molecule DNA sequencing

Body composition changes during 8 weeks of military training are not accurately captured by circumference-based assessments

In 1981, the US military adopted body fat standards to promote physical readiness and prevent obesity. Separate circumference-based equations were developed for women and men. Both predictive equations were known to underestimate %BF. However, it was not known how well these abdominal circumference-based methods tracked changes in %BF. This study examined the validity of the circumference-based %BF equations for assessing changes in %BF in young adult recruits during Army Basic Combat Training (BCT). Dual-energy X-ray absorptiometry (DXA) and circumference-based measures of %BF were obtained in women (n = 481) and men (n = 926) at the start (pre-BCT) and end (post-BCT) of 8 weeks of BCT. Repeated-measure ANOVAs were used to assess differences between DXA and circumference pre-BCT and for the change during BCT. Pre-BCT, circumferences underestimated %BF relative to DXA, with mean errors of −6.0% ± 4.4% for women and −6.0% ± 3.5% for men (both p < 0.01), and no difference between sexes was observed (p = 0.77). DXA detected a −4.0% ± 2.4% and −3.3% ± 2.8% change in %BF for women and men in response to BCT, respectively (both p < 0.01), whereas circumference estimates of %BF indicated a 0.0% ± 3.3% (p = 0.86) change in women and a −2.2% ± 3.3% (p < 0.01) change in men (sex difference by technique p < 0.01). In conclusion, circumference-based measures underestimated %BF at the start of BCT in both sexes as compared to DXA. Circumference measures underestimated changes in %BF during BCT in men and did not detect changes in women. These findings suggest that circumference-based %BF metrics may not be an appropriate tool to track changes in body composition during short duration training

Lentiviral Vectors and Protocols for Creation of Stable hESC Lines for Fluorescent Tracking and Drug Resistance Selection of Cardiomyocytes

Developmental, physiological and tissue engineering studies critical to the development of successful myocardial regeneration therapies require new ways to effectively visualize and isolate large numbers of fluorescently labeled, functional cardiomyocytes.Here we describe methods for the clonal expansion of engineered hESCs and make available a suite of lentiviral vectors for that combine Blasticidin, Neomycin and Puromycin resistance based drug selection of pure populations of stem cells and cardiomyocytes with ubiquitous or lineage-specific promoters that direct expression of fluorescent proteins to visualize and track cardiomyocytes and their progenitors. The phospho-glycerate kinase (PGK) promoter was used to ubiquitously direct expression of histone-2B fused eGFP and mCherry proteins to the nucleus to monitor DNA content and enable tracking of cell migration and lineage. Vectors with T/Brachyury and alpha-myosin heavy chain (alphaMHC) promoters targeted fluorescent or drug-resistance proteins to early mesoderm and cardiomyocytes. The drug selection protocol yielded 96% pure cardiomyocytes that could be cultured for over 4 months. Puromycin-selected cardiomyocytes exhibited a gene expression profile similar to that of adult human cardiomyocytes and generated force and action potentials consistent with normal fetal cardiomyocytes, documenting these parameters in hESC-derived cardiomyocytes and validating that the selected cells retained normal differentiation and function.The protocols, vectors and gene expression data comprise tools to enhance cardiomyocyte production for large-scale applications

52 Genetic Loci Influencing Myocardial Mass.

BACKGROUND: Myocardial mass is a key determinant of cardiac muscle function and hypertrophy. Myocardial depolarization leading to cardiac muscle contraction is reflected by the amplitude and duration of the QRS complex on the electrocardiogram (ECG). Abnormal QRS amplitude or duration reflect changes in myocardial mass and conduction, and are associated with increased risk of heart failure and death. OBJECTIVES: This meta-analysis sought to gain insights into the genetic determinants of myocardial mass. METHODS: We carried out a genome-wide association meta-analysis of 4 QRS traits in up to 73,518 individuals of European ancestry, followed by extensive biological and functional assessment. RESULTS: We identified 52 genomic loci, of which 32 are novel, that are reliably associated with 1 or more QRS phenotypes at p < 1 × 10(-8). These loci are enriched in regions of open chromatin, histone modifications, and transcription factor binding, suggesting that they represent regions of the genome that are actively transcribed in the human heart. Pathway analyses provided evidence that these loci play a role in cardiac hypertrophy. We further highlighted 67 candidate genes at the identified loci that are preferentially expressed in cardiac tissue and associated with cardiac abnormalities in Drosophila melanogaster and Mus musculus. We validated the regulatory function of a novel variant in the SCN5A/SCN10A locus in vitro and in vivo. CONCLUSIONS: Taken together, our findings provide new insights into genes and biological pathways controlling myocardial mass and may help identify novel therapeutic targets