A Simple Protein Precipitation-based Simultaneous Quantification of Lovastatin and Its Active Metabolite Lovastatin Acid in Human Plasma by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry using Polarity Switching

Lovastatin is an anti-cholesterol lactone drug indicated for the treatment of hyperlipidemia and to reduce the risk of coronary heart disease. It is converted to the β-hydroxy acid form (lovastatin acid) in vivo, which is the major pharmacologically active metabolite. Here, we describe the development and validation of an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS)-based method utilizing polarity switching for the simultaneous quantification of lovastatin and lovastatin acid in human plasma. Simple protein precipitation extraction and direct injection of the extracted samples without drying/reconstitution showed good recoveries of both analytes (~70%). The developed method exhibited satisfactory intra-day and inter-day accuracy and precision. The interconversion between lovastatin and lovastatin acid during sample preparation and storage was minimal (< 1.9%). The lower limits of quantification were 0.5 and 0.2 nM (or 0.2 and 0.084 ng/mL) for lovastatin and lovastatin acid, respectively, using only 50 μL of plasma during extraction. The validated method was successfully applied to analyze plasma samples obtained from a healthy human subject who enrolled in a clinical drug interaction study involving lovastatin

Modeling craniofacial development reveals spatiotemporal constraints on robust patterning of the mandibular arch

How does pattern formation occur accurately when confronted with tissue growth and stochastic fluctuations (noise) in gene expression? Dorso-ventral (D-V) patterning of the mandibular arch specifies upper versus lower jaw skeletal elements through a combination of Bone morphogenetic protein (Bmp), Endothelin-1 (Edn1), and Notch signaling, and this system is highly robust. We combine NanoString experiments of early D-V gene expression with live imaging of arch development in zebrafish to construct a computational model of the D-V mandibular patterning network. The model recapitulates published genetic perturbations in arch development. Patterning is most sensitive to changes in Bmp signaling, and the temporal order of gene expression modulates the response of the patterning network to noise. Thus, our integrated systems biology approach reveals non-intuitive features of the complex signaling system crucial for craniofacial development, including novel insights into roles of gene expression timing and stochasticity in signaling and gene regulation

Effect of torso morphology on maximum hydrodynamic resistance in front crawl swimming

The aim of this study was to determine the influence of torso morphology on maximum instantaneous hydrodynamic resistance in front crawl swimming. Outlines of the torso in the frontal and anteroposterior planes were calculated from photographic images to determine continuous form gradients (m/m) for the anterior, posterior and lateral aspects of the torso. Torso cross-sectional areas at each vertical sample (0.001 m) were used to calculate maximal rate of change in cross-sectional area (m2/m) in the chest-waist and waist-hip segments. During the non-propulsive hand phase in middle-long distance front crawl, kicking propulsion is negligible and therefore the net force is equal to the drag. Drag coefficients were calculated at the instant of maximum horizontal deceleration of centre of mass during the non-propulsive hand phase of 400 m pace front crawl. Maximal rate of change in cross-sectional area (r = 0.44, p = 0.014) and posterior form gradient (r = 0.50, p = 0.006) of the waist-hip torso segment had moderate positive correlations with the maximal drag coefficient. A regression model including these variables explained 41% of the variance (p = 0.001). Indentation at the waist and curvature of the buttocks may result in greater drag force and influence swimming performance

Protective Effects of PARP-1 Knockout on Dyslipidemia-Induced Autonomic and Vascular Dysfunction in ApoE−/− Mice: Effects on eNOS and Oxidative Stress

The aims of this study were to investigate the role of poly(ADP-ribose) polymerase (PARP)-1 in dyslipidemia-associated vascular dysfunction as well as autonomic nervous system dysregulation. Apolipoprotein (ApoE)−/− mice fed a high-fat diet were used as a model of atherosclerosis. Vascular and autonomic functions were measured in conscious mice using telemetry. The study revealed that PARP-1 plays an important role in dyslipidemia-associated vascular and autonomic dysfunction. Inhibition of this enzyme by gene knockout partially restored baroreflex sensitivity in ApoE−/− mice without affecting baseline heart-rate and arterial pressure, and also improved heart-rate responses following selective blockade of the autonomic nervous system. The protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction was associated with preservation of eNOS activity. Dyslipidemia induced PARP-1 activation was accompanied by oxidative tissue damage, as evidenced by increased expression of iNOS and subsequent protein nitration. PARP-1 gene deletion reversed these effects, suggesting that PARP-1 may contribute to vascular and autonomic pathologies by promoting oxidative tissue injury. Further, inhibition of this oxidative damage may account for protective effects of PARP-1 gene deletion on vascular and autonomic functions. This study demonstrates that PARP-1 participates in dyslipidemia-mediated dysregulation of the autonomic nervous system and that PARP-1 gene deletion normalizes autonomic and vascular dysfunctions. Maintenance of eNOS activity may be associated with the protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction

Healing of the aponeurosis during recovery from aponeurotomy: morphological and histological adaptation and related changes in mechanical properties

Aponeurotomy, which is the transection of an aponeurosis perpendicular to its length, is performed to lengthen spastic and/or short muscles. During recovery, the cut ends of the aponeurosis are reconnected by new connective tissue bridging both ends. The aim of this study is to investigate the histological features of this new connective tissue as well as its mechanical properties after recovery from aponeurotomy. For this purpose, aponeurotomy was performed on the proximal aponeurosis of rat m. gastrocnemius medialis (GM), which was followed by six weeks of recovery. The lengths of aponeurotic tissues were measured as a function of active muscle length. The results are compared to a control group as well as to the acute effects and a sham operated group. Activation of the muscle at increasing lengths after aponeurotomy caused a gap between the cut ends of the aponeurosis. However, after recovery, new connective tissue is formed bridging the aponeurotic ends, consisting of thin collagen fibres, which are densely packed and generally arranged in the direction of the aponeurosis. The number of fibroblasts was three to five times higher than that of aponeurotic tissue of the intact parts as well as that of the acute and sham operated muscles. The strain of the new connective tissue as a function of active muscle length was shown to be about three times higher than that of the aponeurosis. It is concluded that the inserted new aponeurotic tissue is more compliant and that the aponeurosis becomes 10-15% longer than in untreated muscle. As a consequence, the muscle fibres located distally to the new aponeurotic tissue will become shorter than prior to aponeurotomy. This explains a shift of the length-force curve, which favours the restoration of the range of joint motion. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved

G9a Is Essential for EMT-Mediated Metastasis and Maintenance of Cancer Stem Cell-Like Characters in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma (HNSCC) is a particularly aggressive cancer with poor prognosis, largely due to lymph node metastasis and local recurrence. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics. However, the mechanisms underlying metastasis to lymph nodes in HNSCC is poorly defined. In this study, we show that E-cadherin repression correlates with cancer metastasis and poor prognosis in HNSCC. We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9). Moreover, G9a is required for both lymph node-related metastasis and TGF-β-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers. Our study demonstrates that the G9a protein is essential for the induction of EMT and CSC-like properties in HNSCC. Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC