Cloning and characterization of genes encoding trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2) from Zygosaccharomyces rouxii

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.     

Ectopic expression of active processed form of atrial natriuretic peptide in skeletal myoblasts

Atrial natriuretic peptide (ANP) is a cardiac hormone that elicits a profound diuresis, natriuresis, and hypotension. As a preliminary study toward ANP gene therapy of cardiovascular disorders, we have cloned a cDNA for mouse preproANP and carried out expression studies in muscle cells. The expression cassette, which was flanked by ITRs from AAV-2, consisted of HCMV IE enhancer/promoter, preproANP gene, and polyadenylation signal from bovine growth hormone. We transfected this expression vector into primary skeletal myoblasts and examined the following points: (1) secretion of immunoreactive ANP, (2) biological activity, and (3) nature of secreted ANP(s). The conditioned media from cells transfected with ANP vector had significantly higher levels of irANP in comparison to mock control. The secreted irANP had biological activity as confirmed by the elevated level of intracellular cGMP in human umbilical vein endothelial cells. Reverse-phase HPLC analysis showed that the processed form of ANP was the predominant form. These results demonstrate that preproANP gene could be ectopically expressed and correctly processed in skeletal myoblasts, which has implications for development of muscle-based ANP gene therapy.     

Effects of particulate matter exposure on blood 5-hydroxymethylation: results from the Beijing truck driver air pollution study

Previous studies have reported epigenetic changes induced by environmental exposures. However, previous investigations did not distinguish 5-methylcytosine (5mC) from a similar oxidative form with opposite functions, 5-hydroxymethylcytosine (5hmC). Here, we measured blood DNA global 5mC and 5hmC by ELISA and used adjusted mixed-effects regression models to evaluate the effects of ambient PM₁₀ and personal PM_2.5 and its elemental components—black carbon (BC), aluminum (Al), calcium (Ca), potassium (K), iron (Fe), sulfur (S), silicon (Si), titanium (Ti), and zinc (Zn)—on blood global 5mC and 5hmC levels. The study was conducted in 60 truck drivers and 60 office workers in Beijing, China from The Beijing Truck Driver Air Pollution Study at 2 exams separated by one to 2 weeks. Blood 5hmC level (0.08%) was ∼83-fold lower than 5mC (6.61%). An inter-quartile range (IQR) increase in same-day PM₁₀ was associated with increases in 5hmC of 26.1% in office workers (P = 0.004), 20.2% in truck drivers (P = 0.014), and 21.9% in all participants combined (P < 0.001). PM₁₀ effects on 5hmC were increasingly stronger when averaged over 4, 7, and 14 d preceding assessment (up to 132.6% for the 14-d average in all participants, P < 0.001). PM₁₀ effects were also significant after controlling for multiple testing (family-wise error rate; FWER < 0.05). 5hmC was not correlated with personal measures of PM_2.5 and elemental components (FWER > 0.05). 5mC showed no correlations with PM₁₀, PM_2.5, and elemental components measures (FWER > 0.05). Our study suggests that exposure to ambient PM₁₀ affects 5hmC over time, but not 5mC. This finding demonstrates the need to differentiate 5hmC and 5mC in environmental studies of DNA methylation.     

Strategies for oral delivery of macromolecule drugs

Advances in biotechnology, gene manipulation, and protein engineering for macromolecule drugs, such as insulin, parathyroid hormone (PTH), calcitonin, human growth hormone, erythropoietin (EPO), and peptide YY (PYY) allow commercial production in large scale for diverse therapeutic uses. Other macromolecules, such as mucopolysaccharide heparin, have expanded markets through improvements in their pharmacokinetic and pharmacological effects. However, most products are available only as injectable forms and are limited to patients with no alternative therapeutic choices. Orally available macromolecule formulations are still unmet needs for improving patient compliance and expanding administration paradigms and indications. Oral delivery technologies including carrier systems, absorption enhancers, protease inhibitors, and modification by conjugating transporter or receptor recognition molecules have been developed and some are undergoing clinical studies. In this review, we discuss major obstacles for oral absorption of macromolecule drugs and summarize recent strategies to overcome the huddles related to enhancing intestinal permeation.     

The Role of Elytra in Beetle Flight: I. Generation of Quasi-Static Aerodynamic Forces

We conducted a comprehensive study to investigate the aerodynamic characteristics and force generation of the elytra of abeetle,Allomyrina dichotoma.Our analysis included wind tunnel experiments and three-dimensional computational fluiddynamics simulations using ANSYS-CFX software.Our first approach was a quasi-static study that considered the effect ofinduced flapping flow due to the flapping motion of the fore-wings (elytra) at a frequency of around 30 Hz to 40 Hz.The dihedralangle was varied to represent flapping motion during the upstroke and downstroke.We found that an elytron producespositive lift at 0° geometric angle of attack,negative lift during the upstroke,and always produces drag during both the upstrokeand downstroke.We also found that the lift coefficient of an elytron does not drop even at a very high geometric angle of attack.For a beetle with a body weight of 5 g,based on the quasi-static method,the fore-wings (elytra) can produce lift of less than 1%of its body weight.     

Effect of Chord Flexure on Aerodynamic Performance of a Flapping Wing

Inspired by the fact that a high flexible wing in nature generates high aerodynamic performance, we investigated the aerodynamic performance of the flapping wing with different chord flexures. The unsteady, incompressible, and viscous flow over airfoil NACA0012 in a plunge motion was analyzed by using Navier-Stokes equation. Grid deformation, in which finite element and interpolation ideas are mixed, was introduced for computing large grid deformation caused by the chord flexures. We explored the optimal phase angle for thrust force and propulsive efficiency by varying the chord flexure from 0.05 to 0.7 when reduced frequency and plunge amplitude were fixed. Throughout parametric study on the phase angle and chord flexure amplitude, the maximum thrust force is achieved near at 0° in all given conditions, meanwhile, it is found that the optimal phase angle has dependency of chord flexure amplitude, which achieves higher aerodynamic performance compared to previous studies. These findings will provide a useful guideline for determining wing flexibility in design of a bio-mimetic air vehicle.