Recovery of the mitochondrial COI barcode region in diverse Hexapoda through tRNA-based primers

<p>Abstract</p> <p>Background</p> <p>DNA barcoding uses a 650 bp segment of the mitochondrial cytochrome <it>c </it>oxidase I (COI) gene as the basis for an identification system for members of the animal kingdom and some other groups of eukaryotes. PCR amplification of the barcode region is a key step in the analytical chain, but it sometimes fails because of a lack of homology between the standard primer sets and target DNA.</p> <p>Results</p> <p>Two forward PCR primers were developed following analysis of all known arthropod mitochondrial genome arrangements and sequence alignment of the tRNA-W gene which was usually located within 200 bp upstream of the COI gene. These two primers were combined with a standard reverse primer (LepR1) to produce a cocktail which generated a barcode amplicon from 125 of 141 species that included representatives of 121 different families of Hexapoda. High quality sequences were recovered from 79% of the species including groups, such as scale insects, that invariably fail to amplify with standard primers.</p> <p>Conclusions</p> <p>A cocktail of two tRNA-W forward primers coupled with a standard reverse primer amplifies COI for most hexapods, allowing characterization of the standard barcode primer binding region in COI 5' as well as the barcode segment. The current results show that primers designed to bind to highly conserved gene regions upstream of COI will aid the amplification of this gene region in species where standard primers fail and provide valuable information to design a primer for problem groups.</p

Anti-diabetic effect of Cyclo-His-Pro (CHP)-enriched yeast hydrolysate in streptozotocin-induced diabetic mice

The present study was designed to investigate the hypoglycemic effects of the daily oral dose of 0.50 to 0.75 g/kg of yeast hydrolysate (YH) containing high Cyclo-His-Pro (51.0 mg CHP/g YH) on normal and streptozotocin (STZ)-induced diabetic rats for 14 days. In STZ-induced diabetic rats, after administrations of the YH for 14 days, the body weight gain was significantly increased in dose dependent manner, and the plasma glucose levels were decreased approximately (60%) as compared to the STZ induced diabetic control group. Glucose level showed significant differences between the diabetic control (DC) and the YH administered groups in oral glucose tolerance test (OGTT) (P&lt;0.05). Results of the OGTT showed a significant decrease in the area under curve (AUC) value of YH supplemented groups as compared to the DC group. The present data suggests that the CHP-enriched YH has potential anti-diabetic effect, which can help in the cure and management of diabetes.Keywords: Yeast hydrolysate, Cyclo-His-Pro (CHP), diabetes, streptozotocin.African Journal of Biotechnology Vol. 12(35), pp. 5473-547

Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for (14)C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in (14)C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of (14)C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate (14)C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate (14)C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.These results suggest that AMPK-mediated PLD1 activation is required for (14)C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake

Transcriptome analysis of sputum cells reveals two distinct molecular phenotypes of “asthma and chronic obstructive pulmonary disease overlap” in the elderly

Background Little is known about the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD) overlap (ACO). This study examined the molecular phenotypes of ACO in the elderly. Methods A genome-wide investigation of gene expression in sputum cells from the elderly with asthma, ACO, or COPD was performed using gene set variation analysis (GSVA) with predefined asthma- or COPD-specific gene signatures. We then performed a subsequent cluster analysis using enrichment scores (ESs) to identify molecular clusters in the elderly with ACO. Finally, a second GSVA was conducted with curated gene signatures to gain insight into the pathogenesis of ACO associated with the identified molecular clusters. Results Seventy elderly individuals were enrolled (17 with asthma, 41 with ACO, and 12 with COPD). Two distinct molecular clusters of ACO were identified. Clinically, ACO cluster 1 (N = 23) was characterized by male and smoker dominance, more obstructive lung function, and higher proportions of both neutrophil and eosinophil in induced sputum compared to ACO cluster 2 (N = 18). ACO cluster 1 had molecular features similar to both asthma and COPD, with mitochondria and peroxisome dysfunction as important mechanisms in the pathogenesis of these diseases. The molecular features of ACO cluster 2 differed from those of asthma and COPD, with enhanced innate immune reactions to microorganisms identified as being important in the pathogenesis of this form of ACO. Conclusion Recognition of the unique biological pathways associated with the two distinct molecular phenotypes of ACO will deepen our understanding of ACO in the elderly

Experimental verification of horizontal two-dimensional modified mild-slope equation model

In order to verify modified mild-slope equation models in a horizontal two-dimensional space, a hydraulic experiment is made for surface wave propagation over a circular shoal on which water depth varies substantially. A horizontal two-dimensional numerical model is also constructed based on the hyperbolic equations that have been developed from the modified mild-slope equation to account for the substantial depth variation. Comparison between experimental measurements and numerical results shows that the modified mild-slope equation model is capable of producing accurate results for wave propagation in a region where water depth varies substantially, while the conventional mild-slope equation model gives large errors as the mild-slope assumption is violated.author's final versio

Bubble formation in globe valve and flow characteristics of partially filled pipe water flow

Air bubble entrainment is a phenomenon that can significantly reduce the efficiency of liquid motion in piping systems. In the present study, the bubble formation mechanism in a globe valve with 90% water fraction flow is explained by visualization study and pressure oscillation analysis. The shadowgraph imaging technique is applied to illustrate the unsteady flow inside the transparent valve. This helps to study the effect of bubbles induced by the globe valve on pressure distribution and valve flow coefficient. International Society of Automation (ISA) recommends locations for measuring pressure drop of the valve to determine its flow coefficient. This paper presents the comparison of the pressures at different locations along with the upstream and the downstream of the valve with the values at recommended positions by the ISA standard. The results show that in partially filled pipe flow, the discrepancies in pressure between different measurement locations in the valve downstream are significant at valve openings less than 30%. The aerated flow induces the oscillation in pressure and flow rate, which leads to the fluctuation in the flow coefficient of the valve. The flow coefficients have a linear relationship with the Reynolds number. For the same increase of Reynolds number, the flow coefficients grow faster with larger valve openings and level off at the opening of 50%

Pumpless, selective docking of yeast cells inside a microfluidic channel induced by receding meniscus

We present a simple cell docking method induced by receding meniscus to capture non-adherent yeast cells onto microwells inside a microfluidic channel. Microwells were fabricated either by capillary moulding of UV curable polyurethane acrylate (PUA) onto glass substrate or direct replica moulding of poly(dimethyl siloxane) (PDMS). A cell suspension of the budding yeast, Saccharomyces cerevisiae, was introduced into the microfluidic channel by surface tension driven capillary flow and a receding meniscus was subsequently generated by evaporation. As the meniscus progressed, one to multiple yeast cells were spontaneously captured onto microwells by lateral capillary force created at the bottom of the meniscus. Using this cell-based platform, we observed the response of yeast cells upon stimulation by a mating pheromone (alpha-factor) by monitoring the expression of green fluorescent protein (GFP) with time. It was observed that alpha-factor triggered the expression of GFP at 60 min after stimulation and the fluorescence intensity was sustained for an additional 60 min without changes.This work was supported by the Micro Thermal System Research Center of Seoul National University and the Ministry of Science and Technology through Bio Tool R&D Project for Cell Research. This work was also supported in part by the SRC program of MOST/KOSEF (R11-2005-009-02004-0) to S.-H. P