Surrogate reporters for enrichment of cells with nuclease-induced mutations

Zinc-finger nucleases (ZFNs) and TAL-effector nucleases (TALENs) are powerful tools for creating genetic modifications in eukaryotic cells and organisms. But wild-type and mutant cells that contain genetic modifications induced by these programmable nucleases are often phenotypically indistinguishable, hampering isolation of mutant cells. Here we show that transiently transfected episomal reporters encoding fluorescent proteins can be used as surrogate genes for the efficient enrichment of endogenous gene-modified cells by flow cytometry.     

Differences of urease activity and expression of associated genes according to gastric topography

Background: We hypothesize that pH difference between acid‐secreting corpus and non‐secreting antrum might influence the activity of H. pylori’s urease and/or related genes. We therefore measured urease activity and the expression of amiE whose encoded protein that hydrolyzes short‐chain amides to produce ammonia. Materials and Methods: Fifty‐four patients were recruited into this study. Each gastroscopic biopsy specimen collected from the antrum and body of each patient was immediately used to measure urease activity using serial changes of urease activity (ammonia levels) during 60 minutes. Probe specific for amiE was labeled with a biotin nick‐translation kit and was used to detect expression of these genes (mRNA) in fresh‐frozen gastroscopic biopsy specimens using fluorescent in situ hybridization (FISH). Results: Urease activity at 60 minutes from the gastric antrum and body of all patients infected with H. pylori was 399.5 ± 490.5 and 837.9 ± 1038.9 μg/dL, respectively (p = .004). Urease activity in the antrum was correlated with H. pylori density. Urease activity or H. pylori density in the antrum was significantly correlated with chronic active inflammation; in contrast, this correlation was not found in the gastric body. The expression level of amiE was 1.5 times higher (p < .05) in the gastric body compared with the antrum. Conclusion: Topographically, the urease activity in body was much higher than in antrum. The expression level of amiE was higher in the gastric body compared with the antrum.     

Molecular identification of AFLP fragments associated with elytra color variation of the Asian ladybird beetle,Harmonia axyridis (Coleoptera: Coccinellidae)

The Asian ladybird beetle, Harmonia axyridis shows polymorphism in elytra color patterns. However, it is uncertain whether these color patterns are regulated by genetic factors. This investigation used amplified fragment length polymorphism (AFLP) analysis to determine any genetic causes of the variability of color patterns. Using four individuals of each group, AFLP analysis produced 37 polymorphic bands. Among several polymorphic bands, six AFLP markers were associated with elytra color patterns after further analysis using six additional individuals of each group. These polymorphic sites were sequenced but did not match DNA sequence data deposited in GenBank. Based on the color-associated AFLP markers, SCAR primers were designed for PCR amplification of genomic DNA. These primers (SCAR 12 and SCAR 44) were used to analyze color-associated loci and/or alleles of H. axyridis DNA. SCAR 12 primers designed from a Spectabilis type-specific fragment (AFLP 12) amplified a specific band of 530 bp in four Spectabilis individuals, but not in the insects with other color patterns.     

Influence of performance level on anaerobic power and body composition in elite male judoists

This study examined the relationship between 30-second anaerobic power and body composition by performance level in elite Judoists. During a 3-month period, 10 male Korean Judo national team athletes (NT), 26 male university varsity team athletes (VT), and 28 male junior varsity team athletes (JT) were assessed for 30-second anaerobic power and body composition at the Youngin University. Anaerobic power was measured using a 30-second Wingate test. Body composition was assessed via bioelectric impedance analysis in standardized conditions using BioSpace (Korean)-specific prediction formulas. All testing occurred at the beginning of the winter nonseason period but excluded a brief weight-loss period before the competition phase. Anaerobic power measures were significantly greater (p < 0.05) in NT and VT than in JT. Fat-free mass (FFM), muscle mass (MM), and total body water in JT were also greater than in VT and JT (p < 0.05). Muscle mass in VT was significantly lower than in NT (p < 0.05). Fat-free mass in NT was strongly correlated to mean and peak anaerobic power (r = 0.77, p = 0.009; r = 0.87, p < 0.001, respectively). Varsity team athletes also indicated a moderate association between FFM and peak and mean anaerobic power (r = 0.63, p < 0.001; r = 0.48, p = 0.013, respectively). However, relationship between FFM and anaerobic power was not statistically significantly correlated in JT (r = 0.14, p = 0.470; r = 0.23, p = 0.232, separately). In conclusion, our data indicated that anaerobic power is closely correlated with increase in FFM and MM and was different dependent among performance levels. Further research in the field is warranted to elucidate the Judo-specific relationship between FFM and performance.     

Angiopoietin-1 protects heart against ischemia/reperfusion injury through VE-cadherin dephosphorylation and myocardiac integrin-β1/ERK/caspase-9 phosphorylation cascade

Early reperfusion after myocardial ischemia that is essential for tissue salvage also causes myocardial and vascular injury. Cardioprotection during reperfusion therapy is an essential aspect of treating myocardial infarction. Angiopoietin-1 is an endothelial-specific angiogenic factor. The potential effects of angiopoietin-1 on cardiomyocytes and vascular cells undergoing reperfusion have not been investigated. We propose a protective mechanism whereby angiopoietin-1 increases the integrity of the endothelial lining and exerts a direct survival effect on cardiomyocytes under myocardial ischemia followed by reperfusion. First, we found that angiopoietin-1 prevents vascular leakage through regulating vascular endothelial (VE)-cadherin phosphorylation. The membrane expression of VE-cadherin was markedly decreased on hypoxia/reoxygenation but was restored by angiopoietin-1 treatment. Interestingly, these effects were mediated by the facilitated binding between SH2 domain-containing tyrosine phosphatase (SHP2) or receptor protein tyrosine phosphatase μ (PTPμ) and VE-cadherin, leading to dephosphorylation of VE-cadherin. siRNA against SHP2 or PTPμ abolished the effect of angiopoietin-1 on VE-cadherin dephosphorylation and thereby decreased levels of membrane-localized VE-cadherin. Second, we found that angiopoietin-1 prevented cardiomyocyte death, although cardiomyocytes lack the angiopoietin-1 receptor Tie2. Angiopoietin-1 increased cardiomyocyte survival through integrin-β1-mediated extracellular signal-regulated kinase (ERK) phosphorylation, which inhibited caspase-9 through phosphorylation at Thr12? and subsequently reduced active caspase-3. Neutralizing antibody against integrin-β1 blocked these protective effects. In a mouse myocardial ischemia/reperfusion model, angiopoietin-1 enhanced cardiac function and reduction in left ventricular-end systolic dimension (LV-ESD) and left ventricular-end diastolic dimension (LV-EDD) with an increase in ejection fraction (EF) and fractional shortening (FS). Our findings suggest the novel cardioprotective mechanisms of angiopoietin-1 that are achieved by reducing both vascular leakage and cardiomyocyte death after ischemia/reperfusion injury.     

A DsRed fluorescent protein marker under <Emphasis Type="Italic">polyubiquitin</Emphasis> promoter regulation allows visual and amplified gene detection of transgenic Caribbean fruit flies in field traps

Field population surveillance of a targeted insect pest species is critical in evaluating management programs such as the sterile insect technique. Fluorescent powder dyes currently used to distinguish released tephritids from the field population are not optimal in terms of reliability and human health issues. Genetically transformed tephritid species present the possibility of using fluorescent transgenes for marking. Here we studied the stability of DsRed fluorescence in transgenic flies maintained in aqueous torula yeast borax and propylene glycol. DsRed was stable in both solutions for three weeks by visual microscopic observations and could be used to unambiguously distinguish them from non-fluorescent wild type flies. To compensate for any potential ambiguity in visual identification a diagnostic PCR was developed that could specifically amplify the exotic heterologous marker gene. Therefore, the use of sterile transgenic insect strains carrying stably integrated fluorescent protein marker genes in biologically-based control programs could greatly improve released fly identification in pest management programs.