Overexpression of Arabidopsis ABF3 gene confers enhanced tolerance to drought and heat stress in creeping bentgrass

The Arabidopsis, abscisic acid responsive element-binding factor 3, ABF3 is known to play an important role in stress responses via regulating the expression of stress-responsive genes. In this study, we introduced pCAMBIA3301 vector harboring the ABF3 gene into creeping bentgrass (Agrostis stolonifera) through Agrobacterium-mediated transformation in order to develop a stress-tolerant variety of turfgrass. After transformation, putative transgenic plants were selected using the herbicide resistance assay. Genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by northern blot analysis. Under drought-stressed condition, the transgenic plants overexpressing ABF3 displayed significantly enhanced drought tolerance with higher water content and slower water loss rate than the control plants. Furthermore, the stomata of the ABF3 transgenic plants closed more than those of wild-type creeping bentgrass plants, under both non-stressed and ABA treatment conditions. In addition, the transgenic plants showed enhanced tolerance to heat stress. These results suggest that the overexpression of the ABF3 gene in creeping bentgrass might enhance survival in water-limiting and high temperature environments through increased stomatal closure and reduced water losses.     

Development of cyan fluorescent protein (CFP) reporter system in green alga Chlamydomonas reinhardtii and macroalgae Pyropia sp.

Various fluorescent proteins have been developed for in vivo reporter systems in diverse prokaryotes and eukaryotes. However, few in vivo imaging systems have been reported for the model algae Chlamydomonas reinhardtii or Pyropia sp. In this study, an effective imaging system using cyan fluorescent protein (CFP) was developed for the green alga C. reinhardtii, and its application was also successful in the red macroalgae Pyropia tenera and P. yezoensis. For optimization of CFP expression in C. reinhardtii and Pyropia sp., we modified codon usage in the CFP gene (CFP), generating PtCrCFP (Pyropia tenera/Chlamydomonas reinhardtii CFP). PtCrCFP was successfully expressed in PtCrCFP-expressing UVM11 transgenic lines, and high accumulation levels of PtCrCFP were found by western blotting. Consistent with these results, PtCrCFP fluorescence was clearly detected with a low level of chlorophyll background fluorescence in PtCrCFP-expressing UVM11 transgenic lines. In Pyropia sp. gametophytic cells, transient expression of PtCrCFP fluorescence was distinctly visualized. PtCrCFP fluorescence was also observed during the regeneration of monospores and young gametophytes from PtCrCFP-expressing P. yezoensis gametophytic cells. These results suggest that PtCrCFP may be useful as an in vivo reporter in green algae due to the short emission wavelength of CFP, which provides a low level of chlorophyll background fluorescence. This study also presents the possibility of PtCrCFP’s use as a visible selection marker for the generation of transgenic lines in the red algae Pyropia sp. Thus, PtCrCFP as an in vivo visualization tool may offer new opportunities for the functional analysis of genetic studies in both green and red algae.     

Effects of GC Bias in Next-Generation-Sequencing Data on De Novo Genome Assembly

Next-generation-sequencing (NGS) has revolutionized the field of genome assembly because of its much higher data throughput and much lower cost compared with traditional Sanger sequencing. However, NGS poses new computational challenges to de novo genome assembly. Among the challenges, GC bias in NGS data is known to aggravate genome assembly. However, it is not clear to what extent GC bias affects genome assembly in general. In this work, we conduct a systematic analysis on the effects of GC bias on genome assembly. Our analyses reveal that GC bias only lowers assembly completeness when the degree of GC bias is above a threshold. At a strong GC bias, the assembly fragmentation due to GC bias can be explained by the low coverage of reads in the GC-poor or GC-rich regions of a genome. This effect is observed for all the assemblers under study. Increasing the total amount of NGS data thus rescues the assembly fragmentation because of GC bias. However, the amount of data needed for a full rescue depends on the distribution of GC contents. Both low and high coverage depths due to GC bias lower the accuracy of assembly. These pieces of information provide guidance toward a better de novo genome assembly in the presence of GC bias.     

Gene Expression Profiling of Ampullary Carcinomas Classifies Ampullary Carcinomas into Biliary-Like and Intestinal-Like Subtypes That Are Prognostic of Outcome

Background

Adenocarcinomas of the ampulla of Vater are classified as biliary cancers, though the exact epithelium of origin for these cancers is not known. We sought to molecularly classify ampullary adenocarcinomas in comparison to known adenocarcinomas of the pancreas, bile duct, and duodenum by gene expression analysis.

Methods

We analyzed 32 fresh-frozen resected, untreated periampullary adenocarcinomas (8 pancreatic, 2 extrahepatic biliary, 8 duodenal, and 14 ampullary) using the Affymetrix U133 Plus 2.0 genome array. Unsupervised and supervised hierarchical clustering identified two subtypes of ampullary carcinomas that were molecularly and histologically characterized.

Results

Hierarchical clustering of periampullary carcinomas segregated ampullary carcinomas into two subgroups, which were distinctly different from pancreatic carcinomas. Non-pancreatic periampullary adenocarcinomas were segregated into two subgroups with differing prognoses: 5 year RFS (77% vs. 0%, p = 0.007) and 5 year OS (100% vs. 35%, p = 0.005). Unsupervised clustering analysis of the 14 ampullary samples also identified two subgroups: a good prognosis intestinal-like subgroup and a poor prognosis biliary-like subgroup with 5 year OS of 70% vs. 28%, P = 0.09. Expression of CK7+/CK20- but not CDX-2 correlated with these two subgroups. Activation of the AKT and MAPK pathways were both increased in the poor prognostic biliary-like subgroup. In an independent 80 patient ampullary validation dataset only histological subtype (intestinal vs. pancreaticobiliary) was significantly associated with OS in both univariate (p = 0.006) and multivariate analysis (P = 0.04).

Conclusions

Gene expression analysis discriminated pancreatic adenocarcinomas from other periampullary carcinomas and identified two prognostically relevant subgroups of ampullary adenocarcinomas. Histological subtype was an independent prognostic factor in ampullary adenocarcinomas.     

Risk Factors for Progressive Visual Field Loss in Primary Angle-Closure Glaucoma: A Retrospective Cohort Study

Purpose

To investigate risk factors associated with progressive visual field (VF) loss in primary angle closure glaucoma (PACG).

Methods

We retrospectively reviewed medical record of PACG patients who had ≥5 reliable VF examinations (central 24-2 threshold test, Humphrey Field Analyzer) and ≥2 years of follow-up. Each VF was scored using Collaborative Initial Glaucoma Treatment Study system. Progression was defined if 3 consecutive follow-up VF tests had an increased score of ≥3 above the mean of the first 2 VF scores. Factors associated with VF progression were evaluated by Cox proportional hazards models.

Results

A total of 89 eyes from 89 patients (mean age, 69.8 ± 7.9 years), who received a mean of 6.9 ± 2.3 VF tests (mean deviation at initial, -8.1 ± 4.4 dB) with a mean follow-up of 63.9 ± 23.9 months were included. VF progression was detected in 9 eyes (10%). The axial length (AL), anterior chamber depth, and intraocular pressure (IOP) in patients with and without progression were 22.5 ± 0.6 and 23.1 ± 0.9 mm, 2.5 ± 0.3 and 2.5 ± 0.3 mm, 14.8 ± 2.4 and 14.3 ± 2.3 mm Hg, respectively. AL was the only factor associated with progression in both Cox proportional hazards univariate (p = 0.031) and multivariate models (p = 0.023).

Conclusion

When taking into account age, IOP, follow-up period, and number of VF tests, a shorter AL is the only factor associated with VF progression in this cohort of Chinese patients with PACG. Further studies are warranted to verify the role of AL in progressive VF loss in PACG.     

A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP^32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP^32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP^32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP^32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP^32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP^32–41 internalization routes. ECP^32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.     

Sarcopenia Is Independently Associated with Cardiovascular Disease in Older Korean Adults: The Korea National Health and Nutrition Examination Survey (KNHANES) from 2009

Background

The association between sarcopenia and cardiovascular disease (CVD) in elderly people has not been adequately assessed. The aim of this study was to investigate whether CVD is more prevalent in subjects with sarcopenia independent of other well-established cardiovascular risk factors in older Korean adults.

Method

This study utilized the representative Korean population data from the Korea National Health and Nutrition Examination Survey (KNHANES) which was conducted in 2009. Subjects older than 65 years of age with appendicular skeletal muscle mass (ASM) determined by dual energy X-ray absorptiometry were selected. The prevalence of sarcopenia in the older Korean adults was investigated, and it was determined whether sarcopenia is associated with CVD independent of other well-known risk factors.

Results

1,578 subjects aged 65 years and older with the data for ASM were selected, and the overall prevalence of sarcopenia was 30.3% in men and 29.3% in women. Most of the risk factors for CVD such as age, waist circumference, body mass index, fasting plasma glucose and total cholesterol showed significant negative correlations with the ratio between appendicular skeletal muscle mass and body weight. Multiple logistic regression analysis demonstrated that sarcopenia was associated with CVD independent of other well-documented risk factors, renal function and medications (OR, 1.768; 95% CI, 1.075–2.909, P = 0.025).

Conclusions

Sarcopenia was associated with the presence of CVD independent of other cardiovascular risk factors after adjusting renal function and medications.     

Genetic Variation in the NOC Gene Is Associated with Body Mass Index in Chinese Subjects

Circadian clock genes are critical regulators of energy homeostasis and metabolism. However, whether variation in the circadian genes is associated with metabolic phenotypes in humans remains to be explored. In this study, we systemically genotyped 20 tag single nucleotide polymorphisms (SNPs) in 8 candidate genes involved in circadian clock, including CLOCK, BMAL1(ARNTL), PER1, PER2, CRY1, CRY2, CSNK1E,, and NOC(CCRN4L) in 1,510 non-diabetic Chinese subjects in Taipei and Yunlin populations in Taiwan. Their associations with metabolic phenotypes were analyzed. We found that genetic variation in the NOC gene, rs9684900 was associated with body mass index (BMI) (P = 0.0016, Bonferroni corrected P = 0.032). Another variant, rs135764 in the CSNK1E gene was associated with fasting glucose (P = 0.0023, Bonferroni corrected P = 0.046). These associations were consistent in both Taipei and Yunlin populations. Significant epistatic and joint effects between SNPs on BMI and related phenotypes were observed. Furthermore, NOC mRNA levels in human abdominal adipose tissue were significantly increased in obese subjects compared to non-obese controls.

Conclusion

Genetic variation in the NOC gene is associated with BMI in Chinese subjects.     

Divergent Selection and Local Adaptation in Disjunct Populations of an Endangered Conifer,Keteleeria davidiana var. formosana (Pinaceae)

The present study investigated the genetic diversity, population structure, F _ST outliers, and extent and pattern of linkage disequilibrium in five populations of Keteleeria davidiana var. formosana, which is listed as a critically endangered species by the Council of Agriculture, Taiwan. Twelve amplified fragment length polymorphism primer pairs generated a total of 465 markers, of which 83.74% on average were polymorphic across populations, with a mean Nei’s genetic diversity of 0.233 and a low level of genetic differentiation (approximately 6%) based on the total dataset. Linkage disequilibrium and HICKORY analyses suggested recent population bottlenecks and inbreeding in K. davidiana var. formosana. Both STRUCTURE and BAPS observed extensive admixture of individual genotypes among populations based on the total dataset in various clustering scenarios, which probably resulted from incomplete lineage sorting of ancestral variation rather than a high rate of recent gene flow. Our results based on outlier analysis revealed generally high levels of genetic differentiation and suggest that divergent selection arising from environmental variation has been driven by differences in temperature, precipitation, and humidity. Identification of ecologically associated outliers among environmentally disparate populations further support divergent selection and potential local adaptation.