Identification of genes related to floral organ development in pak choi by expression profiling.

Pak choi is a highly nutritious vegetable that is widely grown in China, Southeast Asia, and other parts of the world. Because it reproduces by seed, it is very important to understand the mechanism of floral organ development. Therefore, using the Chinese cabbage genome as a reference, this study analyzed the expression profiles of shoot apex genes at flower bud differentiation stages 1 and 5, in order to identify genes related to floral organ development. The results showed that the proportion of mapped genes was high, with 84.25 and 83.80% of clean reads from the two sample saligned to the reference genome, respectively. A total of 525 differentially expressed genes (DEGs) were identified, 224 of which were upregulated and 301 were downregulated. The expression levels of genes homologous to Chinese cabbage flowering genes were also analyzed at stages 1 and 5; the expression levels of Bra012997 (ap1), Bra000393 (SOC1), and Bra004928 (SOC1) were significantly upregulated at stage 5, suggesting that these three genes positively regulate floral development in pak choi. DEGs involved in floral organ development were analyzed with homologous genes from Arabidopsis thaliana; the homologous genes Bra029281 (AGL42), Bra026577 (ARPN), Bra022954 (SPL3), Bra029293 (ARF2), Bra007978 (AtRLP12), Bra033221 (SPL8), Bra008037 (LOX4), Bra001598 (IAA19), Bra003892 (PATL1), Bra038778 (AT4G21323), Bra025315 (KLCR2), and Bra013906 (DTX35) are directly related to floral organ development in Arabidopsis, suggesting that these genes have corresponding functions during flower organ development in pak choi, and could be candidates for further genetic research. These results provide a foundation for research on the molecular mechanism of flower organ development in pak choi and other Brassica rapa vegetables.

A 425 T>C polymorphism in complement C7 association with resistance to Aeromonas hydrophila in grass carp.

Aeromonas hydrophila, a widespread bacterium in the aquatic environment, causes hemorrhagic septicemia in fish. In the last decade, the disease has caused mass mortalities and tremendous economic loss in cultured fish. The complement component C7 is a terminal component of complement that interacts in a sequence of polymerization reactions with other terminal complement components to form a membrane attack complex. The formation of the membrane attack complex creates a pore in the membranes of certain pathogen that can lead to their death. The objective of this study was to identify single nucleotide polymorphisms (SNPs) in the C7 gene and to assess their association with A. hydrophila resistance in grass carp. A resource population consisting of 186 susceptible and 191 resistant grass carp was constructed. We sequenced a total of 7826 bp of the C7 gene and identified 6 SNPs that were genotyped in the resource population. The SNP -1575 A>C was positioned in the promoter region of the gene. The SNP 425 C>T identified in the coding exon was a synonymous substitution in the fourth exon. Statistical analysis showed that SNP 425 C>T was associated with the incidence of hemorrhagic septicemia. The SNPs -1575 A>C, -688 T>C, and -266 A>C were highly linked together (r(2) > 0.85). No haplotypes generated with these 3 SNPs were associated with resistance to A. hydrophila in grass carp. These findings suggest that the 425 C>T polymorphism in C7 gene may be a significant molecular marker for resistance to A. hydrophila in grass carp.

Telomere-associated factor expression in replicative senescence of human embryonic lung fibroblasts.

The objective of this study was to find the key regulatory molecules in the cell senescence process through observing the expression of telomere-associated factor during the normal cell replicative senescence process. Based on the established cell replicative senescence model, reverse transcription-polymerase chain reaction and western blot analyses were used to detect telomere-associated factor expression at the mRNA and protein levels, including that of human telomere binding protein 1, tankyrase 1, telomerase RNA, telomere protection protein 1 (POT1), and p53 during the process of human embryonic lung fibroblast replicative senescence. The results showed that transcription of human telomere binding protein 1 did not change with cell senescence, whereas the protein expression of human telomere binding protein 1 increased gradually and then decreased rapidly; there was no change in the mRNA and protein expression of POT1; with the replicative senescence of human embryonic lung fibroblasts, expression of POT1 decreased gradually; TRF1 showed an increasing trend with cell senescence; and p53 protein expression did not change. Together, the results from this study suggest that human telomere binding protein 1, POT1, and TRF1 played important roles in cell senescence.

Characterization of FeDREB1 promoter involved in cold- and drought-inducible expression from common buckwheat (Fagopyrum esculentum).

C-repeat-binding factor (CBF)/dehydration-responsive element (DREB) transcription factors play key roles in plant stress responses. However, little information is available on the regulation of CBF/DREB expression. In this study, we isolated and characterized the FeDREB1 promoter sequence from the common buckwheat accession Xinong 9976. To identify the upstream region of the FeDREB1 gene required for promoter activity, we constructed a series of FeDREB1 promoter deletion derivatives. Each deletion construct was analyzed through Agrobacterium-mediated transient transformation in tobacco leaves treated with 4°C cold or drought stress. Promoter-beta-glucuronidase fusion assays revealed that the pCD1 (-270 bp) deletion in the upstream region of FeDREB1 could activate expression of the GUS gene at 4°C. The pCD1 (-270 bp), pCD2 (-530 bp), and pCD3 (-904 bp) deletion induced low-level GUS expression under drought stress. However, the pCD4 (-1278 bp) deletion clearly activated GUS gene expression. Our results suggest that sections pCD1 (-270 bp) and pCD4 (-1278 bp) in the FeDREB1 gene promoter are new sources of induced promoters for adversity-resistance breeding in plant genetic engineering.

Association of interleukin-1ß -511C/T promoter polymorphism with COPD risk: a meta-analysis.

Studies examining the role of interleukin (IL)-1ß -511C/T promoter polymorphism in the pathogenesis of chronic obstructive pulmonary disease (COPD) have shown inconsistent results. This meta-analysis was performed to assess the association between the IL-1ß-511C/T promoter polymorphism and COPD susceptibility. Published case-control, cross-sectional, and cohort studies from Pubmed, Embase, and China National Knowledge Infrastructure databases were retrieved. Data were extracted and pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated. Twelve studies with 1692 cases and 2009 controls were included in this meta-analysis. Pooled effect size showed an overall but not significantly decreased risk of IL-1ß-511 C/T with COPD susceptibility (OR = 0.89, 95%CI = 0.78-1.01) in a complete overdominant genetic model (TT+CC vs CT), indicating that homozygous individuals (CC and TT) have a decreased risk for COPD compared with heterozygotes (CT). In subgroup analysis by ethnicity, IL-1ß-511C/T was significantly correlated with a decreased risk of COPD in Asians (OR = 0.73, 95%CI = 0.60-0.88, P = 0.001), but not in Caucasians (OR = 1.02, 95%CI = 0.83- 1.24, P = 0.46), confirming a protective role of IL-1ß-511C/T in COPD in Asians. Moreover, after excluding studies that included populations not in Hardy-Weinberg equilibrium, the pooled results were robust and no publication bias was observed. This meta-analysis suggests that the IL-1ß-511C/T promoter polymorphism deceases the risk of COPD in Asians.

Differential expression of interleukin-12 p35 and p40 subunits in response to Aeromonas hydrophila and Aquareovirus infection in grass carp, Ctenopharyngodon idella.

The grass carp (Ctenopharyngodon idella) aquaculture industry in Asia is prone to bacterial and viral hemorrhagic diseases. Effective adjuvants for vaccine formulation are the need of the hour for control of these diseases and long-term sustainability of grass carp farming. In this study, the involvement of interleukin-12 (IL-12) from grass carp (gcIL­12) in anti-bacterial and anti-viral immune responses was demonstrated via expression profiles of gcIL-12 subunits in immune tissues of the fish, following infection by Aeromonas hydrophila and Aquareovirus. Additionally, cDNA of the gcIL-12 subunits, p35 and p40 was cloned and characterized. We found that most of the structurally and functionally important features of vertebrate orthologues were conserved in gcIL-12 subunits, p35 and p40, with some features specific to grass carp. High levels of gcIL-12 p35 expression in the brain and gills suggest that IL-12 plays an important role in neural and immune systems. High expression levels in the heart, blood, and immune-related tissues suggest an important role in circulation and the immune system as well. Infections by both, A. hydrophila and Aquareovirus stimulated the mRNA expression of gcIL-12 subunits, p35 and p40 in most immune tissues. Significant upregulation or downregulation of gcIL-12 subunits, p35 and p40 by bacterial and viral infection confirms their potential role in anti-bacterial and anti-viral immune responses in fish.

Linker length affects expression and bioactivity of the onconase fusion protein in Pichia pastoris.

The aim of this study was to analyze the effect of linker length on the expression and biological activity of recombinant protein onconase (ONC) in fusion with human serum albumin (HSA) in Pichia pastoris. Four flexible linkers with different lengths namely Linker L0, L1: (GGGGS)1, L2: (GGGGS)2, and L3:(GGGGS)3 were inserted into the fusion gene and referred to as HSA-n-ONC, where N = 0, 5, 10, or 15. The sequence of the fusion gene HSA-ONC was designed based on the GC content and codon bias in P. pastoris; the signal peptide of albumin was used as the secretion signal. Gene sequences coding for the fusion protein with different linkers were inserted into pPICZα-A to form recombinant plasmids pPICZα-A/HSA-n-ONC, which were then transformed into P. pastoris X-33 for protein expression. Ideal conditions for expression of the fusion proteins were optimized at a small scale, using shake flasks before proceeding to mass production in 10-L fermenters. The recombinant fusion proteins were purified by aqueous two-phase extraction coupled with DEAE anion exchange chromatography, and their cytotoxic effect on the tumor cell was evaluated by the sulforhodamine B assay. The results showed that the expressed amount of fusion proteins had no significant relationship with the length of different linkers and rHSA-0-ONC had no cytotoxic effect on the tumor cells. While rHSA-5-ONC and rHSA-10-ONC had a weak cytotoxic effect, rHSA-15-ONC could kill various tumor cells in vitro. In summary, the biological activity of the fusion protein gradually improved with increasing length of the linker.

Clinical significance of microRNA-34a in esophageal squamous cell carcinoma.

MicroRNA-34a (miR-34a) has been found to be downregulated in esophageal squamous cell carcinoma (ESCC) tissues compared with that in normal tissues (P < 0.05), and miR-34a overexpression increased apoptosis and decreased clonogenic formation. However, the clinical significance and prognostic value of miR-34a in ESCC has not yet been investigated. In total, 111 patients with ESCC diagnosed and treated at the Department of Thoracic Surgery of Fujian Provincial Hospital between March 2008 and February 2014 were included in this retrospective study. Quantitative real-time PCR was performed to detect expression levels of miR-34a. The associations between miR-34a expression and clinicopathological features were analyzed using χ(2) tests. For analysis of survival data, Kaplan-Meier curves were constructed, and the log-rank test was performed. The expression levels of miR-34a in ESCC tissues were significantly decreased (P < 0.01), compared with those in adjacent normal esophageal tissues. Low miR-34a expression in ESCC tissues was significantly associated with tumor differentiation (P = 0.013), lymph node status (P = 0.038), and advanced clinical stage (P < 0.001). The Kaplan-Meier analysis and log-rank test revealed that low miR-34a levels had a significant impact on overall survival of patients with ESCC (P = 0.006). Multivariate analyses showed that the expression of miR-34a was an independent prognostic factor for ESCC (P = 0.018). Our findings indicate that there is reduced expression of miR-34a in human ESCC tissues and suggest a crucial role for miR-34a downregulation in ESCC progression and prognosis.

Decreased expression of tumor suppressive miR-874 and its clinical significance in human osteosarcoma.

Dysregulation of microRNAs (miRs) is associated with cancer development and progression and aberrant expression of miR-874 have been found in some types of cancer. However, the expression and function of miR-874 in osteosarcoma remain unclear. The aim of this study was to explore the effects of miR-874 in osteosarcoma tumorigenesis and development. The expression level of miR-874 was quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR) in human osteosarcoma cell lines and tissues. Using a miR-874 mimic, cell proliferation and migration assays were performed in an osteosarcoma cell line and tumorigenicity was observed in vivo in order to determine the effects of miR-874 in osteosarcoma cell lines and tissues. MiR-874 was significantly downregulated in osteosarcoma cell lines and clinical specimens. Decreased miR-874 expression was significantly associated with large tumor size, distant metastasis, and advanced clinical stage, and was an independent predictor of poor survival. Overexpression of miR-874 inhibited cell proliferation, invasion and migration in vitro, promoted cell apoptosis in vitro, and suppressed tumorigenicity in vivo. These findings indicate that miR-874 may act as a tumor suppressor in osteosarcoma and could serve as a novel therapeutic agent for miR-based therapy.

Identification of sequence-related amplified polymorphism and insertion-deletion markers linked to the male fertility restorer gene of pol-like CMS06J45 in heading Chinese cabbage (Brassica rapa subsp pekinensis).

In order to map the restorer gene BrRfp of the polima (pol)-like cytoplasmic male sterility (CMS) 06J45 line in heading Chinese cabbage, an F2 segregating population with 258 individuals of CMS06J45 and the restorer line 01S325 were tested by sequence-related amplified polymorphism (SRAP) and insertion-deletion (InDel) technologies combined with the bulked segregant analysis method. As a result, two SRAP markers, me3em3.366 and pm88bg5.263, that were linked with the BrRfp gene were identified from 463 SRAP primer pairs. By cloning, sequencing, and basic local alignment search tool analysis, the two markers were targeted to the BGIScaffold000053 of Brassica rapa in the Brassica database. Using the BGIScaffold000053 sequence, four InDel primer pairs were designed and identified to be linked with the BrRfp gene in this population. Linkage analysis showed that these markers were distributed on both sides of the BrRfp gene, the linkage distances of two nearest markers InDel878.1125 and InDel920.713 were 0.82 and 0.46 cM, respectively, and the BrRfp gene was restricted to a 243-kb genomic region of B. rapa. These specific markers provided basic information for map-based cloning of the BrRfp gene and will be very valuable for the marker-assisted selection of a new restorer line in heading Chinese cabbage.

Unruptured saccular aneurysm presenting migraine.

Headache can be attributed to cranial or cervical vascular disorders including ischemic stroke or transient ischemic attack, non-traumatic intracranial hemorrhage, unruptured vascular malformation, arteritis, carotid-vertebral artery pain, and cerebral venous thrombosis. Here, we present a case report of unruptured saccular aneurysm with migraine. The patient was a previously healthy 32-year-old man with repeated episodes of headache for 6 years. Findings for computed tomography and magnetic resonance imaging of the head were normal. Head magnetic resonance angiography revealed a small, nodule-like protuberance seen in the anterior communicating artery. Based on his clinical features and ancillary examinations, the patient was diagnosed with migraine without aura. The patient subsequently underwent digital subtraction angiography (DSA), which revealed a 2-mm cystic protuberance in the superoposterior anterior communicating artery. The patient underwent stent-assisted coil embolization of the aneurysm. Subsequent DSA results indicated no recurrence of aneurysm and no recurrence of headache was reported after surgical treatment.

Polymorphisms of the angiotensin II type 1 receptor gene affect antihypertensive response to angiotensin receptor blockers in hypertensive Chinese.

The renin-angiotensin-aldosterone system plays a key role in regulating blood pressure by maintaining vascular tone and the water/sodium balance. Many antihypertensive drugs target the renin-angiotensin-aldosterone system, but the effect differs considerably among hypertensive patients. We investigated whether genetic variants of the angiotensin II type 1 receptor are associated with blood pressure response to angiotensin II receptor blockers in hypertensive Chinese patients. After a 2-week single-blind placebo run-in period, 148 patients with mild-to-moderate primary hypertension received monotherapy with 80 mg/day telmisartan and then were followed up for 8 weeks. The 1166A/C, 573T/C, -810A/T, and -521C/T polymorphisms of the AT1R gene were determined through PCR and RFLP analysis. The relationship between these polymorphisms and changes in blood pressure was observed and evaluated after 8 weeks of treatment. Patients with the AT1R -521CC genotype had a significant reduction in diastolic blood pressure compared to those carrying the T allele. No significant reduction in blood pressure was found in individuals with the 1166A/C, 573T/C, or -810A/T polymorphisms of the AT1R gene. We conclude that only the AT1R -521CC genotype is associated with a significant decrease in blood pressure in response to telmisartan treatment in Chinese hypertensive patients.