[Association among urinary polycyclic aromatic hydrocarbons metabolites, SAHH activity and H19 expression in coke oven workers].

Objective: To investigate the relationship of polycyclic aromatic hydrocarbons (PAHs) exposure, S-adenosylhomocysteine hydrolase (SAHH) activity and long noncoding RNA H19 gene expression in the urine of coke oven workers. Methods: In September 2019, in a coking plant in Taiyuan City, 146 male workers who had worked in coke oven operations for one year were selected through a completely random sampling method, and their basic personal information was collected by questionnaire survey, and blood and urine samples were collected. The levels of 4 PAHs metabolites 2-hydroxfluorene (2-FLU), 2- hydroxynaphthalene (2-NAP), 9-hydroxyphenanthren (9-PHE), and 1-hydroxypyrene (1-OHP) in urine were detected by high performance liquid chromatography (HPLC) -fluorescence detection method. HPLC-UV detection method was used to detect the content of S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) in plasma, and the SAHH activity value was obtained by calculating the ratio. Reverse transcription PCR method was used to determine the H19 gene expression level. Urine levels of 2-FLU, 2-NAP, 9-PHE, and 1-OHP were divided into Q(1), Q(2), Q(3), and Q(4) groups according to quartiles (P(25), P(50), P(75)). Regression, trend test and restricted cubic splines were used to analyze the relationship among PAHs metabolites, SAHH activity, H19 gene expression and their dose-response. Results: The median age of coke oven workers was 39.60 years old, the median length of service was 20.38 years, and the urinary levels of 2-FLU, 2-NAP, 9- PHE, and 1-OHP were 0.29, 0.74, 0.09, and 0.06 µg/mmol Cr, respectively. The levels of 2-FLU, 2-NAP and 9-PHE in the urine of workers were significantly different between groups with different 1-OHP levels (P<0.05). After adjusting for age, length of service, smoking, drinking, and levels of 2-FLU, 2-NAP and 9-PHE, SAHH activity decreased with the increase of urinary 1-OHP level (OR=0.63, 95%CI: 0.41-0.98, P=0.038), showing a nonlinear relationship (P(nonlinear)= 0.030). H19 gene expression increased with the increase of urinary 1- OHP level (OR=1.51, 95%CI: 1.03-2.19, P=0.033), there was a linear relationship (P(trend)= 0.058). The relationship between the other three metabolites in urine and SAHH activity and H19 gene expression was not statistically significant (P>0.05) . Conclusion: Urinary 1-OHP level may be a risk factor for decreased SAHH activity and increased H19 gene expression in coke oven workers.

miRNA expression analysis of the sheep follicle during the prerecruitment, dominant, and mature stages of development under FSH stimulation.

Follicle stimulating hormone (FSH) has been widely reported to influence ovarian follicular development, and miRNAs play a significant role in mammalian follicular development by regulating their target genes. Therefore, it is of interest to explore the roles of miRNAs in sheep follicular development during FSH stimulation. In the current study, we constructed miRNA expression profiles of small follicles (SFs, prerecruitment stage), medium follicles (MFs, dominance stage), and large follicles (LFs, maturation stage). Three and 50 significant differentially expressed miRNAs (DEMs) were identified in the MF vs. SF and LF vs. SF comparisons, respectively, and none were identified in the LF vs. MF comparison. Oar-miR-10a was significantly downregulated in MFs compared with SFs. In LFs compared with SFs, miR-212-3p, miR-212-5p and miR-202-5p were significantly upregulated, and miR-27a-3p, miR-181a-5p, miR-204-5p, and miR-182-5p were significantly downregulated. Furthermore, we predicted the target genes of significant DEMs and performed functional enrichment analyses of these target genes. Analyses of KEGG pathways and GO terms showed that the putative target genes were significantly enriched in ovarian steroidogenesis, glutathione metabolism, positive regulation of cell differentiation, positive regulation of cell development, and cellular response to oxygen-containing compounds. Analyses of miRNA-gene regulatory networks suggested that miR-181a-5p-CYP11A1, (miR-27a-3p and miR-129-5p)-LDLR, (miR-212-3p and miR-212-5p)-EFNA5, (miR-181a-5p, miR-182-5p, and miR-27a-3p)-INHBA, and miR-182-5p-SOD2 might be involved in follicular development. The present study provides basic data and suggests research directions for further exploration of the roles of miRNAs in sheep follicular development under FSH stimulation.

Transcriptome analysis of sheep follicular development during prerecruitment, dominant, and mature stages after FSH superstimulation.

Sheep is usually a monovular animal; superovulation technology is used to increase the number of offspring per individual and shorten generation intervals. To date, mature FSH superstimulatory treatments have been successfully used in sheep breeding, but much remains unknown about genes, pathways, and biological functions involved in follicular development. Therefore, in this study, we performed transcriptome profiling of small follicles (SFs; 2-2.5 mm), medium follicles (MFs; 3.5-4.5 mm), and large follicles (LFs; > 6 mm) in Mongolian ewes after FSH superstimulation. Furthermore, we identified differentially expressed genes and performed Kyoto Encyclopedia of Genes and Genomes pathway and Gene Ontology enrichment analyses in 3 separate pairwise comparisons. We found that ovarian steroidogenesis was significantly enriched in the SFs versus MFs analysis; the associated genes, cytochrome P450 family 19 (CYP19) and Hydroxy-delta-5-steroid dehydrogenase 3 beta- and steroid delta-isomerase 1 (HSD3B1), were significantly upregulated. Moreover, proline metabolism, glutathione metabolism, and PPAR signaling pathways were significantly enriched in the LFs versus SFs analysis; the associated genes, glutamate-cysteine ligase modifier subunit (GCLM) and cystathionine gamma-lyase (CTH), were significantly upregulated, whereas peroxisome proliferator-activated receptor gamma (PPARγ) was significantly downregulated. In summary, our study provides basic data and possible biological direction to further explore the molecular mechanism of sheep follicular development after FSH superstimulation.

Long noncoding RNA SNHG14 accelerates cell proliferation, migration, invasion and suppresses apoptosis in colorectal cancer cells by targeting miR-944/KRAS axis through PI3K/AKT pathway.

OBJECTIVE: Colorectal cancer (CRC) is a gastrointestinal tract cancer, which threatens the well-being of million of patients due to high metastasis. Recently, numerous studies have recognized nuclear RNA host gene 14 (SNHG14) as a remarkable oncogene in different cancers. However, the regulatory mechanism of SNHG14 in CRC development is mostly unclear. PATIENTS AND METHODS: The expression of SNHG14, miR-944 and Kirsten rat sarcoma (KRAS) in tissues and cells was measured by quantitative Real-time polymerase chain reaction (qRT-PCR). Cell viability and apoptosis were evaluated by cell counting kit-8 (CCK-8) and flow cytometry assay, respectively. Cell migration and invasion were assessed using transwell assay. Protein expression of KRAS, AKT, phosphorylated AKT (p-AKT), phosphatidylinositol-3-kinase (PI3K) and phosphorylated PI3K (p-PI3K) was detected by Western blot. Animal models were constructed by subcutaneously injecting SW620 cells stably transfected with sh-SNHG14 and sh-NC. The interaction among SNHG14, miR-944 and KRAS was determined by luciferase reporter assay and RIP assay. RESULTS: The expression of SNHG14 and KRAS was up-regulated whereas miR-944 was down-regulated in CRC tumors and cells compared with normal tissues and cells. In addition, SNHG14 silencing attenuated cell proliferation, migration and invasion, while accelerated apoptosis in CRC cells by suppressing PI3K/AKT pathway. Consistently, SNHG14 knockdown hindered tumor growth in vivo. MiR-944 was a target of SNHG14 and directly targeted KRAS. Moreover, miR-944 inhibitor abrogated silenced SNHG14-mediated inhibition on proliferation, migration and invasion, as well as promotion on apoptosis in CRC cells. Similarly, miR-944 regulated CRC cell progression by targeting KRAS through PI3K/AKT pathway. CONCLUSIONS: SNHG14 contributed to cell proliferation, migration and invasion, while suppressed apoptosis in CRC cells by targeting miR-944/KRAS axis through PI3K/AKT pathway, representing novel biomarkers for CRC therapy.

Overexpression of miR-770 indicates a favorable prognosis and suppresses tumorigenesis by modulating PI3K-AKT pathway in glioma.

OBJECTIVE: Previous studies showed that miR-770 expression was deregulated in many tumors. However, the effect of miR-770 function on glioma remains as a mystery. The present study aimed to explore its expression, cellular function and clinic features in glioma. PATIENTS AND METHODS: We analyzed RNA sequencing data to explore abnormally expressed miRNAs in glioma. Glioma tissue specimens and their matched normal tissues were collected to test miR-770 expression using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) analysis. The correlation between miR-770 and the clinicopathological factors and the prognostic value of miR-770 was statistically analyzed. We then investigated alterations in a series of cancer-related phenotypes, including cell viability, apoptosis, colony formation and metastasis capacities. Western blot analysis was performed to examine the expression changes of EMT-related proteins and PI3K/Akt signaling pathway proteins. RESULTS: We identified a novel glioma-related miRNA miR-770, which was significantly down-regulated in human glioma tissues. The results of RT-PCR further showed that miR-770 expression was significantly down-regulated in both glioma tissues and cell lines. Furthermore, decreased miR-770 expression was significantly associated with advanced WHO grade, KPS score and shorter five-year overall survival. Then, functional assays indicated that overexpression of miR-770 suppressed proliferation, migration, invasion and EMT pathway, and induced the apoptosis of glioma cells in vitro. Moreover, we further illustrated that the up-regulation of miR-770 suppressed the PI3K-AKT signaling pathway. CONCLUSIONS: Our present findings firstly reported the roles and mechanisms associated with miR-770 in glioma progression, highlighting miR-770 as a potential therapeutic target for glioma patients.

[Prevalence of dyslipidemia and risk factors among coal miners under different work conditions].

Objective: To understand the prevalence of dyslipidemia and risk factors among coal miners under different work conditions. Methods: The survey was conducted from April 2016 to June 2016. 759 mine workers were divided into three groups (group of the front line miner, underground auxiliary and ground) . Questionnaire and physical examination were used to collect related information of workers. Logistic regression model was used to analyze relative factors. Results: The overall prevalence of dyslipidemia was 43.2% in coal miners. The prevalence rate of the front line miner and underground auxiliary miners was 46.6%. Ground workers had the lowest prevalence rate of 36.4%. Multiple Logistic regression analysis showed that higher body mass index (BMI) was risk factors for underground workers (OR=2.18, 95%CI:1.51~3.13) . Smoking (OR=1.99, 95%CI:1.17~3.38) , drinking (OR=1.85, 95%CI:1.11~3.06) , hypertension (OR=1.79, 95%CI:1.00~3.22) and higher waist and hip ratio (OR=1.06, 95%CI:1.04~1.09) were risk factors for underground auxiliary workers. For ground workers, those with higher BMI (OR=2.64, 95%CI:1.68~4.16) were at higher risk of dyslipidemia and female workers had lower risk (OR=0.35, 95%CI:0.18~0.65) than male workers. Conclusion: The dyslipidemia rate of coal mine workers is related to work environment and behavior. Health education may be needed to reduce the dyslipidemia rate of coal mine workers.

[Correlative factors on prevalence rate of dislipidemia among 1 337 coal miners in Shanxi province].

Objective: To understand the prevalence rate and correlative factors of dislipidemia among Shanxi coal miners and to provide evidence for the development of programs on dislipidemia prevention. Methods: We investigated 1 337 mine workers from a Coal Group in April 2016 and collected data related to their blood biochemistry. We then classified the types in accordance with the diagnostic criteria of " Guidelines for prevention and treatment of dyslipidemia in Chinese adults (2007)" , using χ(2) test and unconditional logistic regression model for analysis. Results: The overall prevalence rate of Dislipidemia was 59.1% (790/1 337), with males as 60.4% (708/1 173) and females as 50.0%(82/164) while males appeared higher (χ(2)=6.386, P<0.05). Among the 20-34, 35-49, 50 and above year-old groups, the rates were 68.8%, 58.7%, 49.5%, respectively. Results from the χ(2) test showed that gender, age and body mass index were the influencing factors on dislipidemia (χ(2)=7.117, P<0.01; χ(2)=37.135, P<0.01; χ(2)=7.009, P<0.05), while logistic regression analysis showed that sex, age, body mass index level, systolic blood pressure were significantly associated with dislipidemia (P<0.05). Male miners appeared 1.501 times (OR=1.501, 95%CI: 1.895-2.516) higher than female miners in suffering from the risk of dyslipidemia. In different age groups, risks of dyslipidemia in the 35-49, 20-34 year-old groups were 1.672 (OR=1.672, 95%CI: 1.501-2.392) and 2.369 times (OR= 2.369, 95% CI: 1.275-3.469) higher than the 50 year-old. Group that with high BMI, the risk of dyslipidemia was 1.443 times (OR=1.443, 95%CI: 1.139-1.828) higher than the normal BMI group. Group with abnormal systolic pressure was 1.829 times (OR=1.829, 95%CI: 1.152-2.906) higher than normal systolic pressure group. However, diastolic blood pressure, blood sugar, uric acid, and electrocardiogram findings did not seem to show statistically significant meanings on dislipidemia. Conclusion: Among the coal mine workers, those who were males, aged from 20 to 34, having high blood pressure (systolic blood pressure abnormalities) or with high BMI (≥24.0 kg/m(2)) need to be taken special attention on care and prevention of dislipidemia.

[A bibliometric analysis of literature on hand-transmitted vibration in China, 1990-2016].

Objective: To investigate the features of literature on hand-transmitted vibration in China, 1990-2016. Methods: In September 2017, the studies on hand-transmitted vibration in China, which were published in Chinese or English during 1990-2016, with "China" and "Taiwan" as the places where author affiliations were located, were retrieved. A bibliometric analysis was performed to investigate the type of articles, publication time, the journals in which articles were published, author affiliations, author regions, and funding. Results: A total of 205 articles on hand-transmitted vibration were retrieved. There were 7.59 articles on average published annually from 1990 to 2016. In the 205 articles, 114 (55.61%) were published in the journals indexed in one or two core journal databases. In the 64 journals, 22 (34.38%) were indexed in one or two core journal databases. The first authors were from 22 provincial regions (provinces, autonomous regions, or centrally administered municipalities) in China, with 152 articles (74.15%) by the authors in the top five regions. There were a total of 876 authors, and the co-authorship degree was 4.27 (876/205). Most of the first authors (136 articles, 66.34%) were affiliated with universities or institutes for prevention and control of occupational diseases. Among the 205 articles, 103 (50.24%) were original articles or investigations, and 72 (35.12%) were funded. Conclusion: The studies on hand-transmitted vibration fluctuated and increased from 1990 to 2016, with a relatively concentrated distribution in terms of sources, regions, and institutions. Interregional and international academic exchange should be strengthened.

Effect of melatonin supplementation on cryopreserved sperm quality in mouse.

BACKGROUND: Antioxidants protect spermatozoa against cell damage during cryopreservation. OBJECTIVE: To investigate whether melatonin supplement in the extender may improve the quality of cryopreserved mouse sperm. METHODS: Kunming mice sperm frozen in extender R18S3 (18% (w/v) raffinose and 3% (w/v) skim milk) supplemented with melatonin were thawed and evaluated. RESULTS: Mouse spermatozoa were cryopreserved in the freezing extender R18S3 that contained melatonin at 0, 0.125, 0.25 and 0.5 mg/mL melatonin. The extender without melatonin supplement was associated with increased formation of reactive oxygen species (ROS) and decreased sperm motility. Melatonin supplement at 0.125 mg/mL significantly increased the progressive motility of sperm in comparison to other melatonin concentration or control. The percentage of thawed viable sperm with ROS was lower in the melatonin-treated groups than in untreated group. Melatonin supplement also increased antiapoptotic gene Bcl-xl expression in the thawed sperm. CONCLUSION: Supplement of 0.125 mg/mL melatonin could reduce oxidative damage and apoptosis.

Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression.

Disturbed cell autophagy is found in various cardiovascular disease conditions. Biomechanical stimuli induced by laminar blood flow have important protective actions against the development of various vascular diseases. However, the impacts and underlying mechanisms of shear stress on the autophagic process in vascular endothelial cells (ECs) are not entirely understood. Here we investigated the impacts of shear stress on autophagy in human vascular ECs. We found that shear stress induced by laminar flow, but not that by oscillatory or low-magnitude flow, promoted autophagy. Time-course analysis and flow cessation experiments confirmed that this effect was not a transient adaptive stress response but appeared to be a sustained physiological action. Flow had no effect on the mammalian target of rapamycin-ULK pathway, whereas it significantly upregulated Sirt1 expression. Inhibition of Sirt1 blunted shear stress-induced autophagy. Overexpression of wild-type Sirt1, but not the deacetylase-dead mutant, was sufficient to induce autophagy in ECs. Using both of gain- and loss-of-function experiments, we showed that Sirt1-dependent activation of FoxO1 was critical in mediating shear stress-induced autophagy. Shear stress also induced deacetylation of Atg5 and Atg7. Moreover, shear stress-induced Sirt1 expression and autophagy were redox dependent, whereas Sirt1 might act as a redox-sensitive transducer mediating reactive oxygen species-elicited autophagy. Functionally, we demonstrated that flow-conditioned cells are more resistant to oxidant-induced cell injury, and this cytoprotective effect was abolished after inhibition of autophagy. In summary, these results suggest that Sirt1-mediated autophagy in ECs may be a novel mechanism by which laminar flow produces its vascular-protective actions.

Cloning and expression analysis of terpenoid metabolism-related gene NsyCMS in Nicotiana sylvestris.

Terpenoids constitute the main class of secondary metabolites produced in plants with industrial, pharmacological, and agricultural interests. Nicotiana sylvestris has been widely adopted as a diploid model system in plant biology for studies of terpenoid biosynthesis. In this paper, we report the isolation and analysis of the 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (CMS) gene of the MEP (methylerythritol 4-phosphate) pathway from N. sylvestris. We used homologous-based cloning with a RACE method to obtain the full-length coding sequence of the NsCMS. Then, the physical and chemical properties, function, and three-dimensional structure of the NsyCMS protein were predicted. Fluorogenic quantitative PCR was used to conduct an expression analysis at different developmental stages of various tissues of the NsyCMS. The sequence of the NsyCMS consists of a 954-bp open reading frame and encodes a predicted protein of 317 amino acids, with a molecular weight of approximately 49.6 kDa and pi of 6.92. The in vivo localization of the encoded protein was cytoplasmic with no signal peptide, whereas 2 transmembrane regions were found in NsyCMS. The conserved domains of typical 2-C-methyl-d-erythritol 2,4-cyclodiphosphate synthase, aminotransferase, and pyridoxal phosphate-dependent transferase were found in NsyCMS. Differential expression patterns of the NsyCMS were observed throughout the different developmental stages and tissues. NsyCMS messenger RNA was expressed in all tissues, with the highest level of expression in the seedling leaves. NsyMK was expressed at a higher level in the resettling roots. The results from our study set the foundation for exploring the terpenoid biosynthetic pathways in N. sylvestris.

Paclitaxel and gemcitabine combinational drug-loaded mucoadhesive delivery system in the treatment of colon cancers.

The combination of two different types of chemo-therapeutic drugs via nanocarriers is emerged as a promising strategy for treating multiple cancers. Such a co-delivery system will synchronize the drug exposure and synergize the therapeutic effects. Herein, we prepared a paclitaxel (PTX) and gemcitabine (GEM)-loaded N-succinyl chitosan nanoparticles (NSC NP) to target colon cancer. NSC NP showed a pH sensitive swelling at colonic pH and exhibited a sequential release pattern for both the drugs. Binary drug combination exhibited a synergistic cytotoxicity against HT-29 colon cancer cells with a remarkable G2/M phase arrest. Specifically, in vivo antitumor efficacy study showed that NSC NP prolonged the survival time of tumor-bearing mice up to 45 days wherein 50% of mice were still alive. Therefore, these results suggest that co-delivery of drugs with a suitable delivery system could potentially improve the therapeutic efficacy in colon cancers. The study can be further continued by using different types of chemotherapeutic drugs that targets different molecular targets using pH-sensitive nanocarriers.

Identification of apple cultivars on the basis of simple sequence repeat markers.

DNA markers are useful tools that play an important role in plant cultivar identification. They are usually based on polymerase chain reaction (PCR) and include simple sequence repeats (SSRs), inter-simple sequence repeats, and random amplified polymorphic DNA. However, DNA markers were not used effectively in the complete identification of plant cultivars because of the lack of known DNA fingerprints. Recently, a novel approach called the cultivar identification diagram (CID) strategy was developed to facilitate the use of DNA markers for separate plant individuals. The CID was designed whereby a polymorphic maker was generated from each PCR that directly allowed for cultivar sample separation at each step. Therefore, it could be used to identify cultivars and varieties easily with fewer primers. In this study, 60 apple cultivars, including a few main cultivars in fields and varieties from descendants (Fuji x Telamon) were examined. Of the 20 pairs of SSR primers screened, 8 pairs gave reproducible, polymorphic DNA amplification patterns. The banding patterns obtained from these 8 primers were used to construct a CID map. Each cultivar or variety in this study was distinguished from the others completely, indicating that this method can be used for efficient cultivar identification. The result contributed to studies on germplasm resources and the seedling industry in fruit trees.

Breed-specific transcriptome response of spleen from six to eight week old piglet after infection with Streptococcus suis type 2.

Different pig breeds have shown differential susceptibility to the pathogen infection; however, molecular mechanisms of the infection susceptibility are not fully understood. Streptococcus suis type 2 (SS2) is an important zoonotic pathogen. To identify the genes responsible for infection susceptibility, pigs from two different breeds (Enshi black and Landrace) were inoculated with SS2 and their spleen transcriptome profiles were investigated in the present study. The differentially expressed genes (DEGs) were analyzed from infected versus control pigs in each breed, and then compared between both pig breeds. Enshi black pig showed more DEGs than Landrace (830 vs. 611) and most of these were due to down-regulated genes (543 vs. 387). However some DEGs were uniquely expressed in one breed, some were expressed in opposite direction in both breeds. A number of candidate genes and pathways are identified which might be involved in susceptibility to SS2, for example, MMP9 and Resistin were only significantly expressed in Landrace. NPG3 and PMAP23 were up-regulated in Landrace whereas down-regulated in Enshi black. LENG8 in control Landrace have inherently higher expression than control Enshi black. IGKV6 is down-regulated in Landrace but up-regulated in Enshi black. Overall, the transcriptome profiles are consistent with the clinical signs, i.e. the Enshi black is more susceptible to SS2 infection than Landrace. This is the first study to identify differential gene expression between indigenous and modern commercial pigs after in vivo SS2 infection using RNA-seq. The significant DEGs in splenic profiles between two pig breeds suggested considerable involvement of genetic background in susceptibility to the SS2 infection in pigs.

Characterization of EST-derived and non-EST simple sequence repeats in an F1 hybrid population of Vitis vinifera L.

Among different classes of molecular markers, expressed sequence tags (ESTs) are a new resource for developing simple sequence repeat (SSR) functional markers for genotyping and genetic mapping in F1 hybrid populations of Vitis vinifera L. Recently, because of the availability of an enormous amount of data for ESTs in the public domain, the emphasis has shifted from genomic SSRs to EST-SSRs, which belong to transcribed regions of the genome and may have a role in gene expression or function. The objective of this study was to assess the polymorphisms among 94 F1 hybrids from "Early Rose" and "Red Globe" using 25 EST-derived and 25 non-EST SSR markers. A total collection of 362,375 grape ESTs that were retrieved from the National Center for Biotechnology Information (NCBI) and 2522 EST-SSR sequences were identified. From them, 205 primer pairs were randomly selected, including 176 pairs that were EST-derived and 29 non-EST SSR primer pairs, for polymerase chain reaction amplification. A total of 131 alleles were amplified using 50 pairs of primers; 78 alleles were amplified using EST-derived SSR primers and 53 were from non-EST SSR primers. At most, 6 and 5 alleles were amplified by EST-derived and non-EST SSR primers, respectively. The EST-derived SSR markers showed a maximum polymorphic information content (PIC) value of 1 and a minimum of 0.33 while non-EST SSR markers had maximum and minimum PIC values of 1 and 0.25, respectively. The average PIC value was 0.56 for EST-derived SSR markers and 0.45 for non-EST SSR markers.

Homology-based cloning and expression analysis of Rf genes encoding PPR-containing proteins in tobacco.

As a model plant, mechanisms of the cytoplasmic male sterility/restoration of fertility (CMS/Rf) system in tobacco are seldom studied. Using Rf gene sequences from other Solanaceae plants and the draft genome of Nicotiana benthamiana, degenerate primers were designed to amplify the cDNA pool of N. tomentosiformis. In total, six possible Rf sequences were identified, two of which contained base-deletion mutations. The other four were intact open reading frames, of which NtomPPR5 harbored a 3-pentatricopeptide repeat (PPR) motif deletion. Structure analysis revealed that they all encoded a PPR-containing protein with putative mitochondrial targeting signals at their N-terminus, and they all belong to the P subfamily. Phylogenetic analysis showed that all of the Rf-coding PPRs clustered together, and recent duplication events might have occurred in tobacco after the divergence of the species. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that the NtomRfs were expressed in all tissues of N. tomentosiformis and (CMS) K326, although the expression levels varied with gene, organ, and developmental stage. Furthermore, the expression levels of Rf sequences in K326 were lower than those in CMS K326. The molecular basis of the CMS/Rf system in tobacco requires further investigation.

Development of highly polymorphic EST-SSR markers and segregation in F1 hybrid population of Vitis vinifera L.

The objectives of this investigation were to develop and validate the expressed sequence tag (EST)-simple sequence repeat (SSR) markers from large EST sequences, and to study the segregation and distribution of SSRs within two grapevine parental lines. In total, 94 F1 lines crossed between "Early Rose" and "Red Globe" were studied. Approximately 2100 EST-SSR sequences of Vitis vinifera L. were searched for SSRs and analyzed for the design of polymerase chain reaction (PCR) primers amplifying the SSR-rich regions. Trinucleotide repeats were found to be the most abundant, followed by other nucleotide repeats. A total of 182 SSR primer pairs were first developed for the study on the parental polymorphism. Among the 182 SSR primers, 142 primer pairs (78%) could amplify the anticipated PCR products, among which only 52 primer pairs (36.62%) showed polymorphism between the two parents. These polymorphic bands were further surveyed among the 94 F1 lines, and the results showed that a total of 162 bands were amplified, and 98 of them were polymorphic in both parents (60.86% polymorphism), with an average of 1.88 polymorphic DNA bands for each primer pair. After testing with the chi-square test, 33 of the clearly amplified polymorphic bands followed a 3:1 ratio, and 37 followed a 1:1 ratio. The rest showed distorted segregation ratios.

High glucose level induces cardiovascular dysplasia during early embryo development.

The incidence of gestational diabetes mellitus (GDM) has increased dramatically amongst multiethnic population. However, how gestational diabetes mellitus damages the developing embryo is still unknown. In this study, we used yolk sac membrane (YSM) model to investigate angiogenesis in the developing chick embryo. We determined that in the presence of high glucose, it retarded the growth and extension of the embryonic vascular plexus and it also reduced the density of the vasculature in yolk sac membrane model. Using the same strategy, we used the chorioallantoic membrane (CAM) as a model to investigate the influence of high glucose on the vasculature. We established that high glucose inhibited development of the blood vessel plexus and the blood vessels formed had a narrower diameter than control vessels. Concurrent with the abnormal angiogenesis, we also examined how it impacted cardiogenesis. We determined the myocardium in the right ventricle and left atrium were significantly thicker than the control and also there was a reduction in glycogen content in cardiomyocytes. The high glucose also induced excess reactive oxygen species (ROS) production in the cardiomyocytes. We postulated that it was the excess reactive oxygen species that damaged the cardiomyocytes resulting in cardiac hyperplasia.