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There is now biochemical and genetic evidence that oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. To understand the expression characteristics of NCED during ABA biosynthesis in apple (Malus), two NCED genes cDNA sequence were cloned from Malus prunifolia using RT-PCR techniques, named MpNCED1 and MpNCED2. The two cDNA sequences have full-length open reading frame, encoding a polypeptide of 607 and 614 amino acids, respectively. Sequences analysis showed that the deduced two apple NCED proteins were highly homologous to other NCED proteins from different plant species. Real-time PCR analysis revealed MpNCED2 were expressed continuously during the whole period of apple fruit development with the pattern of "higher-low-highest", while the expression of MpNCED1 clearly declined to a steady low level in the mid-later period of fruit development. Expression of the MpNCED2 increased under the drought stress, high temperature and low temperature strongly and rapidly, whereas expression of the MpNCED1 was detected in response to temperature stress, but did not detected under drought stress. These results revealed that MpNCED1 and MpNCED2 may play different roles in regulation of the ABA biosynthesis in fruit development and various stresses response.

Transgene outflow from genetically modified (GM) rice to its wild relatives may cause undesirable ecological consequences. Understanding the level of transgene expression in wild rice following gene flow is important for assessing such consequences, providing that transgene escape from GM rice cannot be prevented. To determine the expression of a transgene in common wild rice (Oryza rufipogon), we analyzed the content of Cry1Ac protein in three GM rice lines containing a Bt transgene, their F(1) hybrids with common wild rice and F(2) progeny at different growth stages, using the sandwich enzyme-linked immunosorbent assay. The average content of Cry1Ac protein in leaf samples of the wild rice lines ranged between 0.016 and 0.069% during the entire growth period, whereas that in stems varied between 0.12 and 0.39%. A great variation in Cry1Ac protein content was detected among individuals of F(1) hybrids and F(2) progeny, with some wild individuals showing higher level of Bt toxin than the cultivated GM rice. The results suggest that the Bt transgene can express normally in the interspecific hybrids between insect-resistant GM rice and common wild rice, and may have similar effects on the target insects as in GM rice.

Histone acetylation and methylation, governed by various histone modification (HM) gene families, are vital for plant biological processes. However, there are limited studies that have explored HMs in ornamental horticultural trees, including sweet osmanthus (Osmanthus fragrans). We performed genome-wide search and identified 208 OfHMs, encompassing 81 histone methyltransferases (OfHMTs), 51 histone demethylases (OfHDMs), 49 histone acetyltransferases (OfHATs) and 27 histone deacetylases (HDACs). Our comprehensive analysis covered chromosome locations, gene structures, conserved domains, cis-acting elements, phylogenetic comparisons, protein interaction networks and functional enrichment pathways for these gene families. Additionally, tandem and fragment replications were unveiled as contributors to the expansion of OfHMs, with some genes exhibiting positive selection. Furthermore, we examined OfHM expression profiles across various tissues and flowering stages, and under 5′-azacytidine (Aza) and ethylene treatments. Most OfHMs displayed heightened expression in leaves, and were downregulated during the flower opening and senescence stages, including OfPRMTs, OfHDTs, OfHDAs, OfSRTs, OfJMJs and OfHAGs; 75.86% and 80.77% of the differentially expressed OfHMs were upregulated after Aza and ethylene treatments, including OfHAGs, OfHDAs and OfSDGs. This study offers a comprehensive analysis of the OfHM gene family, which indicated their potential involvement in ethylene and Aza responses, and in the flowering process. These findings provide valuable insights into the role of OfHMs in flowering and stress responses. [ABSTRACT FROM AUTHOR]

Plums are good sources of various bioactive phytochemical compounds such as vitamins, anthocyanins, and carotenoids, whereby all of which are noted for multiple potential health benefits. However, knowledge regarding plum carotenoid profiles remains limited. Hence, the total and individual carotenoids in the edible parts (skin and flesh) of ten plum cultivars were determined using a spectrophotometer and high-performance liquid chromatography–diode array detection, respectively. Total and individual carotenoid contents in skin were significantly higher (p < 0.05) than those in flesh among all plum cultivars tested. The cultivars with the highest content of total carotenoids in skin were Naili (36.73 μg/g FW), followed by Yinhongli (21.81 μg/g FW) and Yuhuangli (19.70 μg/g FW), with the lowest in Angeleno (8.97 μg/g FW). Lutein, zeaxanthine, β-cryptoxanthin, α-carotene, and β-carotene were the major types of carotenoids detected, with lutein and β-carotene being the predominant constituents of the skin and flesh tissues, respectively. Lutein, zeaxanthine, and total carotenoid contents were positively correlated with the expressions of PSY, LCYB, and LCYE, and negatively correlated with the expressions of PDS and CRTISO. Characterizing the carotenoid profiles and investigating variations in carotenoid biosynthetic gene expressions among plum cultivars are crucial for advancing genetic improvements in plums. [ABSTRACT FROM AUTHOR]

Background: Abnormal miRNA and mRNA expression and dysregulated immune microenvironment have been found to frequently induce the progression of hepatocellular carcinoma (HCC) in recent reports. In particular, the immune-related competing endogenous RNAs (ceRNA) mechanism plays a crucial role in HCC progression. However, the underlying mechanisms remain unclear. Methods: Differentially expressed immune-related genes were obtained from the Immport, GEO, and TCGA databases. The mRNA and protein expression levels in HCC tissues and adjacent normal tissues were confirmed, and we further investigated the methylation levels of these biomarkers to explore their function. Then, the TIMER and TISCH databases were used to assess the relationship between immune infiltration and hub genes. Survival analysis and univariate and multivariate Cox models were used to evaluate the association between hub genes and HCC diagnosis. Hub gene expression was experimentally validated in six HCC cell lines and 15 HCC samples using qRT-PCR and immunohistochemistry. The hub genes were uploaded to DSigDB for drug prediction enrichment analysis. Results: We identified that patients with abnormal miRNAs (hsa-miR-125b-5p and hsa-miR-21-5p) and their targeted genes (NTF3, PSMD14, CD320, and SORT1) had a worse prognosis. Methylation analysis of miRNA-targeted genes suggested that alteration of methylation levels is also a factor in the induction of tumorigenesis. We also found that the development of HCC progression caused by miRNA-mRNA interactions may be closely correlated with the infiltration of immunocytes. Moreover, the GSEA, GO, and KEGG analysis suggested that several common immune-related biological processes and pathways were related to miRNA-targeted genes. The results of qRT-PCR, immunohistochemistry, and western blotting were consistent with our bioinformatics results, suggesting that abnormal miRNAs and their targeted genes may affect HCC progression. Conclusions: Briefly, our study systematically describes the mechanisms of miRNA-mRNA interactions in HCC and predicts promising biomarkers that are associated with immune filtration for HCC progression. [ABSTRACT FROM AUTHOR]

Loquats have gained increasing attention from consumers and growers for their essential nutrients and unusual phenology, which could help plug a gap period at market in early spring. Fruit acid is a critical contributor to fruit quality. The dynamic changes in organic acid (OA) during fruit development and ripening of common loquat (Dawuxing, DWX) and its interspecific hybrid (Chunhua, CH) were compared, as well as the corresponding enzyme activity and gene expression. At harvest, titratable acid was significantly lower (p ≤ 0.01) in CH (0.11%) than in DWX loquats (0.35%). As the predominant OA compound, malic acid accounted for 77.55% and 48.59% of the total acid of DWX and CH loquats at harvest, followed by succinic acid and tartaric acid, respectively. PEPC and NAD-MDH are key enzymes that participate in malic acid metabolism in loquat. The OA differences in DWX loquat and its interspecific hybrid could be attributed to the coordinated regulation of multiple genes and enzymes associated with OA biosynthesis, degradation, and transport. The data obtained in this work will serve as a fundamental and important basis for future loquat breeding programs and even for improvements in loquat cultural practices. [ABSTRACT FROM AUTHOR]

Inadequate potassium (K) availability is a common abiotic stress that limits the growth and quality of fruit trees. Few studies have investigated the physiological and molecular responses of grapes at different potassium levels. In this study, an integrated approach was developed for grapevines grown at four different potassium fertilization levels [0 (K0-CK), 150 (K150), 300 (K300), and 450 (K450) g/plant] in combination with metabolite measurements and transcript analysis. The results showed that different K levels affected the accumulation of sugars and anthocyanins in the fruit. At 78 days after bloom (DAB), the K150, K300, and K450 treatments increased soluble sugar content by 37.39%, 31.10% and 32.59%, respectively, and anthocyanin content by 49.78%, 24.10%, and 13.06%, respectively, compared to K0. Weighted gene co-expression network analysis (WGCNA) of DEGs identified a network of 11 grapevines involved. During fruit development, potassium application promoted the accumulation of anthocyanins and sugars in fruit by regulating the up-regulation of GST, AT, UFGT and SPS, HT, PK gene expressions. These results suggest that potassium deficiency inhibits anthocyanin and sugar metabolism. In addition, it promotes the up-regulation of KUP expression, which is the main cause of K accumulation in fruits. Together, our data revealed the molecular mechanism in response to different K levels during fruit quality formation and provides the scientific foundation for the improvement of fruit quality by adding K fertilizer. [ABSTRACT FROM AUTHOR]

Loquats have drawn much attention due to their essential nutrients and unusual phenology, which fills a market gap in early spring. Fruit firmness (FF) is one of the most important quality attributes. Dynamic changes in FF, cell wall (CW) polysaccharides, CW hydrolase activity, and expression of CW metabolism-related genes during the fruit development and ripening stages of two contrasting loquat cultivars were compared. Although the two cultivars possessed similar FF at the initial fruitlet stage, Dawuxing was significantly firmer than Ninghaibai at all subsequent time points. FF was positively correlated with the contents of covalent-soluble pectin and hemicellulose, activity of peroxidase, and gene expressions of PME, EG, CAD6, and POD; and negatively correlated with the contents of water-soluble pectin, activities of polygalacturonase, endo-glucanase, cellobiohydrolase, and xylanase, and gene expressions of PG, EG2, PAL1, PAL3, and CAD5. Identifying molecular mechanisms underlying the differences in FF is useful for fundamental research and crop improvement in future. [ABSTRACT FROM AUTHOR]

MicroRNAs play an important role in gene regulation for many biological systems, including nicotine and alcohol addiction. However, the underlying mechanism behind miRNAs and mRNA interaction is not well characterized. Microarrays are commonly used to quantify the expression levels of mRNAs and/or miRNAs simultaneously. In this study, we performed a Bayesian network analysis to identify mRNA and miRNA interactions following perinatal exposure to nicotine and/or alcohol. We utilized three sets of microarray data to predict the regulation relationship between mRNA and miRNAs. Following perinatal alcohol exposure, we identified two miRNAs: miR-542-5p and miR-874-3p, that exhibited a strong mutual influence on several mRNA in gene regulatory pathways, mainly Axon guidance and Dopaminergic synapses. Finally, we confirmed our predicted addiction pathways based on the Bayesian network analysis with the widely used Kyoto Encyclopedia of Genes and Genomes (KEGG)-based database and identified comparable relevant miRNA-mRNA pairs. We believe the Bayesian network can provide insight into the complexity biological process related to addiction and can potentially be applied to other diseases. [ABSTRACT FROM AUTHOR]

Background: Lung cancer is one of the most lethal malignant tumors that endangers human health. Lung adenocarcinoma (LUAD) has increased dramatically in recent decades, accounting for nearly 40% of all lung cancer cases. Increasing evidence points to the importance of the competitive endogenous RNA (ceRNA) intrinsic mechanism in various human cancers. However, behavioral characteristics of the ceRNA network in lung adenocarcinoma need further study. Methods: Groups based on SLC2A1 expression were used in this study to identify associated ceRNA networks and potential prognostic markers in lung adenocarcinoma. The Cancer Genome Atlas (TCGA) database was used to obtain the patients' lncRNA, miRNA, and mRNA expression profiles, as well as clinical data. Informatics techniques were used to investigate the effect of hub genes on prognosis. The Cox regression analyses were performed to evaluate the prognostic effect of hub genes. The methylation, GSEA, and immune infiltration analyses were utilized to explore the potential mechanisms of the hub gene. The CCK-8, transwell, and colony formation assays were performed to detect the proliferation and invasion of lung cancer cells. Results: We eventually identified the ITGB1-DT/ARNTL2 axis as an independent fact may promote lung adenocarcinoma progression. Furthermore, methylation analysis revealed that hypo-methylation may cause the dysregulated ITGB1-DT/ARNTL2 axis, and immune infiltration analysis revealed that the ITGB1-DT/ARNTL2 axis may affect the immune microenvironment and the progression of lung adenocarcinoma. The CCK-8, transwell, and colonu formation assays suggested that ITGB1-DT/ARNTL2 promotes the progression of lung adenocarcinoma. And hsa-miR-30b-3p reversed the ITGB1/ARNTL2-mediated oncogenic processes. Conclusion: Our study identified the ITGB1-DT/ARNTL2 axis as a novel prognostic biomarker affects the prognosis of lung adenocarcinoma. [ABSTRACT FROM AUTHOR]

Citrate is one of the most important metabolites determining the flavour of citrus fruit. It has been reported that nitrogen supply may have an impact on acid level of fruit. Here, the relationship between nitrogen metabolism and citrate catabolism was studied in pumelo juice sacs. Differences in metabolites, gene expression and flux distributions were analyzed in juice sacs incubated in medium with and without NH 4 +. Compared with those incubated with NH 4 +, juice sacs under nitrogen deficiency exhibited enhanced flux through phosphoenolpyruvate carboxykinase (PEPCK) and accelerated consumption of citrate, while the other two TCA cycle efflux points, through malic enzyme (ME) and glutamate dehydrogenase (GDH), were both repressed. Consistent with the estimated fluxes, the expression of PEPCK1 was upregulated under nitrogen deficiency, while that of GDH1 , GDH2 , NAD-ME1 and NADP-ME2 were all repressed. Thus, we propose that PEPCK1 contributes to citrate degradation under nitrogen limitation. • Metabolism in pomelo juice sacs was analyzed systematically and quantitatively with stoichiometric metabolic flux analysis. • Nitrogen deficiency accelerated decrease in citrate content of juice sacs. • Nitrogen deficiency enhanced reaction of PEPCK and suppressed other TCA cycle exporting reactions. • CitPEPCK1 gene expression was activated under different conditions where inorganic nitrogen is absent. [ABSTRACT FROM AUTHOR]

Background: Malaria caused by Plasmodium spp. is still a major threat to public health globally. The various approaches to developing new antimalarial agents rely on the understanding of the complex regulatory mechanisms of dynamic gene expression in the life-cycle of these malaria parasites. The nuclear members of the evolutionarily conserved actin-related protein nuclear (ARP) superfamily are the major components of nucleosome remodelling complexes. In the human malaria parasite Plasmodium falciparum, bioinformatics analysis has predicted three ARP orthologues: PfArp1, PfArp4 and PfArp6. However, little is known about the biological functions of putative PfArp4. In this study, we aimed to investigate the function and the underlying mechanisms of PfArp4 gene regulation. Methods: A conditional gene knockdown approach was adopted by incorporating the glucosamine-inducible glmS ribozyme sequence into the 3' UTR of the PfArp4 and PfArp6 genes. The transgenic parasites PfArp4-Ty1-Ribo, PfArp6-Ty1-Ribo and pL6-PfArp4-Ty1::PfArp6-HA were generated by the CRISPR-Cas9 technique. The knockdown effect in the transgenic parasite was measured by growth curve assay and western blot (WB) analysis. The direct interaction between PfArp4 and PfArp6 was validated by co-IFA and co-IP assays. The euchromatic gene expression mediated through H2A.Z (histone H2A variant) deposition and H3K9ac modification at promoters and regulated by PfArp4, was determined by RNA-seq and ChIP-seq. Results: The inducible knockdown of PfArp4 inhibited blood-stage development of P. falciparum. PfArp4 and PfArp6 were colocalized in the nucleus of P. falciparum parasites. PfArp4 gene knockdown altered the global transcriptome. PfArp4 protein colocalized with the histone variant H2A.Z and euchromatic marker H3K9ac in intergenic regions. The inducible downregulation of PfArp4 resulted in the depletion of H2A.Z and lower H3K9ac levels at the upstream regions of eukaryotic genes, thereby repressing the transcriptional abundance of H2A.Z-dependent genes. Conclusions: Our findings suggest that PfArp4 regulates the cell cycle by controlling H2A.Z deposition and affecting centromere function, contributing to the understanding the complex epigenetic regulation of gene expression and the development of P. falciparum. [ABSTRACT FROM AUTHOR]

As transcriptional factors, nuclear receptors (NRs) function as major regulators of gene expression. In particular, dysregulation of NR activity has been shown to significantly alter metabolic homeostasis in various contexts leading to metabolic disorders and cancers. The orphan estrogen-related receptor (ERR) subfamily of NRs, comprised of ERRα, ERRβ, and ERRγ, for which a natural ligand has yet to be identified, are known as central regulators of energy metabolism. If AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) can be viewed as sensors of the metabolic needs of a cell and responding acutely via post-translational control of proteins, then the ERRs can be regarded as downstream effectors of metabolism via transcriptional regulation of genes for a long-term and sustained adaptive response. In this review, we will focus on recent findings centered on the transcriptional roles played by ERRα in hepatocytes. Modulation of ERRα activity in both in vitro and in vivo models via genetic or pharmacological manipulation coupled with chromatin-immunoprecipitation (ChIP)-on-chip and ChIP-sequencing (ChIP-seq) studies have been fundamental in delineating the direct roles of ERRα in the control of hepatic gene expression. These studies have identified crucial roles for ERRα in lipid and carbohydrate metabolism as well as in mitochondrial function under both physiological and pathological conditions. The regulation of ERRα expression and activity via ligand-independent modes of action including coregulator binding, post-translational modifications (PTMs) and control of protein stability will be discussed in the context that may serve as valuable tools to modulate ERRα function as new therapeutic avenues for the treatment of hepatic metabolic dysfunction and related diseases. [ABSTRACT FROM AUTHOR]

Background: Capn4 and ZEB1 play important roles in the metastasis of several types of cancer. However, the roles and relationship of Capn4 and ZEB1 in esophageal squamous cell carcinoma (ESCC) remain unclear. Methods: ESCC tumor tissues and corresponding normal esophageal epithelial tissues were obtained from 86 patients undergoing resection surgery at the Department of General Surgery, First Affiliated Hospital of Chinese PLA General Hospital from 2012 to 2017. Cell migration and invasion were examined via quantitative real‐time PCR and Western blot assay. Results: Our results indicate that both Capn4 and ZEB1 are significantly upregulated in ESCC tissues compared to corresponding adjacent tissues, and a positive correlation between expression and associated malignant characteristics was found. Silencing of Capn4 expression markedly inhibited ESCC invasion and metastasis in vitro and in vivo, and was accompanied by decreased ZEB1 expression. Furthermore, the anti‐metastasis role of Capn4 silencing was reversed by ZEB1 overexpression, whereas knockdown of ZEB1 decreased ESCC metastasis driven by the upregulation of Capn4. Mechanistically, Capn4 regulated ZEB1 expression via activation of the Wnt/β‐catenin signaling pathway in ESCC cells. Conclusion: Overall, our results show that enhanced Capn4 expression activates the Wnt/β‐catenin signaling pathway, resulting in increased ZEB1 expression and the promotion of ESCC cell metastasis. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate the association between serum carbohydrate antigen (CA)19-9 and CA125 levels, and human epidermal growth factor receptor 2 (HER2) expression in patients with gastric cancer, as well as to identify any correlation between them and the risk of recurrence and metastasis. A total of 256 patients were enrolled in the present study, and 219 patients were followed-up to investigate recurrence and metastasis of gastric cancer. Immunohistochemistry was used to detect HER-2 in gastric adenocarcinoma and paracancerous tissues. The positive rate of CA19-9 and CA125 in stages III/IV was higher compared with that in stages I/II. The positive rate of HER2 in distinct stages of gastric cancer was not statistically different. Serum CA19-9 and CA125 were not associated with the positive expression of HER2. The recurrence and metastasis of CA19-9, CA125 and HER2-positive gastric cancer were increased compared with those experienced by CA19-9, CA125 and HER2-negative patients. Age, stage and preoperative tumor markers were associated with 3-year prognosis of gastric cancer. HER2 [odds ratio (OR)=2.55] and CA19-9 (OR=1.22) were independent prognostic factors in patients with gastric cancer. CA19-9, CA125 and HER2 may be used to predict the recurrence or metastasis of gastric cancer. The combined detection may be able to improve the sensitivity and efficiency of predicting the recurrence or metastasis of gastric cancer. Preoperative positive serum for CA19-9 and CA125 were associated with poor prognosis in patients with gastric cancer. CA19-9 and HER2 were independent prognostic factors of gastric cancer. [ABSTRACT FROM AUTHOR]

Fruits provide abundant carotenoid nutrients for humans, whereas the understanding of the transcriptional regulatory mechanisms of carotenoids in fruits is still limited. Here, we identified a transcription factor AcMADS32 in kiwifruit, which was highly expressed in the fruit, correlated with carotenoid content and localized in the nucleus. The silencing expression of AcMADS32 significantly reduced the content of β-carotene and zeaxanthin and expression of β-carotene hydroxylase gene AcBCH1/2 in kiwifruit, while transient overexpression increased the accumulation of zeaxanthin, suggesting that AcMADS32 was an activator involved in the transcriptional regulation of carotenoid in fruit. When AcMADS32 was further stably transformed into kiwifruit, the content of total carotenoid and components in the leaves of transgenic lines significantly increased, and the expression level of carotenogenic genes was up-regulated. Moreover, Y1H and dual luciferase reporter experiments confirmed that AcMADS32 directly bound the AcBCH1/2 promoter and activated its expression. Through Y2H assays, AcMADS32 can interact with other MADS transcription factor AcMADS30, AcMADS64 and AcMADS70. These findings will contribute to our understanding of the transcriptional regulation mechanisms underlying carotenoid biosynthesis in plants. • Transcription factor AcMADS32 was highly expressed in fruit of kiwifruit and correlated with carotenoid content. • The silencing expression of AcMADS32 significantly reduced the content of zeaxanthin and expression of AcBCH1/2. • AcMADS32 overexpression significantly increased carotenoids content and related gene expression in transgenic kiwifruits. • AcMADS32 directly bound the AcBCH1/2 promoter and activated its expression confirmed by Y1H and dual luciferase assays. • AcMADS32 can interact with AcMADS30, AcMADS64 and AcMADS70 revealed by Y2H. [ABSTRACT FROM AUTHOR]

To elucidate metabolism of ascorbic acid (AsA) in sweet cherry fruit (Prunus avium ‘Hongdeng’), we quantified AsA concentration, cloned sequences involved in AsA metabolism and investigated their mRNA expression levels, and determined the activity levels of selected enzymes during fruit development and maturation. We found that AsA concentration was highest at the petal-fall period (0 days after anthesis) and decreased progressively during ripening, but with a slight increase at maturity. AsA did nevertheless continue to accumulate over time because of the increase in fruit fresh weight. Full-length cDNAs of 10 genes involved in the L-galactose pathway of AsA biosynthesis and 10 involved in recycling were obtained. Gene expression patterns of GDP-L-galactose phosphorylase (GGP2), L-galactono-1, 4-lactone dehydrogenase (GalLDH), ascorbate peroxidase (APX3), ascorbate oxidase (AO2), glutathione reductase (GR1), and dehydroascorbate reductase (DHAR1) were in accordance with the AsA concentration pattern during fruit development, indicating that genes involved in ascorbic acid biosynthesis, degradation, and recycling worked in concert to regulate ascorbic acid accumulation in sweet cherry fruit. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the performance of the Sensitire MYCOTB MIC Plate (MYCOTB) which could measure the twelve anti-tuberculosis drugs susceptibility on one 96-wells plate. Methods: A total of 140 MDR-TB strains and 60 non-MDR strains were sub-cultured and 193 strains were finally tested for drug resistance using MYCOTB and agar proportion method (APM) and another 7 strains failed of subculture. The drugs included ofloxacin (Ofx), moxifloxacin (Mfx), rifampin (RFP), amikacin (Am), rifabutin (Rfb), para-aminosalicylic acid (PAS), ethionamide (Eth), isoniazid (INH), kanamycin (Km), ethambutol (EMB), streptomycin (Sm), and cycloserine(Cs). The categorical agreement, conditional agreement, sensitivity and specificity of MYCOTB were assessed in comparison with APM. For strains with inconsistent results between MYCOTB and APM, the drug resistance related gene fragments were amplified and sequenced: gyrA for Ofx and Mfx; rpoB for RFP and Rfb; embB for EMB; rpsl for Sm; katG and the promoter region of inhA for INH, ethA and the promoter region of inhA for Eth. The sequence results were compared with results of MYCOTB and APM to analyze the consistency between sequence results and MYCOTB or APM. Results: The categorical agreement between two methods for each drug ranged from 88.6% to 100%. It was the lowest for INH (88.6%). The sensitivity and specificity of MYCOTB ranged from 71.4% to 100% and 84.3% to 100%, respectively. The sensitivity was lowest for Cs(71.4%), EMB at 10μg/ml (80.0%) and INH at 10.0μg/ml (84.6%). The specificity was lowest for Rfb (84.3%). Overall discordance between the two phenotypic methods was observed for 96 strains, of which 63 (65.6%) were found susceptible with APM and resistant with MYCOTB and the remaining 33(34.4%) strains were resistant by APM and susceptible with MYCOTB. 34/52 (65.4%) sequenced APM susceptible and MYCOTB resistant(APM-S/MYCOTB-R) strains had mutations or insertions in the amplified regions. 20/30 (66.7%) sequenced APM resistant and MYCOTB susceptible strains had mutations in the sequenced genes. MICs of twenty-nine of these thirty isolates were equal to or within 1 doubling dilution of the critical concentration. Conclusion: MYCOTB had good performance for most of tested drugs and could be used as an alternative to the more labor demanding and longer turnaround time solid culture based DST method for detection of drug susceptibility in China. [ABSTRACT FROM AUTHOR]

The stress-induced epimutations could be inherited over generations and play important roles in plant adaption to stressful environments. Upland rice has been domesticated in water-limited environments for thousands of years and accumulated drought-induced epimutations of DNA methylation, making it epigenetically differentiated from lowland rice. To study the epigenetic differentiation between upland and lowland rice ecotypes on their drought-resistances, the epigenetic variation was investigated in 180 rice landraces under both normal and osmotic conditions via methylation-sensitive amplified polymorphism (MSAP) technique. Great alterations (52.9~54.3% of total individual-locus combinations) of DNA methylation are recorded when rice encountering the osmotic stress. Although the general level of epigenetic differentiation was very low, considerable level of ΦST (0.134~0.187) was detected on the highly divergent epiloci (HDE). The HDE detected in normal condition tended to stay at low levels in upland rice, particularly the ones de-methylated in responses to osmotic stress. Three out of four selected HDE genes differentially expressed between upland and lowland rice under normal or stressed conditions. Moreover, once a gene at HDE was up-/down-regulated in responses to the osmotic stress, its expression under the normal condition was higher/lower in upland rice. This result suggested expressions of genes at the HDE in upland rice might be more adaptive to the osmotic stress. The epigenetic divergence and its influence on the gene expression should contribute to the higher drought-resistance in upland rice as it is domesticated in the water-limited environment. [ABSTRACT FROM AUTHOR]

The annexins are a multifamily of calcium-regulated phospholipid-binding proteins. To investigate the roles of annexins in fiber development, four genes encoding putative annexin proteins were isolated from cotton ( Gossypium hirsutum) and designated AnnGh3, AnnGh4, AnnGh5, and AnnGh6. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results indicated that AnnGh3, AnnGh4, and AnnGh5 were preferentially expressed in fibers, while the transcripts of AnnGh6 were predominantly accumulated in roots. During fiber development, the transcripts of AnnGh3/4/5 genes were mainly accumulated in rapidly elongating fibers. With fiber cells further developed, their expression activity was dramatically declined to a relatively low level. In situ hybridization results indicated that AnnGh3 and AnnGh5 were expressed in initiating fiber cells (0-2 DPA). Additionally, their expression in fibers was also regulated by phytohormones and [Ca2+]. Subcellular localization analysis discovered that AnnGh3 protein was localized in the cytoplasm. Overexpression of AnnGh3 in Arabidopsis resulted in a significant increase in trichome density and length on leaves of the transgenic plants, suggesting that AnnGh3 may be involved in fiber cell initiation and elongation of cotton. [ABSTRACT FROM AUTHOR]

Analysis of gene expression using RNA from the paraffin-embedded tissues is becoming an important way to study cancer pathogenesis. In this article, total RNA was extracted from tissue of 158 cases of paraffin-embedded tongue cancer, and expression of angiopoietin-like 4, tenascin-C and cathepsin-C were detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Our results demonstrated that high expression level of angiopoietin-like 4 or tenascin-C was predictive of poor prognosis of tongue cancer patients ( p = 0.024 and p = 0.011, respectively), especially when expression levels of both genes were concomitantly high ( p = 0.001). Additionally, high expression of angiopoietin-like 4 and tenascin-C, or concomitant high expression of angiopoietin-like 4 and tenascin-C were independent prognostic factors of poor survival in patients with tongue cancer. These results suggest that the improved method of RNA extraction is suitable for analysing gene expression of paraffin-embedded solid tumours. Angiopoietin-like 4 and tenascin-C, especially the combination of angiopoietin-like 4 and tenascin-C, are useful for predicting the prognosis of the patients with tongue cancer, independent of lymph node metastasis status. [ABSTRACT FROM AUTHOR]

Reference genes are used for the correction of qRT-PCR data, and it is necessary to investigate the optimum reference gene under certain conditions. The expression levels of seven traditional reference genes ACT1, ACT2, GAPDH, 18S rRNA, UBQ, TUB and CYP were analyzed using qRT-PCR in different varieties, tissues, developmental stages and hormone (or pollen polysaccharide) treatments in kiwifruit. Gene expression stability was assessed with the help of three common software (geNorm, NormFinder, BestKeeper), and the minimum number of reference genes necessary for normalization was also determined. GAPDH, ACT1 and ACT2 were selected as reference genes for different genotypes of kiwifruit. GAPDH and UBQ were the best combinations of reference genes for root, stem, leaf, flower and fruit. GAPDH and ACT1 could be the preferred reference genes for normalization of qRT-PCR data during fruit development. The pairing of ACT1 and UBQ constituted the optimal combination of reference genes in kiwifruit treated with different hormones (or pollen polysaccharide). This study provides a new and reliable option for the use of reference genes in the analysis of gene expression patterns of interest in kiwifruit. [ABSTRACT FROM AUTHOR]

• Flesh color were compared between two yellow-fleshed kiwifruit cultivars. • 'Jinshi 1′ has stronger yellow flesh color than 'Hort16A'. • 'Jinshi 1′ has higher carotenoids/chlorophyll ratio. • β-cryptoxanthin is specific detected in 'Jinshi 1′. To identify the biochemical and molecular factors causing flesh-color variation between two yellow-flesh kiwifruit cultivars 'Jinshi 1′ and 'Jinyan', the composition of carotenoids and chlorophyll, and associated gene expression profile in their pulp were investigated during fruit development. At harvest, 'Jinshi 1′ exhibited brighter yellow flesh than 'Hort16A' with a lower color hue angle, although they had a similar total carotenoid content. At the molecular level, 'Jinshi 1′ exhibited higher expression level of chlorophyll catabolic genes (CBR , PPH , and PAO), leading to lower accumulation of chlorophyll in the flesh. Meanwhile, higher expression levels of NCED1 resulted in greater carotenoid degradation in 'Hort16A'. In addition, 'Hort16A' had a higher content of lutein, zeaxanthin and β-carotene, whereas 'Jinshi 1′ had specific β-cryptoxanthin on ripening, accordingly, the expression of CHYB1, which encodes the enzyme for the formation of zeaxanthin and β-cryptoxanthin, was higher during the last developmental stage in 'Jinshi 1′ than in 'Hort16A'. These results suggested that the higher carotenoid/chlorophyll ratio, as well as the higher β-cryptoxanthin proportion are the important reasons for the brighter yellow flesh color. [ABSTRACT FROM AUTHOR]

In the study, a gene encoding a putative ethylene response factor of AP2/EREBP family was isolated from cotton (Gossypium hirsutum) and designated as GhERF12. Sequence alignment showed that GhERF12 protein contains a central AP2/ERF domain (58 amino acids) with two functional conserved amino acid residues (ala14 and asp19). Transactivation assay indicated that GhERF12 displayed strong transcription activation activity in yeast cells, suggesting that this protein may be a transcriptional activator in cotton. Quantitative RT-PCR analysis showed that GhERF12 expression in cotton was induced by ACC and IAA. Overexpression of GhERF12 in Arabidopsis affected seedling growth and development. The GhERF12 transgenic plants grew slowly, and displayed a dwarf phenotype. The mean bolting time of the transgenic plants was delayed for about 10 days, compared with that of wild type. Further study revealed that some ethylene-related and auxin-related genes were dramatically up-regulated in the transgenic plants, compared with those of wild type. Collectively, we speculated that GhERF12, as a transcription factor, may be involved in regulation of plant growth and development by activating the constitutive ethylene response likely related to auxin biosynthesis and/or signaling. [ABSTRACT FROM AUTHOR]

As the second largest family of transcription factors in plants, bHLH is widely involved in the growth and development of organisms and their response to stress. In the past, there was no systematic division of this gene family in kiwifruit. In this study, we identified 206 AcbHLH genes, divided into 22 subfamilies, and analyzed chromosomal localization, physicochemical properties, conserved domains, and intron/exon composition. In addition, the repeat events of the AcbHLH gene in kiwifruit and the evolutionary relationship with Arabidopsis thaliana and Oryza sativa were further analyzed. We analyzed the expression levels of the AcbHLH genes under biotic and abiotic stresses (heat, low temperature, drought, and Bacillus anthracis infection). In addition, we also investigated the biological function of AcbHLH146 under salt stress using a VIGS-mediated gene silencing method. In the AcbHLH146 -VIGS line, the content of H 2 O 2 and O 2 - was lower and the damage was less. SOD, POD, and CAT enzyme activities are higher. These results suggest that AcbHLH146 may play a negative moderating role in the response of kiwifruit to salt stress. Our findings provide new insights into the potential role of AcbHLH146 under abiotic stress, and provide a theoretical basis for the molecular mechanism of Kiwifruit adaptation to salt stress. • 206 AcbHLH members were identified and classified into 22 subclasses in kiwifruit. • AcbHLHs response to abiotic stress including heat, salt, drought and so on. • AcbHLH146 may play a negative role in kiwifruit response to salt stress. [ABSTRACT FROM AUTHOR]

Light is a key environmental factors affecting anthocyanin accumulation in plants. Ubiquitin E3 ligase COP1 has been proved to be a negative regulator involved in light-regulated plant development process, whereas the function and expression specificity of COP1 in anthocyanin biosynthesis in sweet cherry remains unclear. In the present study, we identified a COP1 in sweet cherry, named PacCOP1 , it exhibited apparent different expression patterns in red-colored 'Hongdeng' and bi-colored 'Satonishiki', with increasing trend largely in 'Satonishiki', but decreasing trend in 'Hongdeng' after veraison, which was contrary to their variation tendency of anthocyanin content. While the expression abundance of anthocyanin biosynthesis related genes were largely increased after veraison, in accordance with anthocyanin content. Correlation analysis proved that the expression of PacCOP1 was negative correlated with the major genes on anthocyanin accumulation in 'Hongdeng' and 'Satonishiki' fruit, in especial Pac DFR, Pac ANS, Pac MYBA and Pac bHLH33. Furthermore, over-expression of PacCOP1 in Arabidopsis displayed increased COP1 transcript level with negligible pigmentation and corresponding lower expression level of AtPAP1 , AtDFR , AtLDOX , and AtUFGT. These results revealed the negative regular role of PacCOP1 in anthocyanin biosynthesis by repressing the PacMYBA transcription level, followed by down-regulating the structural genes expression abundance, eventually leading to attenuated anthocyanin accumulation in fruits. • A novel Ubiquitin E3 ligase gene COP1 was identified from sweet cherry, named PacCOP1. • PacCOP1 exhibited apparent different expression patterns in red- and bi-colored cultivars, contrary to that of anthocyanin content. • The expression level of PacCOP1 was negative correlated with mRNA abundance of Pac DFR, Pac ANS, Pac MYBA and Pac bHLH33. [ABSTRACT FROM AUTHOR]