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• MCM10 mRNA expression was significantly upregulated in tumor samples and precancerous lesions compared to normal samples, indicating its involvement in the development and progression of cervical cancer. • MCM10 expression was found to be specific to cervical cancer, with the highest expression level in cervical squamous cell carcinoma compared to other tumor types as evident from onco-database analysis. • The study proposes qRT-PCR based MCM10 signal assessment as a potential method to reduce the number of advanced cervical cancer cases and suggests the development of a kit for detecting MCM10 signals. • HPV 16 and 18 were the most common HPV genotypes found in cervical cancer cases, while HPV 45 was more frequent in precancerous lesions. • Clinical cases of dual HPV infections (HPV16 and HPV33), (HPV6 and HPV16) were detected in our study. Cervical cancer remains a significant health burden worldwide, emphasizing the need for early detection and intervention. DNA replication is perturbed in cancer cells, and the minichromosome maintenance protein 10 plays an important role in origin firing. By analyzing the MCM10 mRNA expression in healthy controls, precancerous lesions, and cervical cancer using qRT-PCR, we can infer if it can be considered a biomarker. We collected cervical smear samples from patients and performed MCM10 expression analysis to set up thresholds for risk stratification. We also investigated the HPV status among the patient samples with precancerous lesions and cervical cancer and found 70 % of them to be positive. Our results demonstrated a significant upregulation of MCM10 mRNA expression in tumor samples (n = 40, 7.83 ± 1.2) and precancerous lesions (n = 54, 5.69 ± 1.4) compared to normal (n = 50, 4.27 ± 0.80) with a R2 value of 0.59, confirming its role in the progression and development of cervical cancer. In conclusion, this study emphasizes the potential role of MCM10 as a biomarker. Our study would improve early detection rates, and we propose MCM10-based community screening for risk stratification, prevention, and prognosis. [ABSTRACT FROM AUTHOR]

[Display omitted] • The human parathyroid glandular cells require higher accuracy for the target gene quantification. • Some of the particular reference genes are not expressed in the parathyroid gland and its glandular cells. • The study provides minor data variability between clinically different individuals. • RPLP0 and GAPDH arethe most stable reference genes among 14 candidates. Identifying a proper reference gene allows us to understand fundamental changes in many biological processes. Normalization during gene expression analyses is essential for every tissue/cell type, including parathyroid tissue glandular cells. Quantitative method of gene expression analyses via qRT-PCR method provides the accurate examination of every target gene. There are limited reports to present commonly used reference genes in human parathyroid tissues rather than for glandular cell types. This study aims to determine and compare the most stable to least stable genes for parathyroid tissue cells. 43 human parathyroid tissue obtained from primary and secondary hyperparathyroidism patients and glandular cells isolated enzymatically by the removal of extracellular matrix components. After extraction of the total RNA, cDNA synthesis was performed, then qRT-PCR evaluated 14 candidate reference genes. Stability was determined by RefFinder software (Delta ct, BestKeeper, Genorm, and NormFinder algorithms), and the outcome was evaluated for five groups. Even if assessed with different groups, the most stable genes were RPLP0 and GAPDH, while the CLTC and RNA 18S were the least stable. We have confirmed the comprehensive ranking of the most stable three genes alone with the NormFinder algorithm to understand intergroup variation and found out that RPLP0>GAPDH>PGK1. Lastly, comparisons of relative target gene (GCM2) expression revealed similar expression patterns for the most stable reference genes. The most stable reference gene is recommended for the stages where stability is evaluated using the results of four different approaches using RefFinder. We aspire for this study to assist future research to conduct thorough assessments of appropriate reference genes before engaging in gene expression analyses for parathyroid tissue. [ABSTRACT FROM AUTHOR]

qRT-PCR requires reliable internal control genes stably expressed in different samples and experimental conditions. The stability of reference genes is rarely tested experimentally, especially in developing tissues given the singularity of these samples. Here we evaluated the suitability of a set of reference genes (Actb , Gapdh , Tbp , Pgk1 and Sdha) using samples from early mouse embryo tissues that are widely used in research (somites, prosencephalon and heart) at different developmental stages. The comparative ΔCq method and five software packages (NormFinder, geNorm, BestKeeper, DataAssist and RefFinder) were used to rank the most stable genes while GenEx and GeNorm programs determined the optimal total number of reference genes for a reliable normalization. The ranking of most reliable reference genes was different for each tissue evaluated: (1) in somite from embryos with 16–18 somite pairs stage, the combination of Pgk1 and Actb provided the best normalization and Actb also presented high stability levels at an earlier developmental stage; (2) Gapdh is the most stable gene in prosencephalon in the two developmental stages tested; and (3) in heart samples, Sdha , Gapdh and Actb were the best combination for qPCR normalization. The analysis of these three tissues simultaneously indicated the combination of Gapdh , Actb and Tbp as the most reliable internal control. This study highlights the importance of appropriate reference genes according to the cell type and/or tissue of interest. The data here described can be applied in future research using mouse embryos as a model for mammalian development. • Standard reference genes (Actb / Gapdh / Tbp/Pgk1 / Sdha) have variable expression stability in early mouse embryo. • In somites the combination of Pgk1 and Actb provided the best normalization. • Gapdh was the most stable gene in prosencephalon. • In the developing heart Sdha , Gapdh and Actb were the top ranked reference genes. • When these 3 tissues were merged the combination of Gapdh , Actb and Tbp was the best normalization. [ABSTRACT FROM AUTHOR]

Cotton fiber initiation is the first step in fiber development, and it determines the yield. Here, genome-wide transcriptome profiling of Gossypium arboreum was performed to determine the molecular basis of cotton fiber initiation. A comparison of the transcriptomes of fiber-bearing ovules at −0.5, 0, 0.5, 1, 1.5, 2, 2.5 and 3 d post-anthesis detected 12,049 differentially expressed genes that mainly participated in ribosome, carbon metabolism and amino acid biosynthesis pathways. Genes encoding alcohol dehydrogenase 1 and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase, involving in fatty acid degradation and flavonoid biosynthesis, were enriched. Furthermore, 1049 differentially expressed transcription factors were identified. Among these, 17 were trihelix family transcription factors, which play important roles in plant development and responses to biotic and abiotic stresses. In total, 52 full-length trihelix genes, named as GaGT s, were identified in G. arboreum and located in 12 of the 13 cotton chromosomes. Transcriptomic data and a quantitative real-time PCR analysis indicated that several GaGT s were significantly induced during fiber initiation in G. arboreum. Thus, the genome-wide comprehensive analysis of gene expression in G. arboreum fiber initiation will serve as a useful resource for unraveling the functions of specific genes. The phylogenetic relationships and expression analyses of the G. arboreum trihelix genes established a solid foundation for future comprehensive functional analyses of the GaGT s. • 12,049 genes and 1,049 TFs were detected by genome-wide transcriptome of G. arboreum at fiber initiation. • Genes of alcohol dehydrogenase 1 and hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase were enriched. • 52 full-length trihelix genes were identified. [ABSTRACT FROM AUTHOR]

Long non coding RNAs (lncRNA) have been previously found to be involved in important cellular activities like epigenetics, implantation, cell growth etc. in pigs. However, comprehensive analysis of lncRNA in back fat tissues at different developmental stages in pigs is still lacking. In this study we conducted transcriptome analysis in the back fat tissue of a F1 crossbred Korean Native Pig (KNP) × Yorkshire Pig to identify lncRNA. We investigated their role in 16 pigs at two different growth stages; stage 1 (10 weeks, n = 8) and stage 2 (26 weeks, n = 8). After quality assessment of sequencing reads, we got a total of 1,641,165 assembled transcripts out of eight paired end read from each stage. Among them, 6808 lncRNA transcripts were identified by filtering on the basis of multiple parameters like read length ≥ 200 nucleotides, exon numbers ≥2, FPKM ≥0.5, coding potential score < 0 etc. PFAM and RFAM were used to filter out all possible protein coding genes and housekeeping RNAs respectively. A total of 103 lncRNAs and 1057 mRNAs were found to be differentially expressed (DE) between the two stages (|log2FC| > 2, q < 0.05). We also identified 306 genes located around 100 kb upstream and 234 genes downstream around these DE lncRNA transcripts. The expression of top eleven DE lncRNAs (COL4A6, LY7S, MYH2, OXCT1, SMPDL3A, TMEM182, TTC36, RFOOOO4, RFOOO15, RFOOO45, CADM2) had been validating by qRT-PCR. Pathway and GO terms analysis showed that, positive regulation of biosynthetic process, Wnt signaling pathway, cellular protein modification process, and positive regulation of nitrogen compound were differentially enriched. Our results suggested that, KEGG pathways such as protein digestion and absorption, Arrhythmogenic right ventricular cardiomyopathy (ARVC) to be significantly enriched in both DE lncRNAs as well as DE mRNAs and involved in back fat tissues development. It also suggests that, identified lncRNAs are involved in regulation of important adipose tissues development pathways. • Analysis of lncRNAs in back fat tissues at different developmental stages in the pigs • A total of 103 lncRNAs and 1057 mRNAs were found to be differentially expressed. • KEGG pathway and GO analysis for functional annotation of lncRNAs • Top DE transcripts were validated by qRTPCR. • Relation between back fat metabolism and lncRNAs were established. [ABSTRACT FROM AUTHOR]

Abstract Whether plasma miRNAs could be used as novel non-invasive biomarkers in diagnosing papillary thyroid carcinoma (PTC) remains unknown. In this study, we designed a four-phase study to identify differentially expressed plasma miRNAs in Chinese PTC patients. Exiqon panel was initially utilized to conduct plasma miRNA profile (3 PTC pools VS. 1 healthy control (HC) pool; each 10 samples were pooled as 1 sample). The dysregulated miRNAs were then analyzed in the training (30 PTC VS. 30 HCs), testing (57 PTC VS. 54 HCs) and external validation phases (33 PTC VS. 30HCs). The identified miRNAs were further affirmed in benign nodules (2 nodular goiter (NG) pool VS. 1 HC pool). We also verified the expression of identified miRNAs in 17 matched malignant and normal tissue samples, NG plasma samples (29 PTC VS. 29 NG) and plasma exosomes (25 PTC VS. 25 HCs). Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic value of the identified miRNAs. As a result, the screening phase demonstrated 30 dysregulated plasma miRNAs in PTC patients compared with HCs. After multiphase experiment processes, miR-346, miR-10a-5p and miR-34a-5p were found significantly elevated in PTC plasma samples relative to HCs. The areas under the ROC curve (AUC) of the three-miRNA panel for the training, testing and validation phases were 0.926, 0.811 and 0.816, separately. The panel could also differentiate PTC from NG with the AUC of 0.877. MiR-346 and miR-34a-5p but not miR-10a-5p were up-regulated in PTC tissues. And the three miRNAs showed consistently up-regulation in PTC plasma exosomes. In conclusion, our study established a three-miRNA panel in plasma with considerable clinical value in discriminating PTC from HC or NG. Highlights • Plasma miR-346, miR-10a-5p and miR-34a-5p were elevated in PTC patients relative to HCs. • The three-miRNA panel could differentiate PTC patients from NG patients or HCs. • MiR-346 and miR-34a-5p but not miR-10a-5p were elevated in tumor tissues to normal tissues. • All the three miRNAs were up-regulated in exosomes from PTC plasma samples compared to HCs. [ABSTRACT FROM AUTHOR]

• In the study on C. grandis , a dioecious Cucurbitaceae species, six reference genes were utilized in qRT-PCR profiling during floral whorls and flower development in male and female plants. • The algorithms geNorm, NormFinder, RefFinder, and BestKeeper were employed to identify the most suitable internal controls from candidate reference genes. • The β-actin combined with β-tubulin emerged as optimal reference genes for qRT-PCR studies on floral samples. • This study represents the first validation of candidate reference genes across flower developmental stages in the dioecious species C. grandis. • The findings provide fundamental data for researching the molecular mechanisms of flower development in C. grandis and lay a foundation for similar investigations in related species. The flowering process is intricate and regulated by a combination of external and internal factors. Delving into gene expression research has the potential to enhance our comprehension of the molecular foundations underlying floral development. Because of its accuracy, specificity, reproducibility, and efficiency, qRT-PCR is now a biological research tool for studying expression pattern of desired genes. The gene expression investigations using qRT-PCR required a reference gene with relatively uniform expression levels in multiple biological samples, including different developmental stages, tissues, and experimental conditions. In this study, experimental sets of floral and floral organ development in the male and female plants of C. grandis , a dioecious Cucurbitaceae species, qRT-PCR profiling was performed using six reference genes as internal control with B-class floral identity gene, PISTILLATA (PI). To analyse the data, algorithms such as geNorm, NormFinder, RefFinder, and BestKeeper were used to pick out the best internal controls from a group of candidates. The optimal reference gene for qRT-PCR studies with floral samples has been recommended as β-actin combined with β-tubulin. This is the first report on the validation of candidate reference genes across flower developmental stages in the dioecious species C. grandis , which will provide basic data for research on the molecular mechanism underlying flower development in this species and lay the groundwork for similar studies in other related species. [ABSTRACT FROM AUTHOR]

[Display omitted] • Five GPX genes were identified in chickpea on a genome-wide scale. • GPX genes were highly conserved among C. arietinum, A. thaliana and M. truncatula. • CaGPX3 could play important role for salt tolerance in chickpea. Chickpea is the second most widely grown legume in the world. Its cultivation is highly affected by saline soils. Salt stress damages its all growth stages from germination to maturity. It has a huge genetic diversity containing adaptation loci that can help produce salt-tolerant cultivars. The glutathione peroxidase (GPX) gene family plays an important role in regulating plant response to abiotic stimuli and protects cells from oxidative damage. In current research, the role of GPX genes is studied for inducing salt tolerance in chickpea. This study identifies the GPX gene family in Cicer arietinum. In response to the NaCl stress, the gene expression profiles of CaGPX3 were examined using real-time qRT-PCR. The results of phylogenetic analysis show that CaGPX genes have an evolutionary relationship with monocots, dicots, chlorophytes, and angiosperms. Gene structure analysis showed that CaGPX3 , CaGPX4 , and CaGPX5 have six, CaGPX2 has five, and CaGPX1 contains 9 exons. According to the Ka and Ks analysis chickpea has one pair of duplicated genes of GPX and the duplication was tandem with negative (purifying) selection Ka < Ks (<1). In-silico gene expression analysis revealed that CaGPX3 is a salt stress-responsive gene among all other five GPX members in chickpea. The qRT-PCR results showed that the CaGPX3 gene expression was co-ordinately regulated under salt stress conditions, confirming CaGPX3 ′s key involvement in salt tolerance. [ABSTRACT FROM AUTHOR]

Abstract Colorectal cancer (CRC) has been one of the most commonly diagnosed cancers in global. The differential expression profiles of microRNAs (miRNAs) in CRC plasma of patients have the potential to serve as a diagnostic biomarker. We conducted a four-stage study to identify the potential plasma miRNAs for CRC detection. In the initial screening phase, Exiqon panel (miRCURY-Ready-to-Use-PCR-Human-panel-I + II-V1.M) including 3 CRC pools and 1 normal controls (NCs) pool were applied to acquire miRNA profiles. In the training stage (30 CRC VS. 30 NCs) and testing stage (79 CRC VS. 76 NCs), quantitative real-time polymerase chain reaction (qRT-PCR) was utilized to conduct candidate miRNA profiles. Then the identified miRNAs were verified in external validation stage (30 CRC VS. 26 NCs). Expression levels of identified miRNAs were assessed in tissue samples (24 pairs) and plasma exosomes (18 CRC VS. 18 NCs). Receiver operating characteristic (ROC) curves were constructed to evaluate the diagnostic accuracy. Seven miRNAs (miR-103a-3p, miR-127-3p, miR-151a-5p, miR-17-5p, miR-181a-5p, miR-18a-5p and miR-18b-5p) were significantly overexpressed in CRC compared with NCs. Area under the ROC curve of the seven-miRNA signature was 0.762, 0.824 and 0.895 for the training, testing and the external validation stages, respectively. Additionally, miR-103a-3p, miR-127-3p, miR-17-5p and miR-18a-5p were discovered significantly up-regulated in CRC tissues; while miR-17-5p, miR-181a-5p, miR-18a-5p and miR-18b-5p were significantly elevated in CRC plasma exosomes. In conclusion, we established a seven-miRNA signature in the peripheral plasma for CRC detection. Highlights • We investigated plasma miRNA profiles of CRC in a four-stage process and present a seven-miRNA signature in discriminating CRC. • We found that miR-103a-3p, miR-127-3p, miR-17-5p and miR-18a-5p were significantly up-regulated in CRC tissues. • We found that miR-17-5p, miR-181a-5p, miR-18a-5p and miR-18b-5p were significantly elevated in CRC plasma exosomes. • We discovered that plasma miR-26a-5p was only up-regulated in colon cancer patients but not in rectal cancer. • We found none of the seven miRNAs demonstrated significant different expression in patients with stage III + IV compared to those with stage I + II. • We found that no difference of the above seven plasma miRNA between left-sided and right-sided CRC. [ABSTRACT FROM AUTHOR]

Abstract In- silico and functional genomics approaches have been used to determine cellular functions of two hypothetical proteins All1122 and Alr0750 of Anabaena sp. PCC 7120. Motif analysis and multiple sequence alignment predicted them as typical α/β ATP binding universal stress family protein-A (UspA) with G-(2×)-G-(9×)-G(S/T) as conserved motif. qRT-PCR data under UV-B, NaCl, heat, As, CdCl 2, mannitol and methyl viologen registered approximately 1.4 to 4.3 fold induction of all1122 and alr0750 thus confirming their multiple abiotic stress tolerance potential. The recombinant E. coli (BL21) cells harboring All1122 and Alr0750 showed 12–41% and 23–41% better growth respectively over wild type control under said abiotic stresses thus revalidating their stress coping ability. Functional complementation on heterologous expression in UspA mutant E. coli strain LN29MG1655 (ΔuspA::Kan) attested their UspA family membership. This study tempted us to suggest that recombinant Anabaena PCC 7120 over expressing all1122 and alr0750 might contribute to the nitrogen economy in paddy fields experiencing array of abiotic stresses including drought and nutrient limitation. Highlights • Molecular characterization of hypothetical proteins All1122 and Alr0750 from Anabaena PCC 7120 as UspA superfamily members. • qRT PCR revealed higher fold expression of all1122 and alr0750 under multiple stresses in Anabaena PCC 7120. • E. coli transformed with recombinant vectors showed tolerance compared to wild type under different abiotic stresses. [ABSTRACT FROM AUTHOR]

Abstract Zinc finger GATA like protein-1 (ZGLP1) is a nuclear zinc finger protein that regulates the interaction between somatic cells and germ cells during gonad developmental process in mammals. In this study, the zglp1 of Chinese tongue sole, Cynoglossus semilaevis (cysezglp1), was cloned and characterized for the first time in fish. Cysezglp1 had an open reading frame with five exons and was located to chromosome 9. The open reading frame of cysezglp1 consisted of 1692 nucleotides and encoded a 583 amino acid polypeptide. The predicted protein contained two zinc finger structures (Znf1 and Znf2), one of which was highly homologous to the GATA-type zinc finger domain. Multiple sequence alignment showed that Znf1 was conserved across different species while Znf2 was more divergent. Through quantitative Real-time PCR (qRT-PCR), we found that cysezglp1 was predominantly expressed in gonads, and the expression level of the ovary was significantly higher than that of the testis. We compared expression level in different embryonic stages and found that cysezglp1 mRNAs were mainly expressed in the fertilized egg to the cleavage stage, subsequently declining in the blastula stage. Cysezglp1 expression was not detected from the gastrulation stage onward. In the ovary, cysezglp1 expression was detected at 120 days after hatching and expression gradually increased with the maturation of the ovary. In situ hybridization showed that the cysezglp1 was mainly expressed in oocytes. Taken together, our results suggest that cysezglp1 may play an important role in the process of oogenesis in Chinese tongue sole. Highlights • Zinc finger GATA like protein-1 (zglp1) was cloned and characterized for the first time in fish. • RT-PCR and ISH demonstrated that cysezglp1 is mainly expressed in oocytes. • Cysezglp1 may play an important role in oogenesis in the Chinese tongue sole. [ABSTRACT FROM AUTHOR]

Abstract Chemoreception is critical for the survival of insects. Insects have a variety of behavioral responses, such as mating, host searching and ovipositing, in response to different odor signals detected in their living environment. Trichogramma chilonis , an egg parasitoid, acts as an efficient and effective biocontrol reagent for many agricultural and forestry insect pests in many parts of China. However, little is known about the molecular mechanism of the olfaction-evoked behavior in T. chilonis. In the present study, we conducted transcriptome profiling analysis of T. chilonis based on the Illumina high-throughput sequencing platform in order to explore differences of chemoreception between male and female T. chilonis. In this study, a total of 85 chemosensory genes were identified from transcriptomic data, including 45 odorant receptors (ORs), 22 odorant binding proteins (OBPs), 14 ionotropic receptors (IRs), 2 sensory neuron membrane proteins (SNMPs) and 2 chemosensory proteins (CSPs). From the analysis of the transcriptome, most of the candidate olfactory genes had similar expression levels in males and females, including a few OR and OBP genes (TchiOR38, TchiOR39, TchiOR40, TchiOR41, TchiOR42, TchiOR43, TchiOR44, TchiOR45, TchiOBP1, TchiOBP4, TchiOBP10, TchiOBP12, TchiOBP18 and TchiOBP19) which showed male-biased expression. Some annotated unigenes were chosen randomly to have qRT-PCR, which verified the correctness of analysis of transcriptome in T. chilonis. This is the first study to obtain and identify candidate genes related to chemoreception in T. chilonis. Our work lays a solid foundation for related future research on the chemosensory system of T. chilonis at the molecular level and helps advance the use of T. chilonis as biological control agents. Highlights • This is the first study to obtain and identify candidate genes related to chemoreception in Trichogramma chilonis. • Chemosensory genes were identified from transcriptomic data of Trichogramma chilonis. • 8 odorant receptors, 6 odorant binding proteins and 1 sensory neuron membrane protein were male-biased expressed in Trichogramma chilonis. [ABSTRACT FROM AUTHOR]

Neuropeptides and peptide hormones play central roles in the regulation of various types of insect physiology and behavior. Artificial light at night, a form of environmental stress, has recently been regarded as a source of light stress on nocturnal insects. Because related genomic information is not available, molecular biological studies on the response of neuropeptides in nocturnal insects to light stress are limited. Based on the de novo sequencing of the Helicoverpa armigera head transcriptome, we obtained 124,960 unigenes. Of these, the number of unigenes annotated as neuropeptides and peptide hormones, neurotransmitter precursor processing enzymes, and neurotransmitter receptors were 34, 17, and 58, respectively. Under light stress, there were sex-specific differences in gene expression measured by qRT-PCR. The IMFamide, leucokinin and sNPF genes were differentially expressed at the mRNA level in males but not in females in response to light stress. The results provide new insights on the diversity of the neuropeptide transcriptional network of H . armigera . In addition, some neuropeptides exhibited sex-specific differential expression in response to light stress. Taken collectively, these results not only expand the catalog of known insect neuropeptides but also provide a framework for future functional studies on the physiological roles they play in the light stress response behavior of nocturnal moths. [ABSTRACT FROM AUTHOR]

Radish is an economically important vegetable crop belonging to the family Brassicaceae. The high anthocyanin content of the ‘Xinlimei’ radish roots has been associated with diverse health benefits. However, there is a lack of transcript-level information regarding anthocyanin biosynthesis. In the present study, the ‘Xinlimei’ radish root transcriptome was analyzed by RNA sequencing at five developmental stages. A total of 222,384,034 clean reads were obtained and 32,253 unigenes were annotated. Expression profiles revealed 10,890 differentially expressed genes (DEGs) among the five analyzed libraries. The DEGs were predominantly involved in KEGG pathways related to the biosynthesis of phenylpropanoids, flavonoids, flavone, and flavonol. The transcriptome data revealed 44 structural and 182 transcription factor genes (TFs) associated with the anthocyanin biosynthetic pathway. Ten structural genes (i.e., 4CL3 , CHSB2 , CHS1 , CHS3 , F3H1 , F3 ′ H , DFR , DFR1 , ANS , and UFGT ) and two MYB genes, which were highly and differentially expressed during root development, may be critical for anthocyanin biosynthesis. Additionally, the co-expression of TFs and structural genes was analyzed. Three structural genes (i.e., DFR , ANS , and UFGT ) were validated by molecular cloning. The qRT-PCR results indicated that the expression profiles of DEGs were generally consistent with the high-throughput sequencing results. These findings helped identify candidate genes involved in anthocyanin biosynthesis and may be useful for clarifying the molecular mechanism underlying the accumulation of anthocyanins in radish roots. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are short, single stranded RNA molecules with approximately 22 nts in length, which regulate the stability and translation of messenger RNAs in several organisms. To increase the repertoire of miRNAs characterized in M. amblycephala, we used the deep sequencing technology to sequence a small RNA library using pooled RNA sample isolated from the 4 different tissues of M. amblycephala . A total of 309 conserved miRNAs that originated from 131 miRNA families were detected. 15 novel candidates miRNA were identified. Randomly selected 6 miRNAs were analyzed by stem-loop qRT-PCR and differential expression patterns were observed in 6 different tissues of M. amblycephala . Furthermore, the potential targets were predicted. GO analysis showed that most of the targets were involved in a broad range of physiological functions including fish growth, development, metabolism, stress responses and so on. Overall, our results significantly increased the number of novel miRNAs in M. amblycephala, which should be useful for further investigation into the role of miRNAs in regulating diverse biological processes. [ABSTRACT FROM AUTHOR]

The appropriate reference genes are important and essential for reliable results of transcript normalization in real-time qRT-PCR. In the current study, we identified 1203 stably expressed genes from 35,286 genes' expression profiles in developmental fruits of Actinidia chinensis . We manually selected six candidate genes and assessed their expression levels, using two sets of fruit samples of A. chinensis : flesh fruits at four developmental stages and post-harvested fruits. The expression stability of these six genes was assessed by three independent algorithms: geNorm, NormFinder, and BestKeeper. Statistical results indicated these six genes can serve as internal control in both developmental and post-harvested fruits. Among these genes, UBQ_CONJ_E2 ( Ubiquitin-conjugating enzyme E2 36 ) and TUB_FCB ( Tubulin folding cofactor B ) were the two best reference genes identified in this study. The identification and validation of these reference genes can be helpful for elucidating the studies of fruit development and post-harvested fruits' storage in A. chinensis and other fruit crops of Actinidiaceae. [ABSTRACT FROM AUTHOR]

In this study, an α-carbonic anhydrase (α-CA), HcCA3 , from Hyriopsis cumingii was characterized. The full-length cDNA of HcCA3 was 1628 bp, including a CA domain and an ORF of 1053 bp which encoded 350 amino acids. Its predicted molecular weight was 39.69 kDa and the pI was 5.92. qRT-PCR was used to determine the expression of the gene in various tissues at 0 h, 3 h, 6 h, 12 h, 24 h, 48 h, 96 h, 7 d, 14 d, 21 d, 28 d and 35 d after inserting the pearl nucleus. The results showed that the HcCA3 was highly expressed in the mantle, whereas its expression was low in other tissues. Expression in the posterior mantle pallial (pMP) was significantly higher than that in the anterior mantle pallial (aMP) and mantle center (MC). Expression in the aMP, pMP and MC was significantly higher in purple mussels compared with that in white mussels. At the same time, during the formation of pearls, expression in the aMP, pMP and pearl sac (PS) decreased and then increased; whereas expression in the MC increased and then decreased. In-situ hybridization showed that the HcCA3 was expressed in both inside and outside epidermal cells. In protein level, Western blot showed that HcCA3 was mainly expressed in the aMP, pMP and MC. Our results suggest that HcCA3 play a role in the formation of shell and pearl sac formation. [ABSTRACT FROM AUTHOR]

Insulin-like growth factor 1 receptor (IGF-1R) is proved to contribute the development of many types of cancers. But, little is known about its roles in radio-resistance of colorectal cancer (CRC). Here, we demonstrated that low IGF-1R expression value was associated with the better radiotherapy sensitivity of CRC. Besides, through Quantitative Real-time PCR (qRT-PCR), the elevated expression value of epidermal growth factor receptor (EGFR) was observed in CRC cell lines (HT29, RKO) with high radio-sensitivity compared with those with low sensitivity (SW480, LOVO). The irradiation induced apoptosis rates of wild type and EGFR agonist (EGF) or IGF-1R inhibitor (NVP-ADW742) treated HT29 and SW480 cells were quantified by flow cytometry. As a result, the apoptosis rate of EGF and NVP-ADW742 treated HT29 cells was significantly higher than that of those wild type ones, which indicated that high EGFR and low IGF-1R expression level in CRC was associated with the high sensitivity to radiotherapy. We next conducted systemic bioinformatics analysis of genome-wide expression profiles of CRC samples from the Cancer Genome Atlas (TCGA). Differential expression analysis between IGF-1R and EGFR abnormal CRC samples, i.e. CRC samples with higher IGF-1R and lower EGFR expression levels based on their median expression values, and the rest of CRC samples identified potential genes contribute to radiotherapy sensitivity. Functional enrichment of analysis of those differential expression genes (DEGs) in the Database for Annotation, Visualization and Integrated Discovery (DAVID) indicated PPAR signaling pathway as an important pathway for the radio-resistance of CRC. Our study identified the potential biomarkers for the rational selection of radiotherapy for CRC patients. [ABSTRACT FROM AUTHOR]

Real-time quantitative reverse transcriptase-PCR (qRT-PCR) is a feasible tool for determining gene expression profiles, but the accuracy and reliability of the results depends on the stable expression of selected housekeeping genes in different samples. By far, researches on stable housekeeping genes in human heart failure samples are rare. Moreover the effect of heart failure on the expression of housekeeping genes in right and left ventricles is yet to be studied. Therefore we aim to provide stable housekeeping genes for both ventricles in heart failure and normal heart samples. In this study, we selected seven commonly used housekeeping genes as candidates. By using the qRT-PCR, the expression levels of ACTB , RAB7A , GAPDH , REEP5 , RPL5 , PSMB4 and VCP in eight heart failure and four normal heart samples were assessed. The stability of candidate housekeeping genes was evaluated by geNorm and Normfinder softwares. GAPDH showed the least variation in all heart samples. Results also indicated the difference of gene expression existed in heart failure left and right ventricles. GAPDH had the highest expression stability in both heart failure and normal heart samples. We also propose using different sets of housekeeping genes for left and right ventricles respectively. The combination of RPL5 , GAPDH and PSMB4 is suitable for the right ventricle and the combination of GAPDH , REEP5 and RAB7A is suitable for the left ventricle. [ABSTRACT FROM AUTHOR]

The rice root-knot nematode, Meloidogyne graminicola , seriously impairs the growth and yield of rice which is an important staple food worldwide. The disruption of neuropeptide signalling leading to attenuation in nematode behaviour and thereby perturbed infection, offers an attractive alternative to control nematodes. In this direction, the present study was aimed at mining of putative FMRFamide-like peptides (FLPs) from the transcriptomic dataset of M. graminicola followed by characterization of those FLPs via sequencing of PCR products, qRT-PCR and Southern hybridization analysis. We have characterized nine flp genes ( flp-1 , flp-3 , flp-6 , flp-7 , flp-11 , flp-12 , flp-14 , flp-16 and flp-18 ) and a partial neuropeptide receptor gene ( flp-18 GPCR) from M. graminicola in the present study. In addition, in situ localization revealed the expression of flp-1 and flp-7 in neurons posterior to the circumpharyngeal nerve ring of M. graminicola . In vitro silencing of nine flp genes and flp-18 GPCR in M. graminicola J2 and their subsequent infection in rice and wheat roots demonstrated the reduced penetration ability of FLP silenced worms which underscores the potential of the FLPergic system as a broad-spectrum target to manage the root-knot nematode problem in rice-wheat cropping system. [ABSTRACT FROM AUTHOR]

• The gene expression of three TRPMLs is abundant in mouse spleen, lung, and kidney tissues. • Three TRPMLs show different responses to pathogen stimuli in lung tissue and A549 cells. • The responses of three TRPMLs to pathogen stimuli that occur similarly in other tissues. • LPS exhibits a significant effect on three overexpressed TRPMLs in A549 cells. • Both TRPML1- and TRPML3-specific activators show dose-dependent upregulation of inflammatory factors. The transient receptor potential mucolipin (TRPML) subfamily in mammalian has three members, namely TRPML1, TRPML2, and TRPML3, who play key roles in regulating intracellular Ca2+ homeostasis, endosomal pH, membrane trafficking and autophagy. Previous studies had shown that three TRPMLs are closely related to the occurrence of pathogen invasion and immune regulation in some immune tissues or cells, but the relationship between TRPMLs expression and pathogen invasion in lung tissue or cell remains elusive. Here, we investigated the expression distribution of three TRPML channels in mouse different tissues by qRT-PCR, and then found that all three TRPMLs were highly expressed in the mouse lung tissue, as well as mouse spleen and kidney tissues. The expression of TRPML1 or TRPML3 in all three mouse tissues had a significant down-regulation after the treatment of Salmonella or LPS, but TRPML2 expression showed a remarkable increase. Consistently, TRPML1 or TRPML3 but not TRPML2 in A549 cells also displayed a decreased expression induced by LPS stimulation, which shared a similar regulation pattern in the mouse lung tissue. Furthermore, the treatment of the TRPML1 or TRPML3 specific activator induced a dose-dependent up-regulation of inflammatory factors IL-1β, IL-6 and TNFα, suggesting that TRPML1 and TRPML3 are likely to play an important role in immune and inflammatory regulation. Together, our study identified the gene expression of TRPMLs induced by pathogen stimulation in vivo and in vitro , which may provide novel targets for innate immunity or pathogen regulation. [ABSTRACT FROM AUTHOR]

Phycocyanin (PC) from Spirulina platensis has inhibitory effects on tumor cell growth. In this research, the transcriptome study was designed to investigate the underlying molecular mechanisms of PC inhibition on human ovarian cancer cell SKOV-3 proliferation. The PC IC 50 was 216.6 μM and 163.8 μM for 24 h and 48 h exposure, respectively, as determined by CCK-8 assay. The morphological changes of SKOV-3 cells after PC exposure were recorded using HE staining. Cells arrested in G 2 /M stages as determined by flow cytometry. The transcriptome analysis showed that 2031 genes (with > three-fold differences) were differentially expressed between the untreated and the PC-treated cells, including 1065 up-regulated and 966 down-regulated genes. Gene ontology and KEGG pathway analysis identified 18 classical pathways that were remarkably enriched, such as neurotrophin signaling pathway, VEGF signaling pathway and P53 signaling pathway. qPCR results further showed that PTPN12 , S100A2 , RPL26 , and LAMA3 increased while HNRNPA1P10 decreased in PC-treated cells. Molecules and genes in those pathways may be potential targets to develop treatments for ovarian cancer. [ABSTRACT FROM AUTHOR]

• BES1 TFs regulate plant growth, development, and stress resistance, and play a pivotal role in the BR signal transduction pathway. • qRT-PCR analysis showed that the expression levels of VvBES1 genes differed in response to BR, MeJA, cold (-4 °C), NaCl, and PEG treatments. • VvBES1-3 overexpression enhances salt stress tolerance in transgenic Arabidopsis. • This study will contribute to further understanding the functions of BES1 TFs in the abiotic stress response. BRI1-EMS-Suppressor 1 (BES1) regulates plant growth, development, and stress resistance, and plays a pivotal role in the brassinosteroid (BR) signal transduction pathway. In this study, a total of 12 BES1 genes were identified in the grape (Vitis vinifera) genome. Phylogenetic, structure, and motif sequence analyses of these genes provided insights into their evolutionary characteristics. Hormone-, stress-, and light-responsive and organ-specific cis -acting elements were identified in VvBES1 gene promoters. Microarray data analysis showed that VvBES1 family members exhibit diverse expression patterns in different organs. Quantitative real-time PCR (qRT-PCR) analysis showed that the expression levels of VvBES1 genes differed in response to BR, methyl jasmonate (MeJA), cold (4 °C), NaCl, and polyethylene glycol (PEG) treatments. The expression of VvBES1-3 was 29-fold higher under salt stress than control at 12 h. Moreover, VvBES1-3 -overexpessing Arabidopsis thaliana plants showed lower malondialdehyde content, higher proline content, enhanced antioxidant enzyme (catalase, superoxide dismutase, peroxidase) activities, and higher salt-responsive gene expression levels than wild-type plants under salt stress, indicating that VvBES1-3 overexpression enhances salt stress tolerance in transgenic Arabidopsis. These results will contribute to further understanding the functions of BES1 transcription factors in the abiotic stress response. [ABSTRACT FROM AUTHOR]

• The SNP of ALB was significantly associated with ADG. • The SNP of AHSG was significantly associated with ADG and FCR. • The SNP of GC was significantly associated with BW, BH and BL. • ALB , AHSG , and GC expression levels were significantly higher in H_BW group. Growth traits are economically important traits in sheep breeding. This study was conducted to evaluate the polymorphisms of ALB , AHSG and GC genes and their association with growth traits in Hu sheep. We measured and recorded the body weight (BW), body height (BH), body length (BL) and feed conversion ratio (FCR) of 1418 male Hu sheep raised in the same environment from 80 to 180 days of age. The total of four SNPs in the ALB , AHSG and GC genes were identified by direct sequencing technology. The results of association analysis showed that two loci (g.8699 A>T and g.13458 T>C) of ALB gene significantly affect average daily gain (ADG; P < 0.05). The genotypes of SNP g.2454 T>C in AHSG gene were significantly associated with ADG and FCR (P < 0.05). There were significant associations between GC g.19484 A>C and BW, BH and BL (P < 0.05). The results of qRT-PCR showed that ALB , AHSG , and GC genes were extremely significantly higher in H_BW sheep compared with those in the L_BW sheep (P < 0.01). These results revealed that ALB-1 g.8699 A>T, ALB-2 g.13458 T>C, AHSG g.2454 T>C and GC g.19484 A>C loci are potential molecular markers for Hu sheep breeding. [ABSTRACT FROM AUTHOR]

NAC (NAM, ATAF, and CUC) is one of the largest transcription factor families in plants, and its members play various roles in plant growth, development, and the response to biotic and abiotic stresses. Currently, 77 NAC genes have been reported in cotton ( Gossypium hirsutum L.). And GhNAC12 showed up-regulation during leaf senescence, but its role in this process is poorly understood. In the present study, a preliminary function analysis of GhNAC12 was performed during leaf senescence. qRT-PCR analysis indicated that GhNAC12 expression increased during the early-aging process and the aging of cotyledons. Additionally, we observed that overexpression of GhNAC12 in Arabidopsis led to early senescence (early aging). Our findings suggest that GhNAC12 is a candidate gene for early aging in upland cotton cultivars. Neutrality tests suggested that there was no selection pressure imposed on GhNAC12 during the domestication of upland cotton. [ABSTRACT FROM AUTHOR]

Phaseolus vulgaris is subjected to serious post-harvest diseases such as grey mold and cottony rot diseases caused by Botrytis cinerea and Pythium aphanidermatum , respectively . In current study, potassium silicate (KSi), potassium thiosulfate (KTS) and potassium sulfate (KS) suppressed moderately the growth of B. cinerea and P. aphanidermatum in vitro . The applied treatments significantly suppressed grey mold and cottony rot of Xera and Valentino snap beans varieties' pods stored at 7 ± 1 °C and 90–95% RH for 20 days. Ethylene responsive factor ( ERF ), polygalacturonase inhibitor protein ( PGIP ), phosphatase associated to defense ( PA ) and pathogenesis-related protein ( PR1 ) defense genes were over-expressed in leaves tissue of both bean varieties responding positively to potassium salts field application. The expression of these genes was influenced by plant genotype and environment as it varied by snap bean varieties. Accumulation of ERF , GIP , PA and PR1 genes transcript under KTS at 4000 ppm treatment were the highest in Xera tissues (3.5-, 4.8-, 4- and 4.8-fold, respectively). In conclusion, pre-harvest potassium salt in vivo application could be used as effective safe alternatives to fungicides against grey mold and cottony rot diseases of snap beans during storage for up to 20 days at 7 ± 1 °C. [ABSTRACT FROM AUTHOR]

As invasive species have successfully colonized a wide range of dramatically different local environments, they offer a good opportunity to study interactions between species and rapidly changing environments. Gene expression represents one of the primary and crucial mechanisms for rapid adaptation to local environments. Here, we aim to select reference genes for quantitative gene expression analysis based on quantitative Real-Time PCR (qRT-PCR) for a model invasive ascidian, Ciona savignyi . We analyzed the stability of ten candidate reference genes in three tissues (siphon, pharynx and intestine) under two key environmental stresses (temperature and salinity) in the marine realm based on three programs (geNorm, NormFinder and delta Ct method). Our results demonstrated only minor difference for stability rankings among the three methods. The use of different single reference gene might influence the data interpretation, while multiple reference genes could minimize possible errors. Therefore, reference gene combinations were recommended for different tissues - the optimal reference gene combination for siphon was RPS15 and RPL17 under temperature stress, and RPL17 , UBQ and TubA under salinity treatment; for pharynx, TubB , TubA and RPL17 were the most stable genes under temperature stress, while TubB , TubA and UBQ were the best under salinity stress; for intestine, UBQ , RPS15 and RPL17 were the most reliable reference genes under both treatments. Our results suggest that the necessity of selection and test of reference genes for different tissues under varying environmental stresses. The results obtained here are expected to reveal mechanisms of gene expression-mediated invasion success using C. savignyi as a model species. [ABSTRACT FROM AUTHOR]

Roche 454 next-generation sequencing was applied to obtain extensive information about the transcriptomes of the bread wheat cultivar Yunong 201 and its EMS mutant line Yunong 3114. Totals of 1.43million and 1.44 million raw reads were generated, 14,432, 17,845 and 27,867 isotigs were constructed using the reads in Yunong 201, Yunong 3114 and their combination, respectively. Moreover, 29,042, 34,722, and 48,486 unigenes were generated in Yunong 201, Yunong 3114, and combined cultivars, respectively. A total of 50,382 and 59,891 unigenes from the Yunong 201 and Yunong 3114 were mapped on different chromosomes. Of all unigenes, 1363 DEGs were identified in Yunong 201 and Yunong 3114. qRT-PCR analysis confirmed the expression profiles of 40 candidate unigenes possibly related to abiotic stresses. The expression patterns of four annotated DEG swere also verified in the two wheat cultivars under abiotic stresses. This study provided useful information for further analysis of wheat functional genomics. [ABSTRACT FROM AUTHOR]

Gentiana straminea is a popular Tibetan medicine that has been used for thousands of years in China to treat various diseases and conditions. Although it has multiple pharmaceutical purposes and important economic plant resource in China, transcriptome and molecular base still known limited. In flowering season, samples were collected from different tissues, using the NGS Illumina. Solexa platform, about 58.85 million sequencing reads were generated and assembled de novo, yielding 78,764 high quality unigenes with an average length of 1090 bp. Gene Ontology (GO), KEGG pathway mapping showed that 49,033 of these were identified as putative homologs of annotated sequences in the protein databases. Among them, candidate genes associated with iridoid, flavonoid and anthocyanin were identified. Further the key enzymes involved to iridoid and flavonoid synthesis pathway were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) on different tissues, the flower and root had the higher expression than leaves. In addition, 7591 SSR markers were identified from the unigenes of the G . straminea transcriptome. The foundation of G . straminea provided the important resource for facilitating to study molecular and functional genomics of it and related this species on the Qinghai–Tibet Plateau. [ABSTRACT FROM AUTHOR]

Quantitative real-time polymerase chain reaction (qRT-PCR) is widely used for the accurate analysis of gene expression. However, high homology among gene families might result in unsuitability of reference genes, which leads to the inaccuracy of qRT-PCR analysis. The release of the Ganoderma lucidum genome has triggered numerous studies to be done on the homology among gene families with the purpose of selecting reliable reference genes. Based on the G. lucdum genome and transcriptome database, 38 candidate reference genes including 28 novel genes were systematically selected and evaluated for qRT-PCR normalization. The result indicated that commonly used polyubiquitin ( PUB ), beta-actin ( BAT ), and glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) were unsuitable reference genes because of the high sequence similarity and low primer specificity. According to the evaluation of RefFinder, cyclophilin 5 ( CYP5 ) was ranked as the most stable reference gene for 27 tested samples under all experimental conditions and eighteen mycelial samples. Based on sequence analysis and expression analysis, our study suggested that gene characteristic, primer specificity of high homologous genes, allele-specificity expression of candidate genes and under-evaluation of reference genes influenced the accuracy and sensitivity of qRT-PCR analysis. This investigation not only revealed potential factors influencing the unsuitability of reference genes but also selected the superior reference genes from more candidate genes and testing samples than those used in the previous study. Furthermore, our study established a model for reference gene analysis by using the genomic sequence. [ABSTRACT FROM AUTHOR]

The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest. [ABSTRACT FROM AUTHOR]

Iron overload is the major cause of morbidity and mortality in transfusion dependent β-thalassemia major patients. There is a sophisticated balance of body iron metabolism of storage and transport which is regulated by several factors including the peptide hepcidin. Hepcidin is the main iron regulatory molecule; it is secreted mainly by the liver and other tissues including monocytes and lymphocytes. Expression of hepcidin in such cells is unclear and has been studied in few reports with controverted result. Peripheral expression of hepcidin was measured using quantitative real time PCR (qRT-PCR) in 50 β-thalassemia major patients, in addition to 20 healthy volunteers as a control group. Hepcidin levels in β-thalassemia major patients showed statistically significant decrease in comparison to the control group, and was correlated to cardiac iron stores (T2*). However, hepcidin level was not different among the patients according to the HCV status or whether splenectomized or not. In conclusion; peripheral expression of hepcidin, in iron overloaded β-thalassemia major patients, is a reflection of hepatic expression. It can be used as a molecular predictor for the severity of cardiac iron overload and can be used as a future target for therapy in β-thalassemia major patients. [ABSTRACT FROM AUTHOR]

Dead end ( dnd ) encodes an RNA-binding protein that is essential for primordial germ cell (PGC) migration and gametogenesis in vertebrates. In this study, a Chinese sturgeon Acipenser sinensis dead end homologue, designated Asdnd , was identified and characterized. The full-length cDNA of Asdnd was 1630 base pairs (bp) and encoded a peptide of 396 amino acid residues. Multiple sequence alignment showed that As Dnd shared six conserved regions of Dnd orthologs, including the RNA recognition motif. Phylogenetic analysis revealed that As Dnd was grouped with teleosts. By quantitative real-time PCR analysis, the Asdnd transcripts were found to originate from the maternal parent and be specifically expressed in gonads of immature Chinese sturgeons of both sexes. Fluorescent in situ hybridization indicated that Asdnd transcripts were restricted to germ cells. In the testis, Asdnd was abundant in spermatogonia and tended to gradually diminish in late spermatogenic stages, while strong signals were found in primary oocytes, as opposed to oogonia, in the ovary. Zebrafish PGCs were clearly visualized at 24 h post-fertilization by co-injecting RFP- Asdnd 3′ UTR and GFP- nos 3 3′ UTR mRNA, indicating that dnd 3′ UTR has a conserved function among teleosts. Therefore, dnd could serve as a germ cell marker in Chinese sturgeon. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) contamination in agricultural soils has become a major environmental problem in China. Ramie, a fiber crop, has frequently been proposed for use as a phytoremediation crop for the restoration of Cd-contaminated farmlands. However, high levels of Cd can greatly inhibit stem growth in ramie, which reduces its economic value as a crop. To understand the potential mechanisms behind this phenomenon, the ramie genes involved in the Cd stress response were identified using Illumina pair-end sequencing on two Cd-stressed plants (CdS1 and CdS2) and two control plants (CO1 and CO2). Approximately 48.7, 51.6, 41.2, and 47.1 million clean sequence reads were generated from the libraries of CO1, CO2, CdS1, and CdS2, respectively, and de novo assembled to yield 56,932 non-redundant unigenes. A total of 26,686 (46.9%) genes were annotated for their function. Comparison of gene expression levels in CO and CdS ramie revealed 155 differentially expressed genes (DEGs) between treatment and control conditions. Sixteen DEGs were further analyzed for expression differences by using real-time quantitative PCR (qRT-PCR). Among these 16 DEGs, 2 genes encoding GA2-oxidase (a major enzyme for deactivating bioactive gibberellins [GAs]) showed markedly up-regulated expression in Cd stressed ramie. This might be responsible for the growth inhibition of Cd-stressed ramie. Pathway enrichment analysis revealed that the cutin, suberine and wax biosynthesis pathway was markedly enriched by DEGs. The discovery of these Cd stress-responsive genes and pathways will be helpful in further understanding the mechanism of Cd-stress response and improving Cd stress tolerance in ramie. [ABSTRACT FROM AUTHOR]

The most common malignancy in women is breast cancer. Drug resistance in the treatment of cancer still remains a major clinical concern. Resistance to tamoxifen is seen in half of the recurrences in breast cancer. The anti-estrogen tamoxifen gains agonistic property by transactivating ERα. PAK1-mediated phosphorylation of serine 305 (S305) of ERα leads to resistance to tamoxifen. In our study, PAK1-induced suggestive tamoxifen resistance was designed. According to our hypothesis, phosphorylation of ERα-S305 by PAK1 may be reversed by PAK1 transcriptional inhibition by miR-221-3p due to miR-221-3p targeting the 3′ UTR of PAK1. For this purpose, we used Real-time PCR (qRT-PCR) to measure the expression level of miR-221-3p in ER-positive breast cancer cell lines (ZR-75-1, MCF7) and breast epithelial cell line, hTERT-HME1, as control in the laboratory in our department. The increase in the expression of PAK1 depending on miR-221-3p may be related to ZR-75-1 cell line which has invasive characteristic but other two ER + cancer cell lines, MCF7 and HCC1500, have milder cancer severity. miR-221-3p may have a role on regulation of PAK1 expression because miR-221-3p expression level decreases while PAK1 expression level increases in SKBR3 cell line. miR-221-3p and PAK1 expressions in MDA-MB-231 cell line are higher than that of hTERT-HME1 cell line. This may mean that miR-221-3p has no regulatory effect on of PAK1 expression in this cell line. According to these results, miR-221-3p may give crucial information about molecular mechanism of the disease upon PAK1 activity or different mechanisms with respect to histopathology and severity of breast cancer. [ABSTRACT FROM AUTHOR]

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is a powerful tool for the measurement of gene expression; however, the accuracy of this approach depends on the stability of reference genes. The objective of the present study was to identify the stable reference genes in orchardgrass ( Dactylis glomerata L.), a principal cool-season forage grass in the world. Ten candidate reference genes were selected in this study including ATP-binding [ ABC ], actin [ ACTIN ], cyclophilin [ CYP2 ], glyceraldehyde 3-phosphate dehydrogenase [ GAPDH ], beta-amylase 4 [ BAM4 ], zeitlupe [ ZTL ], MAP Kinase 4 [ MPK4 ], ubiquitin-conjugating enzyme [ UBC ], S-adenosylmethionine decarboxylase [ SAMDC ], and translationally controlled tumor protein [ TCTP ]. The candidate genes were assessed in orchardgrass leaves and roots under conditions of drought, high salinity, heat, waterlogging, and abscisic acid (ABA) treatments. We used GeNorm, BestKeeper, NormFinder, and RefFinder for qRT-PCR normalization and validation to determine that the expression of these reference genes was stress-dependent. ACTIN , CYP2 , and ABC were found to be the most stably expressed genes for drought stress while ACTIN , TCTP , and ABC were the most stable under salt stress. ACTIN , CYP2 , and ABC were all found to be good reference genes for studying heat stress. Likewise, CYP2 , MPK4 , and ABC were most suitable to study waterlogging, and ACTIN , CYP2 , and MPK4 were determined as the three best reference genes for ABA studies. Our study identified and validated the possible reference genes in orchardgrass that may be used for quantification of target gene expression under various abiotic stresses. [ABSTRACT FROM AUTHOR]

L-type lectin receptor kinase (LecRK) proteins are an important family involved in diverse biological processes such as pollen development, senescence, wounding, salinity and especially in innate immunity in model plants such as Arabidopsis and tobacco. Till date, LecRK proteins or genes of cucumber have not been reported. In this study, a total of 25 LecRK genes were identified in the cucumber genome, unequally distributed across its seven chromosomes. According to similarity comparison of their encoded proteins, the Cucumis sativus LecRK (CsLecRK) genes were classified into six major clades (from Clade I to CladeVI). Expression of CsLecRK genes were tested using QRT-PCR method and the results showed that 25 CsLecRK genes exhibited different responses to abiotic (water immersion) and biotic ( Phytophthora melonis and Phytophthora capsici inoculation) stresses, as well as that between disease resistant cultivar (JSH) and disease susceptible cultivar (B80). Among the 25 CsLecRK genes, we found CsLecRK6.1 was especially induced by P. melonis and P. capsici in JSH plants. All these results suggested that CsLecRK genes may play important roles in biotic and abiotic stresses. [ABSTRACT FROM AUTHOR]

MiRNAs are a novel group of non-coding small RNAs that negatively regulate gene expression. Many miRNAs have been identified and investigated extensively in plant species with sequenced genomes. However, few miRNAs have been identified in foxtail millet ( Setaria italica ), which is an ancient cereal crop of great importance for dry land agriculture. In this study, 271 foxtail millet miRNAs belonging to 44 families were identified using a bioinformatics approach. Twenty-three pairs of sense/antisense miRNAs belonging to 13 families, and 18 miRNA clusters containing members of 8 families were discovered in foxtail millet. We identified 432 potential targets for 38 miRNA families, most of which were predicted to be involved in plant development, signal transduction, metabolic pathways, disease resistance, and environmental stress responses. Gene ontology (GO) analysis revealed that 101, 56, and 23 target genes were involved in molecular functions, biological processes, and cellular components, respectively. We investigated the expression patterns of 43 selected miRNAs using qRT-PCR analysis. All of the miRNAs were expressed ubiquitously with many exhibiting different expression levels in different tissues. We validated five predicted targets of four miRNAs using the RNA ligase mediated rapid amplification of cDNA end (5′-RLM-RACE) method. [ABSTRACT FROM AUTHOR]

• The stability in vitro of reference genes in the stages of proliferation and differentiation of bovine preadipocytes during cells was studied systematically. • The stable reference genes in vitro of bovine preadipocytes during cells proliferation stage and differentiation stage are not completely consistent. • The stable in vitro reference genes of bovine preadipocytes during cells at the proliferation stage and differentiation stage are GAPDH and RPS15A 、 RPLP0 and EIF3K , respectively. The stability of internal reference genes is crucial to the reliability of gene expression results using real-time fluorescence quantitative PCR (qRT-PCR). Inappropriate reference genes may lead to inaccurate results or even wrong conclusions. This study aims to identify stable reference genes for analyzing the expression of proliferation-related and differentiation-inducing genes in bovine primary preadipocytes (BPPs) in vitro. In this study, the stability of 16 candidate internal reference genes (GAPDH , ACTB , PPIA , LRP10 , HPRT1 , YWHAZ , B2M , TBP , EIF3K , RPS9 , UXT , 18S rRNA , RPLP0 , MARVELD , EMD and RPS15A) for qRT-PCR at proliferation and differentiation stages of BPPs was investigated by three different algorithms (geNorm, NormFinder and BestKeeper). The expression of two marker genes, PCNA and LPL , was used to determine the validity of the candidate reference genes (RGs) at the proliferation and differentiation stages, respectively. The results showed that GAPDH and RPS15A were the most stable RGs in the proliferation of bovine primary preadipocyte, while PPIA was the least stable internal reference gene. RPLP0 and EIF3K were the most stable RGs in the differentiation induction of bovine primary preadipocyte, while GAPDH was the least stable internal reference gene. This study of RGs laid the foundation for subsequent research into the mechanism of proliferation and differentiation of BPPs in vitro using qRT-PCR. [ABSTRACT FROM AUTHOR]

Abstract: In both plants and animals, programmed cell death (PCD) is an indispensable process that removes redundant cells. In seedless grapes (Vitis vinifera), abnormal PCD in ovule cells and subsequent ovule abortion play key roles in stenospermocarpy. Metacaspase, a type of cysteine-dependent protease, plays an essential role in PCD. To reveal the characteristics of the metacaspase (MC) gene family and the relationship between metacaspases and the seedless trait, we identified the 6 V. vinifera metacaspases VvMC1–VvMC6, from the grape genome, using BLASTN against the 9 known Arabidopsis metacaspases. We also obtained full-length cDNAs by RT-PCR. Each of the 6 grape metacaspases contains small (p10-like) and a large (p20-like) conserved structural domains. Phylogenetic analysis of 6 grape and 9 Arabidopsis metacaspases showed that all metacaspases could be grouped into two classes: Type I and Type II. Each phylogenetic branch shares a similar exon/intron structure. Furthermore, the putative promoters of the grape metacaspases contained cis-elements that are involved in grape endosperm development. Moreover, expression analysis of metacaspases using real-time quantitative PCR demonstrated that VvMC1 and VvMC2 were able to be detected in any tissue, and VvMC3, VvMC4, VvMC5 and VvMC6 exhibited tissue-specific expression. Lastly, in cv. Thompson seedless grapes VvMC1, VvMC3, and VvMC4 were significantly up-regulated at the 35 DAF during ovule development, roughly same stage as endosperm abortion. In addition, the expression trend of VvMC2 and VvMC5 was similar between cv. Pinot Noir and cv. Thompson grape ovule development and that of VvMC6 was sustained in a relatively low level except the expression of cv. Pinot Noir significantly up-regulated in 25 DAF. Our data provided new insights into PCD by identifying the grape metacaspase gene family and provide a useful reference for further functional analysis of metacaspases in grape. [Copyright &y& Elsevier]

Thioredoxins (Trxs) are a family of small, highly conserved and ubiquitous proteins that are involved in protecting organisms against toxic reactive oxygen species (ROS). In this study, a typical thioredoxin 2 gene was isolated from Apis cerana cerana, AccTrx2. The full-length cDNA sequence of AccTrx2 was composed of 407bp containing a 318bp open reading frame (ORF) that encodes a predicted protein of 105 amino acids, 11.974kDa and an isoelectric point of 4.45. Expression profile of AccTrx2 as determined by a quantitative real-time PCR (qRT-PCR) analysis was higher in brain than in other tissues, with its highest transcript occurring on the 15day post-emergence adult and upregulated by such abiotic stresses as 4°C, 16°C, 25°C, H2O2, cyhalothrin, acaricide, paraquat, phoxime and mercury (HgCl2) treatments. However, AccTrx2 was slightly repressed when exposed to 42°C treatment. Characterisation of the recombinant protein showed that the purified AccTrx2 had insulin disulfide reductase activity and could protect DNA from ROS damage. These results indicate that AccTrx2 functions as an antioxidant that plays an important role in response to oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: The minichromosome maintenance (MCM) family of proteins is conserved from archaea to humans and is required for assembly of pre-replication complexes (pre-RCs) to initiate DNA replication. MCM9 is an uncharacterized member of the eukaryotic MCM protein family that contains conserved ATP binding and hydrolysis motifs. We have identified a novel alternatively spliced isoform of HsMCM9 that results in a medium length protein product (MCM9M) that eliminates a long C-terminal extension of the fully spliced product (MCM9L). Quantitative real-time reverse transcriptase PCR (qRT-PCR) separated and measured the relative mRNA isoform expression levels across a variety of cell lines. Although there is some variability in expression levels, the full length MCM9L transcript is more abundant than the MCM9M variant in all cell lines tested. The expression of both MCM9 isoforms is cell cycle regulated: induced in S-phase, decreases through G2/M, and becomes constant through G1. Consistent with recent reports suggesting MCM9 participates in repair or prevention of double strand breaks, mitomycin C significantly induces the specific expression of MCM9L, while the replication fork inhibitor, hydroxyurea, has no effect. Evolutionary analysis indicates that the MCM9M isoform is a conserved variant, whereas the addition of the terminal exon producing MCM9L appears to be a more recent event present only in the highest order of eukaryotes. [Copyright &y& Elsevier]

Abstract: Histone acetylation is a vital mechanism for the post-translational modifications of chromatin components. Histone acetyltransferases (HATs) are critical elements that determine histone acetylation and regulate chromatin dynamics and gene expression. While histone acetyltransferases have been well studied in mammals and Drosophila melanogaster, information from agriculturally important insect pests is still limited. In our effort to understand the epigenetic mechanisms regulating development in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Geometroidea), a major rice pest in many parts of Asia, two full-length cDNA sequences encoding HAT members of the GNAT and MYST family, namely NlElp3 and NlMof, respectively, were isolated and structurally and phylogenetically characterized. The NlElp3 contains an open reading frame (ORF) of 1656bp encoding a protein of 551 amino acids. The NlMof contains a 1353bp ORF encoding a protein of 450 amino acids. Sequence analysis showed that NlElp3 contains GNAT-type HAT domain and Radical SAM domain, and NlMof contains chromodomain and MOZ-SAS acetyltransferase domain. Multiple sequence alignments showed that NlElp3 and NlMof have high amino acid sequence identity with other insect homologues. Expression analysis of the NlElp3 and NlMof revealed significant differences in mRNA expression levels among N. lugens developmental stages, suggesting that HAT activities of NlElp3 and NlMof may be controlled, at least in part, by their developmental regulation. Remarkably, the mRNA expression levels of NlElp3 and NlMof in female adults were significantly higher than that in male adults, supporting an important role for both genes in female reproductive function in N. lugens. [Copyright &y& Elsevier]

Abstract: In fish, some ABC transporters are implicated in a multixenobiotic resistance (MXR) mechanism to deal with the presence of xenobiotics, by effluxing them, or their metabolites, from inside the cells. These efflux transporters have been considered an integral part of cellular detoxification pathways, acting in coordination with phase I and II detoxification enzymes. However, the full characterization of this detoxification system is still incomplete, especially during the developmental stages of aquatic organisms, which are particularly sensitive periods to the presence of anthropogenic contamination. The goal of this study was to evaluate the mRNA expression dynamics of putatively important MXR proteins (ABCB1b, ABCB11, ABCC1, ABCC2 and ABCG2a) and phase I (CYP1A) and II (GSTα) biotransformation enzymes, during the embryonic and larval developments of the specie Oreochromis niloticus (Nile tilapia). Our results showed that ABCB1b, ABCC1, CYP1A and GSTα transcripts are maternally transmitted. Transcripts for ABCB11, ABCC2 and ABCG2a were only detected after the pharyngula period, which precedes a highly sensitive stage in the embryonic development, the hatching. This study has shown, for the first time, very distinct expression patterns of genes encoding for proteins involved in protection mechanisms against pollutants during the development of Nile tilapia. Moreover, the temporal pattern of gene expression suggests that increased intrinsic protection levels are required at specific developmental stages. [Copyright &y& Elsevier]

Abstract: The microRNAs are a new class of small non-coding endogenous RNAs with lengths of approximately ~21 nt. MicroRNAs perform their biological function via the degradation of the target mRNAs or by inhibiting protein translation. Until recently, only limited numbers of miRNAs were identified in Brassica oleracea, a vegetable widely cultivated around the world. In present study, 193 potential miRNA candidates were identified from 17 expressed sequence tag (ESTs) and 152 genome survey sequences (GSSs) in B. oleracea. These miRNA candidates were classified into 70 families using a well-defined comparative genome-based computational analysis. Most miRNAs belong to the miRNA169, miR5021, miR156 and miR158 families. Of these, 36 miRNA families are firstly found in Brassica species. Around 1393 B. oleracea genes were predicted as candidate targets of 175 miRNAs. The mutual relationship between miRNAs and the candidate target genes was verified by checking differentially expression levels using quantitative real-time polymerase chain reaction (qRT-PCR) and 5′ RLM-RACE analyses. These target genes participate in multiple biological and metabolic processes, including signal transduction, stress response, and plant development. Gene Ontology analysis shows that the 818, 514, and 265 target genes are involved in molecular functions, biological processes, and cellular component respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway enrichment analysis suggests that these miRNAs might regulate 186 metabolic pathways, including those of lipid, energy, starch and sucrose, fatty acid and nitrogen. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are the family of noncoding single-strand RNA molecules of 21–25 nucleotides in length and play a broad and key regulation role in various physiological and pathological processes including differentiation, apoptosis, proliferation, and tumorigenesis. In Bombyx mori, a total of 487 pre-miRNAs and 562 mature miRNAs were identified by experimental or computational approaches, but their functions remain unknown. To carry out the research of gain-of-function of miRNAs in BmN cells, we firstly identified the endogenous expression of miRNAs in BmN cells by microarray and found that only 73 miRNAs could be detected by miRNA microarray. Then three low abundance or undetected miRNAs, pri-mir-1a, pri-mir-8 and pri-mir-133, were selected to express in BmN cells. The eukaryotic expression vector pIEx-1 harboring baculovirus ie1 promoter and hr5 enhancer was screened and used for expressing miRNA in BmN cells. Three miRNA expression vectors pIEx-1-EGFP-pri-mir-1a/8/133 were constructed, which contained the three corresponding pri-miRNA sequences, respectively. The constructed miRNA vectors were successfully transfected into BmN cells and the qRT-PCR analysis showed that relative abundance of bmo-mir-1a, bmo-mir-8 and bmo-mir-133 in BmN cells transfected with the pIEx-1-EGFP-pri-mir-1a/8/133 is as 32, 4.4 and 904 times as that in BmN cells transfected with the control vector pIEx-1-EGFP, respectively. The present work lays a foundation for the further functional studies of miRNAs in silkworm. [Copyright &y& Elsevier]

Abstract: Mitogen-activated protein kinases (MAPKs), a family of Ser/Thr protein kinases, play an essential role in mediating biotic and abiotic stress responses in plants. In this study, we investigated 16 putative SlMAPK genes from tomato genome and compared them with those from Arabidopsis. The full cDNA sequences of 13 novel SlMAPKs in tomato were obtained through PCR ampilification. A comprehensive genome-wide analysis of SlMAPKs in tomato is presented, including their gene structure, phylogeny, genome localization, and expression profiles. Phylogenic analysis of the 16 SlMAPKs and 20 AtMAPKs from Arabidopsis indicated that the SlMAPK genes were clustered into four major groups, and genes within the same groups had similar exon–intron structures. All SlMAPK proteins in groups A, B and C had a Thr-Glu-Tyr (TEY) activation domain, whereas those in group D contained a Thr-Asp-Tyr (TDY) activation domain. The analysis of 5′-upstream region of SlMAPKs revealed a group of putative cis-acting elements related to stress responses. Expression analysis of SlMAPK genes using RT-PCR and real-time quantitative PCR demonstrated that all SlMAPK transcripts were able to be detected in at least one investigated tissue, and some of them exhibited tissue-specific expression patterns. The transcript abundance of nearly all SlMAPK genes was increased in response to heat stress treatment. Our data provided an insight into the evolution of the gene family and a useful reference for further functional analysis of MAPK family genes in tomato. [Copyright &y& Elsevier]

Abstract: MicroRNAs (miRNAs) are small non-coding RNA molecules that play a vital role in the regulation of gene expression. Despite their identification in hundreds of plant species, few miRNAs have been identified in the Asteraceae, a large family that comprises approximately one tenth of all flowering plants. In this study, we used the expressed sequence tag (EST) analysis to identify potential conserved miRNAs and their putative target genes in the Asteraceae. We applied quantitative Real-Time PCR (qRT-PCR) to confirm the expression of eight potential miRNAs in Carthamus tinctorius and Helianthus annuus. We also performed qRT-PCR analysis to investigate the differential expression pattern of five newly identified miRNAs during five different cotyledon growth stages in safflower. Using these methods, we successfully identified and characterized 151 potentially conserved miRNAs, belonging to 26 miRNA families, in 11 genus of Asteraceae. EST analysis predicted that the newly identified conserved Asteraceae miRNAs target 130 total protein-coding ESTs in sunflower and safflower, as well as 433 additional target genes in other plant species. We experimentally confirmed the existence of seven predicted miRNAs, (miR156, miR159, miR160, miR162, miR166, miR396, and miR398) in safflower and sunflower seedlings. We also observed that five out of eight miRNAs are differentially expressed during cotyledon development. Our results indicate that miRNAs may be involved in the regulation of gene expression during seed germination and the formation of the cotyledons in the Asteraceae. The findings of this study might ultimately help in the understanding of miRNA-mediated gene regulation in important crop species. [Copyright &y& Elsevier]