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Circular RNA (circRNA) is a noncoding RNA class with important implications for gene expression regulation, mostly by interaction with other RNA species or RNA-binding proteins. While the commonly applied short-read Illumina RNA-sequencing techniques can be used to detect circRNAs, their full sequence is not revealed. However, the complete sequence information is needed to analyze potential interactions and thus the mechanism of action of circRNAs. Here, we present an improved protocol to enrich and sequence full-length circRNAs by using the Oxford Nanopore long-read sequencing platform. The protocol involves an enrichment of lowly abundant circRNAs by exonuclease treatment and negative selection of linear RNAs. Then, a cDNA library is created and amplified by PCR. This protocol provides enough material for several sequencing runs. The library is used as input for ligation-based sequencing together with native barcoding. Stringent quality control of the libraries is ensured by a combination of Qubit, Fragment Analyzer and qRT-PCR. Multiplexing of up to 4 libraries yields in total more than 1–2 Million reads per library, of which 1–2% are circRNA-specific reads with >99% of them full-length. The protocol works well with human cancer cell lines. We further provide suggestions for the bioinformatic analysis of the created data, as well as the limitations of our approach together with recommendations for troubleshooting and interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a noncoding RNA type involved in a growing number of physiologic and pathologic conditions. Metadata Associated content. https://dx.doi.org/10.17504/protocols.io.rm7vzy8r4lx1/v2. [ABSTRACT FROM AUTHOR]

During the blood feeding, sand fly females inject saliva containing immunomodulatory and anti-haemostatic molecules into their vertebrate hosts. The saliva composition is species-specific, likely due to an adaptation to particular haemostatic pathways of their preferred host. Research on sand fly saliva is limited to the representatives of two best-studied genera, Phlebotomus and Lutzomyia. Although the members of the genus Sergentomyia are highly abundant in many areas in the Old World, their role in human disease transmission remains uncertain. Most Sergentomyia spp. preferentially attack various species of reptiles, but feeding on warm-blooded vertebrates, including humans and domestic animals, has been repeatedly described, especially for Sergentomyia schwetzi, of which salivary gland transcriptome and proteome is analyzed in the current study. Illumina RNA sequencing and de novo assembly of the reads and their annotation revealed 17,293 sequences homologous to other arthropods' proteins. In the sialome, all proteins typical for sand fly saliva were identified–antigen 5-related, lufaxin, yellow-related, PpSP15-like, D7-related, ParSP25-like, and silk proteins, as well as less frequent salivary proteins included 71kDa-like, ParSP80-like, SP16-like, and ParSP17-like proteins. Salivary enzymes include apyrase, hyaluronidase, endonuclease, amylase, lipase A2, adenosine deaminase, pyrophosphatase, 5'nucleotidase, and ribonuclease. Proteomics analysis of salivary glands identified 631 proteins, 81 of which are likely secreted into the saliva. We also compared two S. schwetzi lineages derived from the same origin. These lineages were adapted for over 40 generations for blood feeding either on mice (S-M) or geckos (S-G), two vertebrate hosts with different haemostatic mechanisms. Altogether, 20 and 40 annotated salivary transcripts were up-regulated in the S-M and S-G lineage, respectively. Proteomic comparison revealed ten salivary proteins more abundant in the lineage S-M, whereas 66 salivary proteins were enriched in the lineage S-G. No difference between lineages was found for apyrase activity; contrarily the hyaluronidase activity was significantly higher in the lineage feeding on mice. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM), the most prevalent primary malignant brain tumor in adults, exhibits a dismal 6.9% five-year survival rate post-diagnosis. Thymoquinone (TQ), the most abundant bioactive compound in Nigella sativa, has been extensively researched for its anticancer properties across various human cancers. However, its specific anti-cancer mechanisms and pathways in glioblastoma remain to be completely elucidated. In this study, we assessed the impact of different TQ concentrations on the viability of A172 cells using WST-8 and Toluidine blue assays, followed by RNA sequencing (RNA-Seq) to identify differentially expressed genes (DEGs). We confirmed their expression levels through quantitative RT-PCR and performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for these DEGs. RNA-seq revealed no significant gene expression changes at 2.5 μM and 5 μM TQ concentrations. However, at 25 μM and 50 μM, TQ significantly reduced cell viability dose-dependently. We identified 1548 DEGs at 25 μM TQ (684 up-regulated, 864 down-regulated) and 2797 DEGs at 50 μM TQ (1528 up-regulated, 1269 downregulated), with 1202 DEGs common to both concentrations. TQ inhibited key pathways such as PI3K-Akt signaling, calcium signaling, focal adhesion, and ECM-receptor interaction in A172 cells. It downregulated several potential oncogenes (e.g., AEBP1, MIAT) and genes linked to GBM proliferation and migration (e.g., SOCS2, HCP5) while modulating Wnt signaling and up-regulating tumor suppressor genes (e.g., SPRY4, BEX2). TQ also affected p53 downstream targets, maintaining p53 levels. This study elucidates the anti-cancer mechanisms of TQ in A172 GBM cells, underscoring its effects on multiple signaling pathways and positioning TQ as a promising candidate for innovative glioblastoma treatment strategies. [ABSTRACT FROM AUTHOR]

Brassica juncea is used as a condiment, as vegetables and as an oilseed crop, especially in semiarid areas. In the present study, we constructed a genetic map using one recombinant inbred line (RIL) of B. juncea. A total of 304 ILP (intron length polymorphism) markers were mapped to 18 linkage groups designated LG01-LG18 in B. juncea. The constructed map covered a total genetic length of 1671.13 cM with an average marker interval of 5.50 cM. The QTLs for 2-propenyl glucosinolates (GSLs) colocalized with the QTLs for 3-butenyl GSLs between At1g26180 and BnapPIP1580 on LG08 in the field experiments of 2016 and 2017. These QTLs accounted for an average of 42.3% and 42.6% phenotypic variation for 2-propenyl and 3-butenyl GSLs, respectively. Furthermore, the Illumina RNA-sequencing technique was used to excavate the genes responsible for the synthesis of GSLs in the siliques of the parental lines of the RIL mapping population, because the bulk of the seed GSLs might originate from the siliques. Comparative analysis and annotation by gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) revealed that 324 genes were involved in GSL metabolism, among which only 24 transcripts were differentially expressed genes (DEGs). Among those DEGs, 15 genes were involved in the biosynthesis and transport of aliphatic GSLs, and their expression patterns were further validated by qRT-PCR analysis. Joint QTL mapping and RNA-sequencing analyses reveal one candidate gene of IIL1 (LOC106416451) for GSL metabolism in B. juncea. These results will be helpful for further fine mapping, gene cloning and genetic mechanisms of 2-propenyl and 3-butenyl GSLs in B. juncea. [ABSTRACT FROM AUTHOR]

The mechanisms governing gene regulation in domestic Yuzhong pigeon breast muscle development remain largely elusive. Here, we conducted a comparative analysis using Iso-seq and RNA-seq data from domestic Yuzhong pigeons and European meat pigeons to uncover signaling pathways and genes involved in breast muscle development. The Iso-seq data from domestic Yuzhong pigeons yielded 131,377,075 subreads, resulting in 16,587 non-redundant high-quality full-length transcripts post-correction. Furthermore, utilizing pfam, CPC, PLEK, and CPAT, we predicted 5575, 4973, 2333, and 4336 lncRNAs, respectively. Notably, several genes potentially implicated in breast muscle development were identified, including tropomyosin beta chain, myosin regulatory light chain 2, and myosin binding protein C. KEGG enrichment analysis revealed critical signaling pathways in breast muscle development, spanning carbon metabolism, biosynthesis of amino acids, glycolysis/gluconeogenesis, estrogen signaling, PI3K-AKT signaling, protein processing in the endoplasmic reticulum, oxidative phosphorylation, pentose phosphate pathway, fructose and mannose metabolism, and tight junctions. These findings offer insights into the biological processes driving breast muscle development in domestic Yuzhong pigeon, contributing to our understanding of this complex phenomenon. [ABSTRACT FROM AUTHOR]

Tangeretin (Tan), a citrus flavonoid, possesses a strong anti-tumor efficacy in various human cancers. However, the precise role of Tan in the development of esophageal squamous cell carcinoma (ESCC) remains unclear. RNA sequencing (RNA-seq) analysis was performed to observe the Tan-related genes in Tan-treated TE-1 cells. The direct relationship between GLI family zinc finger 2 (GLI2) and the promoter of glycoprotein non-metastatic melanoma protein B (GPNMB) was predicted by bioinformatics analysis and validated by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Cell survival after Tan treatment was assessed by CCK8 assay. Gene expression levels were evaluated by a qRT-PCR, western blot, or immunofluorescence method. Cell migration and invasion were detected by wound-healing and transwell assays. The function of Tan in vivo was examined using xenograft studies. Our data indicated anti-migration and anti-invasion functions of Tan in ESCC cells in vitro. Tan also diminished tumor growth in vivo. Mechanistically, Tan diminished the expression and transcriptional activity of GLI2 in ESCC cells. Silencing of GLI2 resulted in decreased expression of GPNMB by inhibiting GPNMB transcription via the binding site at the GPNMB promoter at position +(1539–1550). Moreover, Tan down-regulated GPNMB expression in ESCC cells, and re-expression of GPNMB reversed anti-migration and anti-invasion functions of Tan in ESCC cells. Our findings uncover anti-migration and anti-invasion effects of Tan in ESCC cells by down-regulating GPNMB by suppressing GLI2-mediated GPNMB transcription, providing new evidence that Tan can function as a therapeutic agent against ESCC. [ABSTRACT FROM AUTHOR]

The complexity of the tomato (Solanum lycopersicum) transcriptome has not yet been fully elucidated. To gain insights into the diversity and features of coding and non-coding RNA molecules of tomato fruits, we generated strand-specific libraries from berries of two tomato cultivars grown in two open-field conditions with different soil type. Following high-throughput Illumina RNA-sequencing (RNA-seq), more than 90% of the reads (over one billion, derived from twelve dataset) were aligned to the tomato reference genome. We report a comprehensive analysis of the transcriptome, improved with 39,095 transcripts, which reveals previously unannotated novel transcripts, natural antisense transcripts, long non-coding RNAs and alternative splicing variants. In addition, we investigated the sequence variants between the cultivars under investigation to highlight their genetic difference. Our strand-specific analysis allowed us to expand the current tomato transcriptome annotation and it is the first to reveal the complexity of the poly-adenylated RNA world in tomato. Moreover, our work demonstrates the usefulness of strand specific RNA-seq approach for the transcriptome-based genome annotation and provides a resource valuable for further functional studies. [ABSTRACT FROM AUTHOR]

Mycoplasma agalactiae is a worldwide serious pathogen of small ruminants that usually spreads through the mammary route causing acute to subacute mastitis progressing to chronic persistent disease that is hard to eradicate. Knowledge of mechanisms of its pathogenesis and persistence in the mammary gland are still insufficient, especially the host-pathogen interplay that enables it to reside in a chronic subclinical state. This study reports transcriptome profiling of mammary tissue from udders of sheep experimentally infected with M. agalactiae type strain PG2 in comparison with uninfected control animals using Illumina RNA-sequencing (RNA-Seq). Several differentially expressed genes (DEGs) were observed in the infected udders and RT-qPCR analyses of selected DEGs showed their expression profiles to be in agreement with results from RNA-Seq. Gene Ontology (GO) analysis revealed majority of the DEGs to be associated with mycoplasma defense responses that are directly or indirectly involved in host innate and adaptive immune responses. Similar RNA-Seq analyses were also performed with spleen cells of the same sheep to know the specific systemic transcriptome responses. Spleen cells exhibited a comparatively lower number of DEGs suggesting a less prominent host response in this organ. To our knowledge this is the first study that describes host transcriptomics of M. agalactiae infection and the related immune-inflammatory responses. The data provides useful information to further dissect the molecular genetic mechanisms underlying mycoplasma mastitis, which is a prerequisite for designing effective intervention strategies. [ABSTRACT FROM AUTHOR]

Osmotic stress adversely affects the growth, fruit quality and yield of watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai). Increasing the tolerance of watermelon to osmotic stress caused by factors such as high salt and water deficit is an effective way to improve crop survival in osmotic stress environments. Roots are important organs in water absorption and are involved in the initial response to osmosis stress; however, few studies have examined the underlying mechanism of tolerance to osmotic stress in watermelon roots. For better understanding of this mechanism, the inbred watermelon accession M08, which exhibits relatively high tolerance to water deficits, was treated with 20% polyethylene glycol (PEG) 6000. The root samples were harvested at 6 h after PEG treatment and untreated samples were used as controls. Transcriptome analyses were carried out by Illumina RNA sequencing. A total of 5246 differentially expressed genes were identified. Gene ontology enrichment and biochemical pathway analyses of these 5246 genes showed that short-term osmotic stress affected osmotic adjustment, signal transduction, hormone responses, cell division, cell cycle and ribosome, and M08 may repress root growth to adapt osmotic stress. The results of this study describe the watermelon root transcriptome under osmotic stress and propose new insight into watermelon root responses to osmotic stress at the transcriptome level. Accordingly, these results allow us to better understand the molecular mechanisms of watermelon in response to drought stress and will facilitate watermelon breeding projects to improve drought tolerance. [ABSTRACT FROM AUTHOR]

Auxin has been shown to modulate the fruit ripening process. However, the molecular mechanisms underlying auxin regulation of fruit ripening are still not clear. Illumina RNA sequencing was performed on mature green cherry tomato fruit 1 and 7 days after auxin treatment, with untreated fruit as a control. The results showed that exogenous auxin maintained system 1 ethylene synthesis and delayed the onset of system 2 ethylene synthesis and the ripening process. At the molecular level, genes associated with stress resistance were significantly up-regulated, but genes related to carotenoid metabolism, cell degradation and energy metabolism were strongly down-regulated by exogenous auxin. Furthermore, genes encoding DNA demethylases were inhibited by auxin, whereas genes encoding cytosine-5 DNA methyltransferases were induced, which contributed to the maintenance of high methylation levels in the nucleus and thus inhibited the ripening process. Additionally, exogenous auxin altered the expression patterns of ethylene and auxin signaling-related genes that were induced or repressed in the normal ripening process, suggesting significant crosstalk between these two hormones during tomato ripening. The present work is the first comprehensive transcriptome analysis of auxin-treated tomato fruit during ripening. Our results provide comprehensive insights into the effects of auxin on the tomato ripening process and the mechanism of crosstalk between auxin and ethylene. [ABSTRACT FROM AUTHOR]

Paeonia lactiflora is one of the most famous species of herbaceous peonies with gorgeous flowers. Bud dormancy is a crucial developmental process that allows P. lactiflora to survive unfavorable environmental conditions. However, little information is available on the molecular mechanism of the bud dormancy in P. lactiflora. We performed de novo transcriptome sequencing using the Illumina RNA sequencing platform for the underground renewal buds of P. lactiflora ‘Hangbaishao’ to study the molecular mechanism underlying its bud dormancy transition (the period from endodormancy to ecodormancy) and release (the period from ecodormancy to bud elongation and sprouting). Approximately 300 million high-quality clean reads were generated and assembled into 207,827 (mean length = 828 bp) and 51,481 (mean length = 1250 bp) unigenes using two assembly methods named “Trinity” and “Trinity+PRICE”, respectively. Based on the data obtained by the latter method, 32,316 unigenes were annotated by BLAST against various databases. Approximately 1,251 putative transcription factors were obtained, of which the largest number of unique transcripts belonged to the basic helix-loop-helix protein (bHLH) transcription factor family, and five of the top ten highly expressed transcripts were annotated as dehydrin (DHN). A total of 17,705 simple sequence repeat (SSR) motifs distributed in 13,797 sequences were obtained. The budbreak morphology, levels of indole-3-acetic acid (IAA) and abscisic acid (ABA), and activities of guaiacol peroxidase (POD) and catalase (CAT) were observed. The expression of 20 interested unigenes, which annotated as DHN, heat shock protein (HSP), histone, late elongated hypocotyl (LHY), and phytochrome (PHY), and so on, were also analyzed. These studies were based on morphological, physiological, biochemical, and molecular levels and provide comprehensive insight into the mechanism of dormancy transition and release in P. lactiflora. Transcriptome dataset can be highly valuable for future investigation on gene expression networks in P. lactiflora as well as research on dormancy in other non-model perennial horticultural crops of commercial significance. [ABSTRACT FROM AUTHOR]

Jerusalem artichoke (Helianthus tuberosus L.) has long been cultivated as a vegetable and as a source of fructans (inulin) for pharmaceutical applications in diabetes and obesity prevention. However, transcriptomic and genomic data for Jerusalem artichoke remain scarce. In this study, Illumina RNA sequencing (RNA-Seq) was performed on samples from Jerusalem artichoke leaves, roots, stems and two different tuber tissues (early and late tuber development). Data were used for de novo assembly and characterization of the transcriptome. In total 206,215,632 paired-end reads were generated. These were assembled into 66,322 loci with 272,548 transcripts. Loci were annotated by querying against the NCBI non-redundant, Phytozome and UniProt databases, and 40,215 loci were homologous to existing database sequences. Gene Ontology terms were assigned to 19,848 loci, 15,434 loci were matched to 25 Clusters of Eukaryotic Orthologous Groups classifications, and 11,844 loci were classified into 142 Kyoto Encyclopedia of Genes and Genomes pathways. The assembled loci also contained 10,778 potential simple sequence repeats. The newly assembled transcriptome was used to identify loci with tissue-specific differential expression patterns. In total, 670 loci exhibited tissue-specific expression, and a subset of these were confirmed using RT-PCR and qRT-PCR. Gene expression related to inulin biosynthesis in tuber tissue was also investigated. Exsiting genetic and genomic data for H. tuberosus are scarce. The sequence resources developed in this study will enable the analysis of thousands of transcripts and will thus accelerate marker-assisted breeding studies and studies of inulin biosynthesis in Jerusalem artichoke. [ABSTRACT FROM AUTHOR]

The marine medaka (Oryzias melastigma) has been increasingly used as a fish model for detecting environmental stresses and chemical contaminants in the marine environment. Recent mammalian studies have shown that environmental stresses can alter the expression profiles of microRNAs (miRNAs), leading to transgenerational effects. Here, we use high-throughput Illumina RNA sequencing (RNA-Seq) for miRNA transcriptome analysis of brain, liver, and gonads from sexually mature male and female marine medaka. A total of 128,883,806 filtered sequence reads were generated from six small RNA libraries, identifying a total of 2,125,663 non-redundant sequences. These sequences were aligned and annotated to known animal miRNAs (miRBase) using the BLAST method. A total of 223 distinct miRNA types were identified, with the greatest number expressed in brain tissue. Our data suggested that 55 miRNA types from 34 families are common to all tested tissues, while some of the miRNAs are tissue-enriched or sex-enriched. Quantitative real-time PCR analysis further demonstrated that let-7a, miR-122, and miR-9-3p were downregulated in hypoxic female medaka, while miR-2184 was specifically upregulated in the testis of hypoxic male fish. This is the first study to identify miRNAs in O. melastigma using small RNA deep sequencing technology. Because miRNA expression is highly conserved between marine medaka and other vertebrates, marine medaka may serve as a good model for studies on the functional roles of miRNAs in hypoxia stress response and signaling in marine fish. [ABSTRACT FROM AUTHOR]

Potassium (K) deficiency is one of the major factors affecting crop growth and productivity. Development of low-K tolerant crops is an effective approach to solve the nutritional deficiency in agricultural production. Tibetan annual wild barley is rich in genetic diversity and can grow normally under poor soils, including low-K supply. However, the molecular mechanism about low K tolerance is still poorly understood. In this study, Illumina RNA-Sequencing was performed using two Tibetan wild barley genotypes differing in low K tolerance (XZ153, tolerant and XZ141, sensitive), to determine the genotypic difference in transcriptome profiling. We identified a total of 692 differentially expressed genes (DEGs) in two genotypes at 6 h and 48 h after low-K treatment, including transcription factors, transporters and kinases, oxidative stress and hormone signaling related genes. Meanwhile, 294 low-K tolerant associated DEGs were assigned to transporter and antioxidant activities, stimulus response, and other gene ontology (GO), which were mainly involved in starch and sucrose metabolism, lipid metabolism and ethylene biosynthesis. Finally, a hypothetical model of low-K tolerance mechanism in XZ153 was presented. It may be concluded that wild barley accession XZ153 has a higher capability of K absorption and use efficiency than XZ141 under low K stress. A rapid response to low K stress in XZ153 is attributed to its more K uptake and accumulation in plants, resulting in higher low K tolerance. The ethylene response pathway may account for the genotypic difference in low-K tolerance. [ABSTRACT FROM AUTHOR]

Cassava (Manihot esculenta) is a major food staple in sub-Saharan Africa, which is severely affected by cassava brown streak disease (CBSD). The aim of this study was to identify resistance for CBSD as well as to understand the mechanism of putative resistance for providing effective control for the disease. Three cassava varieties; Kaleso, Kiroba and Albert were inoculated with cassava brown streak viruses by grafting and also using the natural insect vector the whitefly, Bemisia tabaci. Kaleso expressed mild or no disease symptoms and supported low concentrations of viruses, which is a characteristic of resistant plants. In comparison, Kiroba expressed severe leaf but milder root symptoms, while Albert was susceptible with severe symptoms both on leaves and roots. Real-time PCR was used to estimate virus concentrations in cassava varieties. Virus quantities were higher in Kiroba and Albert compared to Kaleso. The Illumina RNA-sequencing was used to further understand the genetic basis of resistance. More than 700 genes were uniquely overexpressed in Kaleso in response to virus infection compared to Albert. Surprisingly, none of them were similar to known resistant gene orthologs. Some of the overexpressed genes, however, belonged to the hormone signalling pathways and secondary metabolites, both of which are linked to plant resistance. These genes should be further characterised before confirming their role in resistance to CBSD. [ABSTRACT FROM AUTHOR]

This study aimed to investigate the potential regulatory role of miRNAs in the development of gastrointestinal tract (GIT) during the early life of dairy calves. Rumen and small intestinal (mid-jejunum and ileum) tissue samples were collected from newborn (30 min after birth; n = 3), 7-day-old (n = 6), 21-day-old (n = 6), and 42-day-old (n = 6) dairy calves. The miRNA profiling was performed using Illumina RNA-sequencing and the temporal and regional differentially expressed miRNAs were further validated using qRT-PCR. Analysis of 16S rRNA gene copy numbers was used to quantify total bacteria, Bifidobacterium and Lactobacillus species. The expression of miR-143 was abundant in all three gut regions, at all time points and it targets genes involved primarily in the proliferation of connective tissue cells and muscle cells, suggesting a role in regulating rapid tissue development during the early life of calves. The expression of miR-146, miR-191, miR-33, miR-7, miR-99/100, miR-486, miR-145, miR-196 and miR-211 displayed significant temporal differences (FDR <0.05), while miR-192/215, miR-194, miR-196, miR-205 and miR-31 revealed significant regional differences (FDR <0.05). The expression levels of miR-15/16, miR-29 and miR-196 were positively correlated with the copy numbers of 16S rRNA gene of Bifidobacterium or Lactobacillus species or both (P<0.05). Functional analysis using Ingenuity Pathway Analysis identified the above mentioned differentially expressed miRNAs as potential regulators of gut tissue cell proliferation and differentiation. The bacterial density-associated miRNAs were identified as modulators of the development of lymphoid tissues (miR-196), maturation of dendritic cells (miR-29) and development of immune cells (miR-15/16). The present study revealed temporal and regional changes in miRNA expression and a correlation between miRNA expression and microbial population in the GIT during the early life, which provides further evidence for another mechanism by which host-microbial interactions play a role in regulating gut development. [ABSTRACT FROM AUTHOR]

Sand pear (Pyrus pyrifolia) russet pericarp is an important trait affecting both the quality and stress tolerance of fruits. This trait is controlled by a relative complex genetic process, with some fundamental biological questions such as how many and which genes are involved in the process remaining elusive. In this study, we explored differentially expressed genes between the russet- and green-pericarp offspring from the sand pear (Pyrus pyrifolia) cv. ‘Qingxiang’ × ‘Cuiguan’ F1 group by RNA-seq-based bulked segregant analysis (BSA). A total of 29,100 unigenes were identified and 206 of which showed significant differences in expression level (log2fold values>1) between the two types of pericarp pools. Gene Ontology (GO) analyses detected 123 unigenes in GO terms related to ‘cellular_component’ and ‘biological_process’, suggesting developmental and growth differentiations between the two types. GO categories associated with various aspects of ‘lipid metabolic processes’, ‘transport’, ‘response to stress’, ‘oxidation-reduction process’ and more were enriched with genes with divergent expressions between the two libraries. Detailed examination of a selected set of these categories revealed repressed expressions of candidate genes for suberin, cutin and wax biosynthesis in the russet pericarps.Genes encoding putative cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD) and peroxidase (POD) that are involved in the lignin biosynthesis were suggested to be candidates for pigmentation of sand pear russet pericarps. Nine differentially expressed genes were analyzed for their expressions using qRT-PCR and the results were consistent with those obtained from Illumina RNA-sequencing. This study provides a comprehensive molecular biology insight into the sand pear pericarp pigmentation and appearance quality formation. [ABSTRACT FROM AUTHOR]

Background: The Eastern hive honey bee, Apis cerana cerana is a native and widely bred honey bee species in China. Molecular biology research about this honey bee species is scarce, and genomic information for A. c. cerana is not currently available. Transcriptome and expression profiling data for this species are therefore important resources needed to better understand the biological mechanisms of A. c. cerana. In this study, we obtained the transcriptome information of A. c. cerana by RNA-sequencing and compared gene expression differences between queens and workers of A. c. cerana by digital gene expression (DGE) analysis. Results: Using high-throughput Illumina RNA sequencing we obtained 51,581,510 clean reads corresponding to 4.64 Gb total nucleotides from a single run. These reads were assembled into 46,999 unigenes with a mean length of 676 bp. Based on a sequence similarity search against the five public databases (NR, Swissport, GO, COG, KEGG) with a cut-off E-value of 25 10 using BLASTX, a total of 24,630 unigenes were annotated with gene descriptions, gene ontology terms, or metabolic pathways. Using these transcriptome data as references we analyzed the gene expression differences between the queens and workers of A. c. cerana using a tag-based digital gene expression method. We obtained 5.96 and 5.66 million clean tags from the queen and worker samples, respectively. A total of 414 genes were differentially expressed between them, with 189 up-regulated and 225 down-regulated in queens. Conclusions: Our transcriptome data provide a comprehensive sequence resource for future A. c. cerana study, establishing an important public information platform for functional genomic studies in A. c. cerana. Furthermore, the DGE data provide comprehensive gene expression information for the queens and workers, which will facilitate our understanding of the molecular mechanisms of the different physiological aspects of the two castes. [ABSTRACT FROM AUTHOR]

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology. [ABSTRACT FROM AUTHOR]

The MADS-box gene family controls plant flowering and floral organ development; therefore, it is particularly important in ornamental plants. To investigate the genes associated with the MADS-box family in Clematis courtoisii, we performed full-length transcriptome sequencing on C. courtoisii using the PacBio Sequel third-generation sequencing platform, as no reference genome data was available. A total of 12.38 Gb of data, containing 9,476,585 subreads and 50,439 Unigenes were obtained. According to functional annotation, a total of 37,923 Unigenes (75.18% of the total) were assigned with functional annotations, and 50 Unigenes were identified as MADS-box related genes. Subsequently, we employed hmmerscan to perform protein sequence similarity search for the translated Unigene sequences and successfully identified 19 Unigenes associated with the MADS-box gene family, including MIKC*(1) and MIKCC (18) genes. Furthermore, within the MIKCC group, six subclasses can be further distinguished. [ABSTRACT FROM AUTHOR]

Translation initiation is a complex and highly regulated process that represents an important mechanism, controlling gene expression. eIF2A was proposed as an alternative initiation factor, however, its role and biological targets remain to be discovered. To further gain insight into the function of eIF2A in Saccharomyces cerevisiae, we identified mRNAs associated with the eIF2A complex and showed that 24% of the most enriched mRNAs encode proteins related to cell wall biogenesis and maintenance. In agreement with this result, we showed that an eIF2A deletion sensitized cells to cell wall damage induced by calcofluor white. eIF2A overexpression led to a growth defect, correlated with decreased synthesis of several cell wall proteins. In contrast, no changes were observed in the transcriptome, suggesting that eIF2A controls the expression of cell wall-related proteins at a translational level. The biochemical characterization of the eIF2A complex revealed that it strongly interacts with the RNA binding protein, Ssd1, which is a negative translational regulator, controlling the expression of cell wall-related genes. Interestingly, eIF2A and Ssd1 bind several common mRNA targets and we found that the binding of eIF2A to some targets was mediated by Ssd1. Surprisingly, we further showed that eIF2A is physically and functionally associated with the exonuclease Xrn1 and other mRNA degradation factors, suggesting an additional level of regulation. Altogether, our results highlight new aspects of this complex and redundant fine-tuned regulation of proteins expression related to the cell wall, a structure required to maintain cell shape and rigidity, providing protection against harmful environmental stress. [ABSTRACT FROM AUTHOR]

Modulation of odorant receptors mRNA induced by prolonged odor exposure is highly correlated with ligand-receptor interactions in Drosophila as well as mammals of the Muridae family. If this response feature is conserved in other organisms, this presents an intriguing initial screening tool when searching for novel receptor-ligand interactions in species with predominantly orphan olfactory receptors. We demonstrate that mRNA modulation in response to 1-octen-3-ol odor exposure occurs in a time- and concentration-dependent manner in Aedes aegypti mosquitoes. To investigate gene expression patterns at a global level, we generated an odor-evoked transcriptome associated with 1-octen-3-ol odor exposure. Transcriptomic data revealed that ORs and OBPs were transcriptionally responsive whereas other chemosensory gene families showed little to no differential expression. Alongside chemosensory gene expression changes, transcriptomic analysis found that prolonged exposure to 1-octen-3-ol modulated xenobiotic response genes, primarily members of the cytochrome P450, insect cuticle proteins, and glucuronosyltransferases families. Together, these findings suggest that mRNA transcriptional modulation of olfactory receptors caused by prolonged odor exposure is pervasive across taxa and can be accompanied by the activation of xenobiotic responses. [ABSTRACT FROM AUTHOR]

Myelofibrosis is a myeloproliferative neoplasm (MPN) which typically results in reduced length and quality of life due to systemic symptoms and blood count changes arising from fibrotic changes in the bone marrow. While the JAK2 inhibitor ruxolitinib provides some clinical benefit, there remains a substantial unmet need for novel targeted therapies to better modify the disease process or eradicate the cells at the heart of myelofibrosis pathology. Repurposing drugs bypasses many of the hurdles present in drug development, such as toxicity and pharmacodynamic profiling. To this end we undertook a re-analysis of our pre-existing proteomic data sets to identify perturbed biochemical pathways and their associated drugs/inhibitors to potentially target the cells driving myelofibrosis. This approach identified CBL0137 as a candidate for targeting Jak2 mutation-driven malignancies. CBL0137 is a drug derived from curaxin targeting the Facilitates Chromatin Transcription (FACT) complex. It is reported to trap the FACT complex on chromatin thereby activating p53 and inhibiting NF-kB activity. We therefore assessed the activity of CBL0137 in primary patient samples and murine models of Jak2-mutated MPN and found it preferentially targets CD34+ stem and progenitor cells from myelofibrosis patients by comparison with healthy control cells. Further we investigate its mechanism of action in primary haemopoietic progenitor cells and demonstrate its ability to reduce splenomegaly and reticulocyte number in a transgenic murine model of myeloproliferative neoplasms. [ABSTRACT FROM AUTHOR]

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ (L36), a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. Knockout of ZntA, a zinc efflux pump, abolished the zinc-resistant phenotype of the rpmJ mutant. RNA sequence analysis revealed that the rpmJ mutant exhibited altered gene expression of diverse functional categories, including translation, energy metabolism, and stress response. These findings suggest that knocking out RpmJ alters gene expression patterns and causes zinc resistance by lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance. [ABSTRACT FROM AUTHOR]

Understanding the mechanisms triggering variation of cell wall degradability is a prerequisite to improving the energy value of lignocellulosic biomass for animal feed or biorefinery. Here, we implemented a multiscale systems approach to shed light on the genetic basis of cell wall degradability in maize. We demonstrated that allele replacement in two pairs of near-isogenic lines at a region encompassing a major quantitative trait locus (QTL) for cell wall degradability led to phenotypic variation of a similar magnitude and sign to that expected from a QTL analysis of cell wall degradability in the F271 × F288 recombinant inbred line progeny. Using DNA sequences within the QTL interval of both F271 and F288 inbred lines and Illumina RNA sequencing datasets from internodes of the selected near-isogenic lines, we annotated the genes present in the QTL interval and provided evidence that allelic variation at the introgressed QTL region gives rise to coordinated changes in gene expression. The identification of a gene co-expression network associated with cell wall-related trait variation revealed that the favorable F288 alleles exploit biological processes related to oxidation-reduction, regulation of hydrogen peroxide metabolism, protein folding and hormone responses. Nested in modules of co-expressed genes, potential new cell-wall regulators were identified, including two transcription factors of the group VII ethylene response factor family, that could be exploited to fine-tune cell wall degradability. Overall, these findings provide new insights into the regulatory mechanisms by which a major locus influences cell wall degradability, paving the way for its map-based cloning in maize. [ABSTRACT FROM AUTHOR]

Presently, concern regarding the effects of selenium (Se) on the environment and organisms worldwide is increasing. Too much Se in the soil is harmful to plants. In this study, Illumina RNA sequencing and the untargeted metabolome of control and Se-treated celery seedlings were analyzed. In total, 297,911,046 clean reads were obtained and assembled into 150,218 transcripts (50,876 unigenes). A total of 36,287 unigenes were annotated using different databases. Additionally, 8,907 differentially expressed genes, including 5,319 up- and 3,588 downregulated genes, were identified between mock and Se-treated plants. "Phenylpropanoid biosynthesis" was the most enriched KEGG pathway. A total of 24 sulfur and selenocompound metabolic unigenes were differentially expressed. Furthermore, 1,774 metabolites and 237 significant differentially accumulated metabolites were identified using the untargeted metabolomic approach. We conducted correlation analyses of enriched KEGG pathways of differentially expressed genes and accumulated metabolites. Our findings suggested that candidate genes and metabolites involved in important biological pathways may regulate Se tolerance in celery. The results increase our understanding of the molecular mechanism responsible for celery's adaptation to Se stress. [ABSTRACT FROM AUTHOR]

Perilla frutescens var. crispa (Labiatae) has two chemo-varietal forms, i.e. red and green forms of perilla, that differ in the production of anthocyanins. To facilitate molecular biological and biochemical studies in perilla-specialized metabolism we used Illumina RNA-sequencing technology in our comprehensive comparison of the transcriptome map of the leaves of red and green forms of perilla. Sequencing generated over 1.2 billion short reads with an average length of 101 nt. De novo transcriptome assembly yielded 47,788 and 47,840 unigenes in the red and green forms of perilla plants, respectively. Comparison of the assembled unigenes and existing perilla cDNA sequences showed highly reliable alignment. All unigenes were annotated with gene ontology (GO) and Enzyme Commission numbers and entered into the Kyoto Encyclopedia of Genes and Genomes. We identified 68 differentially expressed genes (DEGs) in red and green forms of perilla. GO enrichment analysis of the DEGs showed that genes involved in the anthocyanin metabolic process were enriched. Differential expression analysis revealed that the transcript level of anthocyanin biosynthetic unigenes encoding flavonoid 3’-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase was significantly higher in red perilla, while the transcript level of unigenes encoding limonene synthase was significantly higher in green perilla. Our data serve as a basis for future research on perilla bio-engineering and provide a shortcut for the characterization of new functional genes in P. frutescens. [ABSTRACT FROM AUTHOR]

A symbiotic relationship is observed between Armillaria and the Chinese herbal medicine Gastrodia elata (G. elata). Armillaria is a nutrient source for the growth of G. elata, and its nutrient metabolism efficiency affects the growth and development of G. elata. Auxin has been reported to stimulate Armillaria species, but the molecular mechanism remains unknown. We found that naphthalene acetic acid (NAA) can also promote the growth of A. gallica. Moreover, we identified a total of 2071 differentially expressed genes (DEGs) by analyzing the transcriptome sequencing data of A. gallica at 5 and 10 hour of NAA treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these unigenes were significantly enriched in the metabolism pathways of arginine, proline, propanoate, phenylalanine and tryptophan. The expression levels of the general amino acid permease (Gap), ammonium transporter (AMT), glutamate dehydrogenase (GDH), glutamine synthetase (GS), Zn(II) 2Cys6 and C2H2 transcription factor genes were upregulated. Our transcriptome analysis showed that the amino acid and nitrogen metabolism pathways in Armillaria were rapidly induced within hours after NAA treatment. These results provide valuable insights into the molecular mechanisms by which NAA promotes the growth of Armillaria species. [ABSTRACT FROM AUTHOR]

Objective: To determine the gene expression profile in individuals with new latent tuberculosis infection (LTBI), and to compare them with people with active tuberculosis (TB) and those exposed to TB but not infected. Design: A prospective cohort study. Recruitment and follow-up were conducted between September 2016 to December 2018. Gene expression and data processing and analysis from April 2019 to April 2021. Setting: Two male Colombian prisons. Participants: 15 new tuberculin skin test (TST) converters (negative TST at baseline that became positive during follow-up), 11 people that continued with a negative TST after two years of follow-up, and 10 people with pulmonary ATB. Main outcome measures: Gene expression profile using RNA sequencing from PBMC samples. The differential expression was assessed using the DESeq2 package in Bioconductor. Genes with |logFC| >1.0 and an adjusted p-value < 0.1 were differentially expressed. We analyzed the differences in the enrichment of KEGG pathways in each group using InterMiner. Results: The gene expression was affected by the time of incarceration. We identified group-specific differentially expressed genes between the groups: 289 genes in people with a new LTBI and short incarceration (less than three months of incarceration), 117 in those with LTBI and long incarceration (one or more years of incarceration), 26 in ATB, and 276 in the exposed but non-infected individuals. Four pathways encompassed the largest number of down and up-regulated genes among individuals with LTBI and short incarceration: cytokine signaling, signal transduction, neutrophil degranulation, and innate immune system. In individuals with LTBI and long incarceration, the only enriched pathway within up-regulated genes was Emi1 phosphorylation. Conclusions: Recent infection with MTB is associated with an identifiable RNA pattern related to innate immune system pathways that can be used to prioritize LTBI treatment for those at greatest risk for developing active TB. [ABSTRACT FROM AUTHOR]

Natural silks crafted by spiders comprise some of the most versatile materials known. Artificial silks–based on the sequences of their natural brethren–replicate some desirable biophysical properties and are increasingly utilized in commercial and medical applications today. To characterize the repertoire of protein sequences giving silks their biophysical properties and to determine the set of expressed genes across each unique silk gland contributing to the formation of natural silks, we report here draft genomic and transcriptomic assemblies of Darwin's bark spider, Caerostris darwini, an orb-weaving spider whose dragline is one of the toughest known biomaterials on Earth. We identify at least 31 putative spidroin genes, with expansion of multiple spidroin gene classes relative to the golden orb-weaver, Trichonephila clavipes. We observed substantial sharing of spidroin repetitive sequence motifs between species as well as new motifs unique to C. darwini. Comparative gene expression analyses across six silk gland isolates in females plus a composite isolate of all silk glands in males demonstrated gland and sex-specific expression of spidroins, facilitating putative assignment of novel spidroin genes to classes. Broad expression of spidroins across silk gland types suggests that silks emanating from a given gland represent composite materials to a greater extent than previously appreciated. We hypothesize that the extraordinary toughness of C. darwini major ampullate dragline silk may relate to the unique protein composition of major ampullate spidroins, combined with the relatively high expression of stretchy flagelliform spidroins whose union into a single fiber may be aided by novel motifs and cassettes that act as molecule-binding helices. Our assemblies extend the catalog of sequences and sets of expressed genes that confer the unique biophysical properties observed in natural silks. [ABSTRACT FROM AUTHOR]

While the importance of random sequencing errors decreases at higher DNA or RNA sequencing depths, systematic sequencing errors (SSEs) dominate at high sequencing depths and can be difficult to distinguish from biological variants. These SSEs can cause base quality scores to underestimate the probability of error at certain genomic positions, resulting in false positive variant calls, particularly in mixtures such as samples with RNA editing, tumors, circulating tumor cells, bacteria, mitochondrial heteroplasmy, or pooled DNA. Most algorithms proposed for correction of SSEs require a data set used to calculate association of SSEs with various features in the reads and sequence context. This data set is typically either from a part of the data set being ''recalibrated'' (Genome Analysis ToolKit, or GATK) or from a separate data set with special characteristics (SysCall). Here, we combine the advantages of these approaches by adding synthetic RNA spike-in standards to human RNA, and use GATK to recalibrate base quality scores with reads mapped to the spike-in standards. Compared to conventional GATK recalibration that uses reads mapped to the genome, spike-ins improve the accuracy of Illumina base quality scores by a mean of 5 Phred-scaled quality score units, and by as much as 13 units at CpG sites. In addition, since the spike-in data used for recalibration are independent of the genome being sequenced, our method allows run-specific recalibration even for the many species without a comprehensive and accurate SNP database. We also use GATK with the spike-in standards to demonstrate that the Illumina RNA sequencing runs overestimate quality scores for AC, CC, GC, GG, and TC dinucleotides, while SOLiD has less dinucleotide SSEs but more SSEs for certain cycles. We conclude that using these DNA and RNA spike-in standards with GATK improves base quality score recalibration. [ABSTRACT FROM AUTHOR]

Understanding the cell molecular changes occurring as a results of climatic circumstances is crucial in the current days in which climate change and global warming are one of the most serious challenges that living organisms have to face. Sperm are one of the mammals' cells most sensitive to heat, therefore evaluating the impact of seasonal changes in terms of its transcriptional activity can contribute to elucidate how these cells cope with heat stress events. We sequenced the total sperm RNA from 64 ejaculates, 28 collected in summer and 36 collected in autumn, from 40 Manchega rams. A highly rich transcriptome (11,896 different transcripts) with 90 protein coding genes that exceed an average number of 5000 counts were found. Comparing transcriptome in the summer and autumn ejaculates, 236 significant differential abundance genes were assessed, most of them (228) downregulated. The main functions that these genes are related to sexual reproduction and negative regulation of protein metabolic processes and kinase activity. Sperm response to heat stress supposes a drastic decrease of the transcriptional activity, and the upregulation of only a few genes related with the basic functions to maintain the organisms' homeostasis and surviving. Rams' spermatozoids carry remnant mRNAs which are retrospectively indicators of events occurring along the spermatogenesis process, including abiotic factors such as environmental temperature. [ABSTRACT FROM AUTHOR]

Despite the worldwide consumption and high economic importance of tea, the plant (Camellia sinensis) is not well studied in molecular biology. Under the few circumstances in which the plant is studied, C. sinensis flowers, which are important for reproduction and cross-breeding, receive less emphasis than investigation of its leaves or roots. Using high-throughput Illumina RNA sequencing, we analyzed a C. sinensis floral transcriptome, and 26.9 million clean reads were assembled into 75,531 unigenes averaging 402 bp. Among them, 50,792 (67.2%) unigenes were annotated with a BLAST search against the NCBI Non-Redundant (NR) database and 10,290 (16.67%) were detected that contained one or more simple sequence repeats (SSRs). From these SSR-containing sequences, 2,439 candidate SSR markers were developed and 720 were experimentally tested, validating 431 (59.9%) novel polymorphic SSR markers for C. sinensis. Then, a consensus SSR-based linkage map was constructed that covered 1,156.9 cM with 237 SSR markers distributed in 15 linkage groups. Both transcriptome information and the genetic map of C. sinensis presented here offer a valuable foundation for molecular biology investigations such as functional gene isolation, quantitative trait loci mapping, and marker-assisted selection breeding in this important species. [ABSTRACT FROM AUTHOR]

Genome sequencing of Tricholoma matsutake revealed its unusually large size as 189.0 Mbp, which is a consequence of extraordinarily high transposable element (TE) content. We identified that 702 genes were surrounded by TEs, and 83.2% of these genes were not transcribed at any developmental stage. This observation indicated that the insertion of TEs alters the transcription of the genes neighboring these TEs. Repeat-induced point mutation, such as C to T hypermutation with a bias over "CpG" dinucleotides, was also recognized in this genome, representing a typical defense mechanism against TEs during evolution. Many transcription factor genes were activated in both the primordia and fruiting body stages, which indicates that many regulatory processes are shared during the developmental stages. Small secreted protein genes (<300 aa) were dominantly transcribed in the hyphae, where symbiotic interactions occur with the hosts. Comparative analysis with 37 Agaricomycetes genomes revealed that IstB-like domains (PF01695) were conserved across taxonomically diverse mycorrhizal genomes, where the T. matsutake genome contained four copies of this domain. Three of the IstB-like genes were overexpressed in the hyphae. Similar to other ectomycorrhizal genomes, the CAZyme gene set was reduced in T. matsutake, including losses in the glycoside hydrolase genes. The T. matsutake genome sequence provides insight into the causes and consequences of genome size inflation. [ABSTRACT FROM AUTHOR]

Selaginella tamariscina (Beauv.) spring, a primitive vascular resurrection plant, can survive extreme drought and recover when water becomes available. To identify drought-inducible genes and to clarify the molecular mechanism of drought tolerance, a comparative transcriptional pattern analysis was conducted between S. tamariscina and Selaginella moellendorffii Hieron (drought sensitive). 133 drought related genes were identified, including 72 functional genes and 61 regulatory genes. And several drought responsive reactions, such as antioxidant activity, osmotic balance, cuticle defense and signal transduction were highlighted in S. tamariscina under drought. Notably, besides peroxidase, catalase and L-ascorbate oxidase genes, DEGs associated with phenylalanine metabolism and polyamine catabolism could be alternative ways to enhance antioxidant ability in S. tamariscina. DEGs related to soluble carbohydrate metabolism, late embryogenesis abundant protein (LEA) and aquaporin protein (AQP) confirmed that osmotic adjustment could resist drought during desiccation. DEGs involved in xyloglucan metabolic process, pectin metabolic process and cutin biosynthesis may also contribute to drought tolerance of S. tamariscina by cuticle defense. Drought-responsive genes encoding protein kinases, calcium sensors, transcription factors (TFs) and plant hormones also help to drought resistance of S. tamariscina. The preliminary validation experiments were performed and the results were consistent with our hypothetical integrated regulatory network. The results of this study provide candidate resurrection genes and an integrated regulatory network for further studies on the molecular mechanisms of stress tolerance in S. tamariscina. [ABSTRACT FROM AUTHOR]

Development of white striping (WS) and wooden breast (WB) in broiler breast meat have been linked to hypoxia, but their etiologies are not fully understood. This study aimed at investigating absolute expression of hypoxia-inducible factor-1 alpha subunit (HIF1A) and genes involved in stress responses and muscle repair using a droplet digital polymerase chain reaction. Total RNA was isolated from pectoralis major collected from male 6-week-old medium (carcass weight ≤ 2.5 kg) and heavy (carcass weight > 2.5 kg) broilers. Samples were classified as “non-defective” (n = 4), “medium-WS” (n = 6), “heavy-WS” (n = 7) and “heavy-WS+WB” (n = 3) based on abnormality scores. The HIF1A transcript was up-regulated in all of the abnormal groups. Transcript abundances of genes encoding 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), lactate dehydrogenase-A (LDHA), and phosphorylase kinase beta subunit (PHKB) were increased in heavy-WS but decreased in heavy-WS+WB. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was up-regulated in non-defective samples. The muscle-specific mu-2 isoform of glutathione S-transferases (GSTM2) was up-regulated in the abnormal samples, particularly in the heavy groups. The genes encoding myogenic differentiation (MYOD1) and myosin light chain kinase (MYLK) exhibited similar expression pattern, of which medium-WS and heavy-WS significantly increased compared to non-defective whereas expression in heavy-WS+WB was not different from either non-defective or WS-affected group. The greatest and the lowest levels of calpain-3 (CAPN3) and delta-sarcoglycan (SCGD) were observed in heavy-WS and heavy-WS+WB, respectively. Based on micrographs, the abnormal muscles primarily comprised fibers with cross-sectional areas ranging from 2,000 to 3,000 μm2. Despite induced glycolysis at the transcriptional level, lower stored glycogen in the abnormal muscles corresponded with the reduced lactate and higher pH within their meats. The findings support hypoxia within the abnormal breasts, potentially associated with oversized muscle fibers. Between WS and WB, divergent glucose metabolism, cellular detoxification and myoregeneration at the transcriptional level could be anticipated. [ABSTRACT FROM AUTHOR]

Self-renewal and differentiation are essential for intestinal epithelium absorptive functioning and adaptation to pathological states such as short gut syndrome, ulcers, and inflammatory bowel disease. The rodent Slfn3 and its human analog Slfn12 are critical in regulating intestinal epithelial differentiation. We sought to characterize intestinal function in Slfn3 knockout (KO) mice. Male and female pair-fed Slfn3KO mice gained less weight with decreased food efficiency than wild type (WT) mice, with more pronounced effects in females. RNA sequencing performed on intestinal mucosa of Slfn3KO and WT mice showed gene ontology decreases in cell adhesion molecule signaling, tumor necrosis factor receptor binding, and adaptive immune cell proliferation/functioning genes in Slfn3KO mice, with greater effects in females. qPCR analysis of fatty acid metabolism genes, Pla2g4c, Pla2g2f, and Cyp3c55 revealed an increase in Pla2g4c, and a decrease in Pla2g2f in Slfn3KO females. Additionally, adipogenesis genes, Fabp4 and Lpl were decreased and ketogenesis gene Hmgcs2 was increased in female Slfn3KO mice. Sequencing did not reveal significant changes in differentiation markers, so qPCR was utilized. Slfn3KO tended to have decreased expression of intestinal differentiation markers sucrase isomaltase, dipeptidyl peptidase 4, villin 1, and glucose transporter 1 (Glut1) vs. WT males, although these trends did not achieve statistical significance unless data from several markers was pooled. Differentiation markers, Glut2 and sodium-glucose transporter 1 (SGLT1), did show statistically significant sex-dependent differences. Glut2 mRNA was reduced in Slfn3KO females, while SGLT1 increased in Slfn3KO males. Notch2 and Cdx2 were only increased in female Slfn3KO mice. Although Slfn3KO mice gain less weight and decreased food efficiency, their biochemical phenotype is more subtle and suggests a complex interplay between gender effects, Slfn3, and another regulatory pathway yet to be identified that compensates for the chronic loss of Slfn3. [ABSTRACT FROM AUTHOR]

Paralogues pairs are more frequently observed in eels (Anguilla sp.) than in other teleosts. The paralogues often show low phylogenetic distances; however, they have been assigned to the third round of whole genome duplication (WGD), shared by all teleosts (3R), due to their conserved synteny. The apparent contradiction of low phylogenetic difference and 3R conserved synteny led us to study the duplicated gene complement of the freshwater eels. With this aim, we assembled de novo transcriptomes of two highly relevant freshwater eel species: The European (Anguilla anguilla) and the Japanese eel (Anguilla japonica). The duplicated gene complement was analysed in these transcriptomes, and in the genomes and transcriptomes of other Actinopterygii species. The study included an assessment of neutral genetic divergence (4dTv), synteny, and the phylogenetic origins and relationships of the duplicated gene complements. The analyses indicated a high accumulation of duplications (1217 paralogue pairs) among freshwater eel genes, which may have originated in a WGD event after the Elopomorpha lineage diverged from the remaining teleosts, and thus not at the 3R. However, very similar results were observed in the basal Osteoglossomorpha and Clupeocephala branches, indicating that the specific genomic regions of these paralogues may still have been under tetrasomic inheritance at the split of the teleost lineages. Therefore, two potential hypotheses may explain the results: i) The freshwater eel lineage experienced an additional WGD to 3R, and ii) Some duplicated genomic regions experienced lineage specific rediploidization after 3R in the ancestor to freshwater eels. The supporting/opposing evidence for both hypotheses is discussed. [ABSTRACT FROM AUTHOR]

We previously analyzed the expression of genes associated with Paris polyphylla var. yunnanensis seed maturation and dormancy release; however, we were unable to clarify the relationship between gene expression levels and these processes. To reveal the molecular mechanisms underlying P. polyphylla var. yunnanensis seed dormancy release during a warm stratification, the transcriptomes of dormant and germinating P. polyphylla var. yunnanensis seeds were separately analyzed by RNA sequencing and were also compared with the transcriptomes of stem-leaf and root tissues harvested during the seed maturation stage. The RNA sequencing of five tissues generated 234,331 unigenes, of which 10,137 (4.33%) were differentially expressed among the analyzed tissues. The 6,619 unigenes whose expression varied among mature dormant, sprouted, and germinated seeds included 95 metabolic and 62 signaling genes related to abscisic acid, gibberellin, auxin, brassinosteroid, cytokinin, ethylene, jasmonic acid and salicylic acid. Additionally, 243 differentially expressed genes were annotated as known seed dormancy/germination-related genes. Among these genes, 109 were regulated by hormones or involved in hormone signal transduction. Finally, 310 transcription factor unigenes, including 71 homologs of known seed dormancy/ germination-related genes, were observed to be differentially expressed during a warm stratification. These results confirm that multiple hormones and transcription factors influence P. polyphylla var. yunnanensis seed dormancy release and germination during a warm stratification. This study identified candidate genes (e.g., ABI5) that should be cloned and functionally characterized regarding their effects on the release of P. polyphylla var. yunnanensis seed morphophysiological dormancy. [ABSTRACT FROM AUTHOR]

Bletilla striata is an endangered orchid that has been used for millennia as a medicinal herb, in cosmetics and as a horticultural plant. To construct the first nucleotide database for this species and to develop abundant EST-SSR markers for facilitating further studies, various tissues and organs of plants in the main developmental stages were harvested for mRNA isolation and subsequent RNA sequencing. A total of 106,054,784 clean reads were generated by using Illumina paired-end sequencing technology. The reads were assembled into 127,261 unigenes by the Trinity package; the unigenes had an average length of 612 bp and an N50 of 957 bp. Of these unigenes, 67,494 (51.86%) were annotated in a series of databases. Of these annotated unigenes, 41,818 and 24,615 were assigned to gene ontology categories and clusters of orthologous groups, respectively. Additionally, 20,764 (15.96%) unigenes were mapped onto 275 pathways using the KEGG database. In addition, 25,935 high-quality EST-SSR primer pairs were developed from the 15,433 unigenes by MISA mining. To validate the accuracy of the newly designed markers, 87 of 100 randomly selected primers were effectively amplified; 63 of those yielded PCR products of the expected size, and 25 yielded products with significant amounts of polymorphism among the 4 landraces. Furthermore, the transferability test of the 25 polymorphic markers was performed in 6 individuals of two closely related genus Phalaenopsis and dendrobium. Which results showed a total of 5 markers can successfully amplified among these populations. This research provides a comprehensive nucleotide database and lays a solid foundation for functional gene mining and genomic research in B. striata. The developed EST-SSR primers could facilitate phylogenetic studies and breeding. [ABSTRACT FROM AUTHOR]

An unusual novel plant virus provisionally named goji berry chlorosis virus (GBCV) was isolated from goji berry plants (Lycium chinense Miller) showing chlorosis symptoms and its complete genome sequence was determined. The viral genome consists of a positive-sense single-stranded RNA of 10,100 ribonucleotides and contains six open reading frames (ORFs). Electron microscopy showed that the viral genome is packaged as a filamentous particle with an average length of approximately 850 nm. Phylogenetic analysis and amino acid similarity analysis of the encoded ORFs revealed that this new virus could be classified in an intermediate position between the families Benyviridae and Virgaviridae. The GBCV 200-kDa replicase (ORF1) is more similar to benyvirus replicases than to virgavirus replicases, while its 17-kDa coat protein (CP, ORF2) is more closely related with virgavirus CPs than benyvirus CPs. ORF3 was predicted to produce a C-terminally extended protein from ORF2 via frameshifting. While ORF4 (45-kDa), ORF5 (44-kDa), and ORF6 (16-kDa) have no apparent sequence homology with other known viruses, ORF5 is predicted to encode a movement protein (MP) that is phylogenetically related to the furovirus MP and ORF6 was experimentally proven to encode a viral suppressor of RNA silencing. These unusual characteristics suggest that GBCV may represent an evolutionary link between the families Benyviridae and Virgaviridae and indicate the existence of a novel, unidentified virus group. [ABSTRACT FROM AUTHOR]

The whooping cough agent Bordetella pertussis coordinately regulates the expression of its virulence factors with the two-component system BvgAS. In laboratory conditions, specific chemical modulators are used to trigger phenotypic modulation of B. pertussis from its default virulent Bvg+ phase to avirulent Bvg- or intermediate Bvgi phases, in which no virulence factors or only a subset of them are produced, respectively. Whether phenotypic modulation occurs in the host remains unknown. In this work, recombinant B. pertussis strains harboring BvgS variants were tested in a mouse model of infection and analyzed using transcriptomic approaches. Recombinant BP-BvgΔ65, which is in the Bvgi phase by default and can be up-modulated to the Bvg+ phase in vitro, could colonize the mouse nose but was rapidly cleared from the lungs, while Bvg+-phase strains colonized both organs for up to four weeks. These results indicated that phenotypic modulation, which might have restored the full virulence capability of BP-BvgΔ65, does not occur in mice or is temporally or spatially restricted and has no effect in those conditions. Transcriptomic analyses of this and other recombinant Bvgi and Bvg+-phase strains revealed that two distinct ranges of virulence gene expression allow colonization of the mouse nose and lungs, respectively. We also showed that a recombinant strain expressing moderately lower levels of the virulence genes than its wild type parent was as efficient at colonizing both organs. Altogether, genetic modifications of BvgS generate a range of phenotypic phases, which are useful tools to decipher host-pathogen interactions. [ABSTRACT FROM AUTHOR]

Firefighting activities appear to increase the risk of acute and chronic lung disease, including malignancy. While self-contained breathing apparatuses (SCBA) mitigate exposures to inhalable asphyxiates and carcinogens, firefighters frequently remove SCBA during overhaul when the firegrounds appear clear of visible smoke. Using a mouse model of overhaul without airway protection, the impact of fireground environment exposure on lung gene expression was assessed to identify transcripts potentially critical to firefighter-related chronic pulmonary illnesses. Lung tissue was collected 2 hrs post-overhaul and evaluated via whole genome transcriptomics by RNA-seq. Although gas metering showed that the fireground overhaul levels of carbon monoxide (CO), carbon dioxide (CO2), hydrogen cyanine (HCN), hydrogen sulfide (H2S) and oxygen (O2) were within NIOSH ceiling recommendations, 3852 lung genes were differentially expressed when mice exposed to overhaul were compared to mice on the fireground but outside the overhaul environment. Importantly, overhaul exposure was associated with an up/down-regulation of 86 genes with a fold change of 1.5 or greater (p<0.5) including the immunomodulatory-linked genes S100a8 and Tnfsf9 (downregulation) and the cancer-linked genes, Capn11 and Rorc (upregulation). Taken together these findings indicate that, without respiratory protection, exposure to the fireground overhaul environment is associated with transcriptional changes impacting proteins potentially related to inflammation-associated lung disease and cancer. [ABSTRACT FROM AUTHOR]

Messenger RNA is modified by the addition of a 5′ methylated cap structure, which protects the transcript and recruits protein complexes that mediate RNA processing and/or the initiation of translation. Two genes encoding mRNA cap methyltransferases have been identified in T. brucei: TbCMT1 and TbCGM1. Here we analysed the impact of TbCMT1 gene deletion on bloodstream form T. brucei cells. TbCMT1 was dispensable for parasite proliferation in in vitro culture. However, significantly decreased parasitemia was observed in mice inoculated with TbCMT1 null and conditional null cell lines. Using RNA-Seq, we observed that several cysteine peptidase mRNAs were downregulated in TbCMT1 null cells lines. The cysteine peptidase Cathepsin-L was also shown to be reduced at the protein level in TbCMT1 null cell lines. Our data suggest that TbCMT1 is not essential to bloodstream form T. brucei growth in vitro or in vivo but that it contributes significantly to parasite virulence in vivo. [ABSTRACT FROM AUTHOR]

Many plant species possess compounds with juvenile hormone disruptor (JHD) activity. In some plant species, such activity has been attributed to diterpene secondary metabolites. Plant JHD diterpenes disrupt insect development by interfering with the juvenile hormone (JH)-mediated formation of JH receptor complexes. Here, we demonstrate that a plant extract and a diterpene from Lindera erythrocarpa (methyl lucidone) interfere with the formation of both methoprene-tolerant (Met)/Taiman and Germ cell-expressed (GCE)/Taiman heterodimer complexes in yeast two-hybrid assays in vitro. In addition to the in vitro JHD activity, the diterpene and the plant extract from L. erythrocarpa also disrupt the development of larvae and pupae in Drosophila melanogaster. Comparing the transcriptomes of juvenile hormone analog (JHA, methoprene)- and JHD (methyl lucidone)-fed wandering third-instar larvae revealed a large number of genes that were coregulated by JHA and JHD. Moreover, most (83%) of the genes that were repressed by methyl lucidone were significantly activated by methoprene, indicating that JHDs and JHAs have opposing effects on the transcriptional regulation of many JH-dependent genes. Gene ontology analysis also suggested that some of the genes activated-by-JHA/repressed-by-JHD play roles in spermatogenesis. Affymetrix microarray-based analysis indicated that the expression of genes activated-by-JHA/repressed-by-JHD was testis-specific. Together, these results suggest that JH is involved in testis-specific gene expression and that plant JHD diterpenes function as JH antagonists in such JHA-mediated gene regulation. [ABSTRACT FROM AUTHOR]

Flounder is a promising model species for environmental monitoring of coastal regions. To assess the usefulness of liver transcriptome profiling, juvenile olive flounder Paralichthys olivaceus were exposed to two pollutants, bisphenol S (BPS) and benzo[a]pyrene (BaP), which have different chemical characteristics and have distinct modes of metabolic action in teleost. Six hours after intraperitoneal injection with BPS (50 mg/kg bw) or BaP (20 mg/kg bw), liver transcriptomes were analyzed using the Illumina Hiseq 3000 platform. Interestingly, the transcriptome was highly sensitive and was distinctively expressed in response to each chemical. The primary effect of BPS was significantly increased transcription of egg process and vitellogenesis related genes, including vitellogenins (vtg1, vtg2), zona pellucida sperm-binding proteins (zp3, zp4), and estrogen receptors (erα, erβ), with increases in plasma 17β-estradiol (E2) and vitellogenin (VTG) concentrations. Following BaP treatment, detoxification- and biotransformation-related genes such as cyp1a1 and UDP-glucuronosyltransferase (ugt1a1) were significantly increased, with an increase in EROD activity. In both transcriptomes, mRNA expression of genes involved in antioxidant defense systems was increased, while genes involved in innate immunity were decreased upon BPS or BaP exposure with a decrease in complement activity. This study provides useful insight into the chemical-specific hepatic transcriptional response of P. olivaceus and suggests a basis for further studies examining biomarker application of liver transcriptomes for environmental pollution. [ABSTRACT FROM AUTHOR]

Metagenomics and next generation sequencing were employed to discover new virus natural enemies of the fire ant, Solenopsis invicta Buren in its native range (i.e., Formosa, Argentina) with the ultimate goal of testing and releasing new viral pathogens into U.S. S. invicta populations to provide natural, sustainable control of this ant. RNA was purified from worker ants from 182 S. invicta colonies, which was pooled into 4 groups according to location. A library was created from each group and sequenced using Illumina Miseq technology. After a series of winnowing methods to remove S. invicta genes, known S. invicta virus genes, and all other non-virus gene sequences, 61,944 unique singletons were identified with virus identity. These were assembled de novo yielding 171 contiguous sequences with significant identity to non-plant virus genes. Fifteen contiguous sequences exhibited very high expression rates and were detected in all four gene libraries. One contig (Contig_29) exhibited the highest expression level overall and across all four gene libraries. Random amplification of cDNA ends analyses expanded this contiguous sequence yielding a complete virus genome, which we have provisionally named Solenopsis invicta virus 5 (SINV-5). SINV-5 is a positive-sense, single-stranded RNA virus with genome characteristics consistent with insect-infecting viruses from the family Dicistroviridae. Moreover, the replicative genome strand of SINV-5 was detected in worker ants indicating that S. invicta serves as host for the virus. Many additional sequences were identified that are likely of viral origin. These sequences await further investigation to determine their origins and relationship with S. invicta. This study expands knowledge of the RNA virome diversity found within S. invicta populations. [ABSTRACT FROM AUTHOR]

Bacterial stem blight caused by Pseudomonas syringae pv. syringae is a common disease of alfalfa (Medicago sativa L). Little is known about host-pathogen interactions and host defense mechanisms. Here, individual resistant and susceptible plants were selected from cultivars Maverick and ZG9830 and used for transcript profiling at 24 and 72 hours after inoculation (hai) with the isolate PssALF3. Bioinformatic analysis revealed a number of differentially expressed genes (DEGs) in resistant and susceptible genotypes. Although resistant plants from each cultivar produced a hypersensitive response, transcriptome analyses indicated that they respond differently at the molecular level. The number of DEGs was higher in resistant plants of ZG9830 at 24 hai than in Maverick, suggesting that ZG9830 plants had a more rapid effector triggered immune response. Unique up-regulated genes in resistant ZG9830 plants included genes encoding putative nematode resistance HSPRO2-like proteins, orthologs for the rice Xa21 and soybean Rpg1-b resistance genes, and TIR-containing R genes lacking both NBS and LRR domains. The suite of R genes up-regulated in resistant Maverick plants had an over-representation of R genes in the CC-NBS-LRR family including two genes for atypical CCR domains and a putative ortholog of the Arabidopsis RPM1 gene. Resistance in both cultivars appears to be mediated primarily by WRKY family transcription factors and expression of genes involved in protein phosphorylation, regulation of transcription, defense response including synthesis of isoflavonoids, and oxidation-reduction processes. These results will further the identification of mechanisms involved in resistance to facilitate selection of parent populations and development of commercial varieties. [ABSTRACT FROM AUTHOR]

The cell cycle coordinates core functions such as replication and cell division. However, cell-cycle-regulated transcription in the control of non-core functions, such as cell identity maintenance through specific transcription factors (TFs) and signalling pathways remains unclear. Here, we provide a resource consisting of mapped transcriptomes in unsynchronized HeLa and U2OS cancer cells sorted for cell cycle phase by Fucci reporter expression. We developed a novel algorithm for data analysis that enables efficient visualization and data comparisons and identified cell cycle synchronization of Notch signalling and TFs associated with development. Furthermore, the cell cycle synchronizes with the circadian clock, providing a possible link between developmental transcriptional networks and the cell cycle. In conclusion we find that cell cycle synchronized transcriptional patterns are temporally compartmentalized and more complex than previously anticipated, involving genes, which control cell identity and development. [ABSTRACT FROM AUTHOR]

Troxerutin, a semi-synthetic derivative of the natural bioflavanoid rutin, has been reported to possess many beneficial effects in human bodies, such as vasoprotection, immune support, anti-inflammation and anti-aging. However, the effects of troxerutin on genome-wide transcription in blood cells are still unknown. In order to find out effects of troxerutin on gene transcription, a high-throughput RNA sequencing was employed to analysis differential gene expression in blood cells consisting of leucocytes, erythrocytes and platelets isolated from the mice received subcutaneous injection of troxerutin. Transcriptome analysis demonstrated that the expression of only fifteen genes was significantly changed by the treatment with troxerutin, among which 5 genes were up-regulated and 10 genes were down-regulated. Bioinformatic analysis of the fifteen differentially expressed genes was made by utilizing the Gene Ontology (GO), and the differential expression induced by troxerutin was further evaluated by real-time quantitative PCR (Q-PCR). [ABSTRACT FROM AUTHOR]