Candidate Transcripts Research Articles

What makes Candida auris pan-drug resistant? Integrative insights from genomic, transcriptomic, and phenomic analysis of clinical strains resistant to all four major classes of antifungal drugs.

The global epidemic of drug-resistant Candida auris continues unabated. We do not know what caused the unprecedented appearance of pan-drug resistant (PDR) Candida auris strains in a hospitalized patient in New York; the initial report highlighted both known and unique mutations in the prominent gene targets of azoles, amphotericin B, echinocandins, and flucytosine antifungal drugs. However, the factors that allow C. auris to acquire multi-drug resistance and pan-drug resistance are not known. Therefore, we conducted a comprehensive genomic, transcriptomic, and phenomic analysis to better understand PDR C. auris . Among 1,570 genetic variants in drug-resistant C. auris , 299 were unique to PDR strains. The whole genome sequencing results suggested perturbations in genes associated with nucleotide biosynthesis, mRNA processing, and nuclear export of mRNA. Whole transcriptome sequencing of PDR C. auris revealed two genes to be significantly differentially expressed - a DNA repair protein and DNA replication-dependent chromatin assembly factor 1. Of 59 novel transcripts, 12 candidate transcripts had no known homology among expressed transcripts found in other organisms. We observed no fitness defects among multi-drug resistant (MDR) and PDR C. auris strains grown in nutrient-deficient or - enriched media at different temperatures. Phenotypic profiling revealed wider adaptability to nitrogenous nutrients with an uptick in the utilization of substrates critical in upper glycolysis and tricarboxylic acid cycle. Structural modelling of 33-amino acid deletion in the gene for uracil phosphoribosyl transferase suggested an alternate route in C. auris to generate uracil monophosphate that does not accommodate 5-fluorouracil as a substrate. Overall, we find evidence of metabolic adaptations in MDR and PDR C. auris in response to antifungal drug lethality without deleterious fitness costs.

PLS-based integration of metabolomics and transcriptomics to reveal the effect of different growth years on Eurycoma longifolia roots and hairy root cultures

Growth years are important factors that affect the accumulation of secondary metabolites in Eurycoma longifolia roots. This study aimed to investigate the effect of different growth years on the metabolites and gene expression of E. longifolia roots and hairy root cultures. To achieve this, metabolite profiles and candidate transcripts of terpenoid backbone biosynthesis and sesquiterpenoid and triterpenoid pathways of 1- and 11-year old E. longifolia roots and hairy root cultures were obtained and integrated using partial least squares (PLS). Integration of eighty-two metabolites and forty-five selected candidate genes using PLS successfully differentiated 1-, 11-year-old E. longifolia roots and hairy root cultures, indicating significant metabolite-gene expression differences in response to different growth years and root types. Amino acids, 2-C-methyl-D-erythritol-4-phosphate cytidylyl-transferase (MCT) and acetyl-CoA C-acetyltransferase (AACT) were responsible for the discrimination of 1-year-old E. longifolia root. Quassinoids of eurycomanone and eurycomalactone and genes linked to the upstream synthesis in mevalonate (MVA) pathway contributed to the discrimination of the11-year-old E. longifolia root. 11-hydroxycanthin-6-one and genes that participate in the methylerythritol phosphate (MEP) pathway upstream of terpenoid biosynthesis were responsible for the discrimination of E. longifolia hairy root cultures.

Potential Diagnostic Value of Abnormal Pyroptosis Genes Expression in Myelodysplastic Syndromes (MDS): A Primary Observational Cohort Study.

Background: Myelodysplastic syndromes (MDS) are determined by ineffective hematopoiesis and bone marrow cytological dysplasia with somatic gene mutations and chromosomal abnormalities. Accumulating evidence has revealed the pivotal role of NLRP3 inflammasome activation and pyroptotic cell death in the pathogenesis of MDS. Although MDS can be diagnosed with a variety of morphologic and cytogenetic tests, most of these tests have limitations or problems in practice. Materials and Methods: In the present study, we evaluated the expression of genes that form the inflammasome (NLRP3, ASC, and CASP1) in bone marrow specimens of MDS patients and compared the results with those of other leukemias to evaluate their diagnostic value for MDS. Primary samples of this observational cohort study were collected from aspiration samples of patients with myelodysplastic syndromes (27 cases) and patients with non-myelodysplastic syndrome hematological cancers (45 cases). After RNA extraction and c.DNA synthesis, candidate transcripts and housekeeping transcripts were measured by real-time PCR method (SYBER Green assay). Using Kruskal-Wallis the relative gene expressions were compared and differences with p value less than 0.05 were considered as significant. Discrimination capability, cut-off, and area under curve (AUC) of all markers were analyzed with recessive operation curve (ROC) analysis. Results: We found that Caspase-1 and ASC genes expressed at more levels in MDS specimens compared to non-MDS hematological malignancies. A relative average expression of 10.22 with a p-value of 0.001 and 1.86 with p=0.019 was detected for Caspase-1 and ASC, respectively. ROC curve analysis shows an AUC of 0.739 with p=0.0001 for Caspase-1 and an AUC of 0.665 with p=0.0139 for ASC to MDS discrimination. Conclusion: Our results show that Caspase-1 and ASC gene expression levels can be used as potential biomarkers for MDS diagnosis. Prospective studies with large sample numbers are suggested.

A mRNA panel for differentiation between acute exacerbation or pneumonia in COPD patients.

Patients suffering from chronic obstructive pulmonary disease (COPD) are prone to acute exacerbations (AECOPD) or community acquired pneumonia (CAP), both posing severe risk of morbidity and mortality. There is no available biomarker that correctly separates AECOPD from COPD. However, because CAP and AECOPD differ in aetiology, treatment and prognosis, their discrimination would be important. This study analysed the ability of selected candidate transcripts from peripheral blood mononuclear cells (PBMCs) to differentiate between patients with AECOPD, COPD & CAP, and CAP without pre-existing COPD. In a previous study, we identified differentially regulated genes between CAP and AECOPD in PBMCs. In the present new cohort, we tested the potential of selected candidate PBMC transcripts to differentiate at early time points AECOPD, CAP+COPD, and CAP without pre-existing COPD. Expression of YWHAG, E2F1 and TDRD9 held predictive power: This gene set predicted diseases markedly better (model accuracy up to 100%) than classical clinical markers like CRP, lymphocyte count and neutrophil count (model accuracy up to 82%). In summary, in our cohort expression levels of YWHAG, E2F1 and TDRD9 differentiated with high accuracy between COPD patients suffering from acute exacerbation or CAP.

Plasma cell-free RNA profiling of Vietnamese Alzheimer's patients reveals a linkage with chronic inflammation and apoptosis: a pilot study.

Circulating cell-free RNA (cfRNA) is a potential hallmark for early diagnosis of Alzheimer's Disease (AD) as it construes the genetic expression level, giving insights into the pathological progress from the outset. Profiles of cfRNA in Caucasian AD patients have been investigated thoroughly, yet there was no report exploring cfRNAs in the ASEAN groups. This study examined the gap, expecting to support the development of point-of-care AD diagnosis. cfRNA profiles were characterized from 20 Vietnamese plasma samples (10 probable AD and 10 age-matched controls). RNA reads were subjected to differential expression (DE) analysis. Weighted gene correlation network analysis (WGCNA) was performed to identify gene modules that were significantly co-expressed. These modules' expression profiles were then correlated with AD status to identify relevant modules. Genes with the highest intramodular connectivity (module membership) were selected as hub genes. Transcript counts of differentially expressed genes were correlated with key AD measures-MMSE and MTA scores-to identify potential biomarkers. 136 genes were identified as significant AD hallmarks (p < 0.05), with 52 downregulated and 84 upregulated in the AD cohort. 45.6% of these genes are highly expressed in the hippocampus, cerebellum, and cerebral cortex. Notably, all markers related to chronic inflammation were upregulated, and there was a significant shift in all apoptotic markers. Three co-expressed modules were found to be significantly correlated with Alzheimer's status (p < 0.05; R2> 0.5). Functional enrichment analysis on these modules reveals an association with focal adhesion, nucleocytoplasmic transport, and metal ion response leading to apoptosis, suggesting the potential participation of these pathways in AD pathology. 47 significant hub genes were found to be differentially expressed genes with the highest connectivity. Six significant hub genes (CREB1, YTHDC1, IL1RL1, PHACTR2, ANKRD36B, RNF213) were found to be significantly correlated with MTA and MMSE scores. Other significant transcripts (XRN1, UBB, CHP1, THBS1, S100A9) were found to be involved in inflammation and neuronal death. Overall, we have identified candidate transcripts in plasma cf-RNA that are differentially expressed and are implicated in inflammation and apoptosis, which can jumpstart further investigations into applying cf-RNA as an AD biomarker in Vietnam and ASEAN countries.

Comprehensive Long-Read Sequencing Analysis Discloses the Transcriptome Features of Papillary Thyroid Microcarcinoma.

Papillary thyroid microcarcinoma (PTMC) is the most common type of thyroid cancer. It has been shown that lymph node metastasis is associated with poor prognosis in patients with PTMC. We aim to characterize the PTMC transcriptome landscape and identify the candidate transcripts that are associated with lateral neck lymph node metastasis of PTMC. We performed full-length transcriptome sequencing in 64 PTMC samples. Standard bioinformatic pipelines were applied to characterize and annotate the full-length expression profiles of 2 PTMC subtypes. Functional open reading frame (ORF) annotation of the known and novel transcripts were predicted by HMMER, DeepLoc, and DeepTMHMM tools. Candidate transcripts associated with the pN1b subtype were identified after transcript quantification and differential gene expression analyses. We found that skipping exons accounted for the more than 27.82% of the alternative splicing events. At least 42.56% of the discovered transcripts were novel isoforms of annotated genes. A total of 39 193 ORFs in novel transcripts and 18 596 ORFs in known transcripts were identified. Distribution patterns of the characterized transcripts in functional domain, subcellular localization, and transmembrane structure were predicted. In total, 1033 and 1204 differentially expressed genes were identified in the pN0 and pN1b groups, respectively. Moreover, novel isoforms of FRMD3, NOD1, and SHROOM4 were highlighted for their association with pN1b subtype. Our data provided the global transcriptome landscape of PTMC and also revealed the novel isoforms that associated with PTMC aggressiveness.

Transcriptome and metabolome analyses revealed the response mechanism of pepper roots to Phytophthora capsici infection

BackgroundPhytophthora root rot caused by the oomycete Phytophthora capsici is the most devastating disease in pepper production worldwide, and current management strategies have not been effective in preventing this disease. Therefore, the use of resistant varieties was regarded as an important part of disease management of P. capsici. However, our knowledge of the molecular mechanisms underlying the defense response of pepper roots to P. capsici infection is limited.MethodsA comprehensive transcriptome and metabolome approaches were used to dissect the molecular response of pepper to P. capsici infection in the resistant genotype A204 and the susceptible genotype A198 at 0, 24 and 48 hours post-inoculation (hpi).ResultsMore genes and metabolites were induced at 24 hpi in A204 than A198, suggesting the prompt activation of defense responses in the resistant genotype, which can attribute two proteases, subtilisin-like protease and xylem cysteine proteinase 1, involved in pathogen recognition and signal transduction in A204. Further analysis indicated that the resistant genotype responded to P. capsici with fine regulation by the Ca2+- and salicylic acid-mediated signaling pathways, and then activation of downstream defense responses, including cell wall reinforcement and defense-related genes expression and metabolites accumulation. Among them, differentially expressed genes and differentially accumulated metabolites involved in the flavonoid biosynthesis pathways were uniquely activated in the resistant genotype A204 at 24 hpi, indicating a significant role of the flavonoid biosynthesis pathways in pepper resistance to P. capsici.ConclusionThe candidate transcripts may provide genetic resources that may be useful in the improvement of Phytophthora root rot-resistant characters of pepper. In addition, the model proposed in this study provides new insight into the defense response against P. capsici in pepper, and enhance our current understanding of the interaction of pepper–P. capsici.

Dynamic interplay of WRKY, GRAS, and ERF transcription factor families in tomato-endophytic fungal symbiosis: insights from transcriptome and genome-wide analysis.

Plant-microbe interactions play a crucial role in shaping plant growth and development, as well as in mediating plant responses to biotic and abiotic stresses. In this study, we used RNA-seq data to examine the expression profiles of SlWRKY, SlGRAS, and SlERF genes during the symbiotic association of Curvularia lunata SL1 with tomato (Solanum lycopersicum) plants. We also conducted functional annotation analysis by comparative genomics studies of their paralogs and orthologs genes, as well as other approaches, such as gene analysis and protein interaction networks, to identify and characterize the regulatory roles of these TFs in the development of the symbiotic association. We found that more than half of the investigated SlWRKY genes exhibited significant upregulation during symbiotic association, including SlWRKY38, SlWRKY46, SlWRKY19, and SlWRKY51. Several SlGRAS and SlERF genes were upregulated, such as SlGLD2, SlGLD1, SlERF.C.5, ERF16, and SlERF.B12. Conversely, a smaller proportion of SlWRKY, SlGRAS, and SlERF genes were significantly downregulated during symbiotic association. Furthermore, we investigated the possible roles of SlWRKY, SlGRAS, and SlERF genes in hormonal regulation during plant-microbe interactions. We identified several upregulated candidate transcripts likely to be involved in plant hormone signaling pathways. Our findings are consistent with previous studies on these genes, providing further evidence of their involvement in hormonal regulation during plant-microbe interactions. To validate the RNA-seq data accuracy, we performed RT-qPCR analyses of selected SlWRKY, SlGRAS, and SlERF genes, which showed similar expression patterns to those observed in the RNA-seq data. These results confirmed the accuracy of our RNA-seq data and provided additional support for the differential expression of these genes during plant-microbe interactions. Taken together, our study provides new insights into the differential expression profiles of SlWRKY, SlGRAS, and SlERF genes during symbiotic association with C. lunata, as well as their potential roles in hormonal regulation during plant-microbe interactions. These findings could be useful for guiding future research on the ways in which plants and microbes interact, and may ultimately lead to the creation of better approaches for promoting plant growth under stressful conditions.

Contact toxicity of eucalyptol and RNA sequencing of Tribolium castaneum after exposure to eucalyptol

AbstractEucalyptol, the main active component of Artemisia vulgaris (A. vulgaris) essential oil, can reduce the activity of Tribolium castaneum (T. castaneum) metabolic detoxification enzymes. However, the molecular mechanism underlying the toxic effect of eucalyptol on T. castaneum is still unclear. Here, results showed that the eucalyptol caused time‐dependent increase in larval mortality. Further, 1669 differential expressed genes (DEGs) expressed between the control group and the experimental group were detected via RNA sequencing, of which 504 were upregulated and 1165 were downregulated. The validity of the RNA‐Seq results was verified by qRT‐PCR. Further, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to analyze the DEGs, focusing on immune system process, response to stimulus, and developmental process in the GO analysis and on the excretory system and the metabolism of terpenoids and polyketides in the KEGG analysis. The results showed that the upregulation of Apolipoprotein D and the downregulation of V‐type proton ATPase subunits, farnesol dehydrogenase, receptor tyrosine‐protein phosphatase ε and α may affect the growth and metabolism of T. castaneum larvae. In the KEGG pathway analysis, we focused on the role of certain metabolic detoxification enzymes in the regulation of eucalyptol‐stimulated T. castaneum. Among them, the expression of metabolic detoxification enzymes OBP10, CYP4C1, CYP6BS1, CYP4g15, CYP301A1, CYP49A1, UGTs2C1, UGTs2B2, UGTs2B16, and Est FE4 was downregulated. The expression of OBPC01, OBPC09, OBPC12, OBPC02, OBPC11, CYP345B1, CYP346B1, Est E4, Est 6, and GST1 increased significantly. These metabolic detoxification enzymes were involved in the detoxification of eucalyptol by T. castaneum, and their downregulation or upregulation may indirectly affect insect survival and death. Our results provide an overview of transcriptomic changes in eucalyptol‐stimulated T. castaneum and enriches the candidate transcripts that can be exploited for pest control initiatives.

Multisensory integration by polymodal sensory neurons dictates larval settlement in a brainless cnidarian larva.

Multisensory integration (MSI) combines information from more than one sensory modality to elicit behaviours distinct from unisensory behaviours. MSI is best understood in animals with complex brains and specialized centres for parsing different modes of sensory information, but dispersive larvae of sessile marine invertebrates utilize multimodal environmental sensory stimuli to base irreversible settlement decisions on, and most lack complex brains. Here, we examined the sensory determinants of settlement in actinula larvae of the hydrozoan Ectopleura crocea (Cnidaria), which possess a diffuse nerve net. A factorial settlement study revealed that photo-, chemo- and mechanosensory cues each influenced the settlement response in a complex and hierarchical manner that was dependent on specific combinations of cues, an indication of MSI. Additionally, sensory gene expression over development peaked with developmental competence to settle, which in actinulae, requires cnidocyte discharge. Transcriptome analyses also highlighted several deep homological links between cnidarian and bilaterian mechano-, chemo-, and photosensory pathways. Fluorescent in situ hybridization studies of candidate transcripts suggested cellular partitioning of sensory function among the few cell types that comprise the actinula nervous system, where ubiquitous polymodal sensory neurons expressing putative chemo- and photosensitivity interface with mechanoreceptive cnidocytes. We propose a simple multisensory processing circuit, involving polymodal chemo/photosensory neurons and mechanoreceptive cnidocytes, that is sufficient to explain MSI in actinulae settlement. Our study demonstrates that MSI is not exclusive to complex brains, but likely predated and contextualized their evolution.

Actively transcribed rDNA and distal junction (DJ) sequence are involved in association of NORs with nucleoli

Although they are organelles without a limiting membrane, nucleoli have an exclusive structure, built upon the rDNA-rich acrocentric short arms of five human chromosomes (nucleolar organizer regions or NORs). This has raised the question: what are the structural features of a chromosome required for its inclusion in a nucleolus? Previous work has suggested that sequences adjacent to the tandemly repeated rDNA repeat units (DJ, distal junction sequence) may be involved, and we have extended such studies by addressing several issues related to the requirements for the association of NORs with nucleoli. We exploited both a set of somatic cell hybrids containing individual human acrocentric chromosomes and a set of Human Artificial Chromosomes (HACs) carrying different parts of a NOR, including an rDNA unit or DJ or PJ (proximal junction) sequence. Association of NORs with nucleoli was increased when constituent rDNA was transcribed and may be also affected by the status of heterochromatin blocks formed next to the rDNA arrays. Furthermore, our data suggest that a relatively small size DJ region, highly conserved in evolution, is also involved, along with the rDNA repeats, in the localization of p-arms of acrocentric chromosomes in nucleoli. Thus, we infer a cooperative action of rDNA sequence—stimulated by its activity—and sequences distal to rDNA contributing to incorporation into nucleoli. Analysis of NOR sequences also identified LncRNA_038958 in the DJ, a candidate transcript with the region of the suggested promoter that is located close to the DJ/rDNA boundary and contains CTCF binding sites. This LncRNA may affect RNA Polymerase I and/or nucleolar activity. Our findings provide the basis for future studies to determine which RNAs and proteins interact critically with NOR sequences to organize the higher-order structure of nucleoli and their function in normal cells and pathological states.

Abstract 3908: M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer

Abstract BACKGROUND: Chemotherapy resistance is recognized to occur not only through selection of pre-existing genetically resistant clones, but also through rapid phenotypic plasticity mechanisms. We previously reported that bladder cancer cells can rapidly transition to and from a chemo-resistant phenotype. Recently, N6-methyladenosine (m6A) RNA modifications were shown to dynamically and reversibly regulate mRNA processing, differentiation, and cell fate. Here, we tested the hypothesis that m6A modifications regulate phenotypic plasticity and transition to cisplatin resistance in bladder cancer (BC). METHODS: We utilized methyl-RNA-immunoprecipitation followed by sequencing (MeRIP-seq) and by RNA-seq to identify transcripts that were both differentially methylated and differentially expressed between cisplatin-sensitive and cisplatin-resistant BC cell lines. Candidate transcripts were annotated using Gene Ontology (GO) enrichment analysis and Gene Set Enrichment Analysis (GSEA). Clinical relevance was evaluated using databases such as The Cancer Genome Atlas (TCGA) and the Oncology Research Information Network (ORIEN) avatar. Cancer-relevant genes were then validated in vitro by targeted immunoprecipitation and PCR (MeRIP-PCR) and qPCR. Candidate genes were then functionally validated in cell lines and in patient derived organoids (PDOs) by siRNA-mediated knockdown and cisplatin treatment, as well as by m6A measurement after short-term cisplatin treatment. RESULTS: MeRIP-seq and RNA-seq revealed that cisplatin-sensitive and cisplatin-resistant BC cells have distinct m6A profiles, with 130 transcripts that were both differentially methylated and differentially expressed. Using clinical and functional database tools, we filtered this list to 37 transcripts and ranked them based on clinical relevance in BC. Of the top 15 candidates, eight were successfully validated in vitro via repeat qPCR and MeRIP-PCR. In cisplatin-resistant cells, one candidate, SLC7A11, was found to have decreased m6A level, which was associated with increased RNA stability and elevated transcript and protein levels. Consistent with this, 48 hr cisplatin treatment of cisplatin-sensitive cells led to SLC7A11 hypomethylation, increased RNA stability, and elevated transcript and protein levels. Furthermore, depletion of SLC7A11 by siRNA-knockdown re-sensitized BC cell lines and PDOs to cisplatin. CONCLUSIONS: Using unbiased transcriptome-wide m6A profiling followed by targeted validation, we demonstrated that m6A modifications regulate expression of clinically relevant gene transcripts in BC, and that a subset of these modifications may promote resistance to chemotherapy Collectively, these results implicate epitranscriptomic plasticity as a driver in potentiating cisplatin resistance in bladder cancer. Citation Format: Emmanuelle Hodara, Aubree Mades, Tong Xu, Amir Goldkorn, Suhn Rhie. M6A RNA modifications regulate expression of transcripts that promote transition to cisplatin resistance in bladder cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3908.

Gene Co-Expression Network Analysis Reveals the Hub Genes and Key Pathways Associated with Resistance to Salmonella Enteritidis Colonization in Chicken

Salmonella negatively impacts the poultry industry and threatens animals' and humans' health. The gastrointestinal microbiota and its metabolites can modulate the host's physiology and immune system. Recent research demonstrated the role of commensal bacteria and short-chain fatty acids (SCFAs) in developing resistance to Salmonella infection and colonization. However, the complex interactions among chicken, Salmonella, host-microbiome, and microbial metabolites remain unelucidated. Therefore, this study aimed to explore these complex interactions by identifying the driver and hub genes highly correlated with factors that confer resistance to Salmonella. Differential gene expression (DEGs) and dynamic developmental genes (DDGs) analyses and weighted gene co-expression network analysis (WGCNA) were performed using transcriptome data from the cecum of Salmonella Enteritidis-infected chicken at 7 and 21 days after infection. Furthermore, we identified the driver and hub genes associated with important traits such as the heterophil/lymphocyte (H/L) ratio, body weight post-infection, bacterial load, propionate and valerate cecal contents, and Firmicutes, Bacteroidetes, and Proteobacteria cecal relative abundance. Among the multiple genes detected in this study, EXFABP, S100A9/12, CEMIP, FKBP5, MAVS, FAM168B, HESX1, EMC6, and others were found as potential candidate gene and transcript (co-) factors for resistance to Salmonella infection. In addition, we found that the PPAR and oxidative phosphorylation (OXPHOS) metabolic pathways were also involved in the host's immune response/defense against Salmonella colonization at the earlier and later stage post-infection, respectively. This study provides a valuable resource of transcriptome profiles from chicken cecum at the earlier and later stage post-infection and mechanistic understanding of the complex interactions among chicken, Salmonella, host-microbiome, and associated metabolites.

Integrative Analysis of the Identified Transcriptome and Proteome Major Metabolism Pathways Involved in the Development of Grafted Apricot Hybrids

Plant distant grafting can produce stable genetic variation, which is a new method for germplasm innovation. Two chimeras, peach/apricot (PA) and apricot/peach (AP), were created through two-way grafting between peach and apricot. The leaves, flowers and fruit phenotypes of chimeras were significantly different to self-rooted rootstock. In order to investigate the causes of such changes, transcriptome and proteome integrative analyses were conducted on apricots from these two chimeras. Many differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) that may be connected to the development of grafted apricot hybrids were identified and explored based on function. Moreover, we found 76 genes in forward-grafted PA and 46 in reverse-grafted AP that overlapped both in DEGs and DEPs (DEGs/DEPs) via transcriptome–proteome integrative analysis. Mapping the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database in PA and AP, the top significant enrichment pathways of DEGs/DEPs included lipid metabolism (fatty acid elongation, cutin, suberine and wax biosynthesis, fatty acid degradation and alpha-linolenic acid metabolism) and carbohydrate metabolism (glycolysis/gluconeogenesis, starch and sucrose metabolism and galactose metabolism), revealing that lipid metabolism and carbohydrate metabolism may play an irreplaceable role in the development of grafted apricot hybrids. Taken together, this work uncovered numerous candidate transcripts and proteins involved in the development of grafted apricot hybrids. The molecular mechanisms provide new insights into this important process in other heterografting hybrids.

Tolerance to dietary linalool primarily involves co-expression of cytochrome P450s and cuticular proteins in Pagiophloeus tsushimanus (Coleoptera: Curculionidae) larvae using SMRT sequencing and RNA-seq

BackgroundPagiophloeus tsushimanus (Coleoptera: Curculionidae), an emerging forest pest exclusively infesting camphor trees, has recently caused severe ecological and economic damage in localized areas in China. Its population outbreak depends largely on the capacity to overcome the pressure of terpenoid-derived metabolites (e.g. linalool) from camphor trees. At present, the molecular basis of physiological adaptation of P. tsushimanus to dietary linalool is poorly understood, and there is no available reference genome or transcriptome.ResultsHerein, we constructed the transcriptome profiling of P. tsushimanus larvae reared on linalool-infused diets using RNA sequencing and single-molecule real-time sequencing. A total of 20,325 high-quality full-length transcripts were identified as a reference transcriptome, of which 14,492 protein-coding transcripts including 130 transcription factors (TFs), and 5561 long non-coding RNAs (lncRNAs) were detected. Also, 30 alternative splicing events and 8049 simple sequence repeats were captured. Gene ontology enrichment of differential expressed transcripts revealed that overall up-regulation of both cytochrome P450s (CYP450s) and cuticular proteins (CPs), was the primary response characteristic against dietary linalool. Other physiological effects possibly caused by linalool exposure, such as increase in Reactive Oxygen Species (ROS) and hormetic stimulation, were compensated by a handful of induced genes encoding antioxidases, heat shock proteins (HSPs), juvenile hormone (JH) epoxide hydrolases, and digestive enzymes. Additionally, based on co-expression networks analysis, a diverse array of hub lncRNAs and TFs co-expressed with CYP450s and CPs were screened as the potential gene regulators. Temporal expression of candidate transcripts determined by quantitative real-time PCR also indicated a cooperative relationship between the inductions of CYP450s and CPs upon exposure to linalool.ConclusionsOur present study provides an important transcriptome resource of P. tsushimanus, and lays a valuable foundation for understanding how this specialist pest copes with chemical challenges in its specific host environments.

Distinct effects of α-linolenic acid and docosahexaenoic acid on the expression of genes related to cholesterol metabolism and the response to infection in THP-1 monocytes and immune cells of obese humans

BackgroundMonocytes play a large role in chronic inflammatory conditions such as obesity, atherosclerosis and infection. Marine-derived omega-3 fatty acids such as docosahexaenoic acid (DHA) beneficially alter immune function and attenuate chronic inflammation in part by modifying gene expression. Comparisons with plant-derived omega-3 α-linolenic acid (ALA) on immune cell gene expression and function are limited. MethodsTranscriptome analysis was performed on THP-1 human monocytes treated with ALA, DHA or vehicle for 48 hr using fold change analysis, principal component analysis (PCA), partial least squares-discriminant analysis (PLS-DA), variable importance analysis (VIP), and ingenuity pathway analysis (IPA). Candidate genes were validated by qPCR. Functional assays evaluated the transcriptomic predictions. Expression of candidate transcripts identified in THP-1 cells were examined in PBMC from clinical trial (OXBIO; NCT 03583281) participants consuming ALA- or DHA-rich oil supplements. FindingsALA and DHA-treated monocytes presented distinct transcriptomic profiles as per VIP and PLS-DA. Both fatty acids were predicted to reduce cellular cholesterol content, while ALA would uniquely increase response to infection and chemotactic signals. Functional assays revealed ALA and DHA decreased cholesterol content. DHA significantly decreased the response to infection and chemotaxis, but ALA had no effect. Candidate transcripts responded similarly in PBMC from n-3 PUFA supplemented women with obesity. ConclusionALA and DHA differentially alter the transcription profiles and functions associated with the response to infection, chemotaxis, and cholesterol metabolism in mononuclear immune cells. Thus, they may uniquely affect related disease processes contributing to obesity, atherosclerosis, and the response to infection.

BSR and Full-Length Transcriptome Approaches Identified Candidate Genes for High Seed Ratio in Camellia vietnamensis.

(1) Background: C. vietnamensis is very suitable for growth in the low hilly areas of southern subtropical regions. Under appropriate conditions, the oil yield of C. vietnamensis can reach 1125 kg/ha (the existing varieties can reach 750 kg/ha). Moreover, the fruit of C. vietnamensis is large and the pericarp is thick (&gt;5 cm). Therefore, a high seed ratio has become the main target economic trait for the breeding of C. vietnamensis. (2) Methods: A half-sibling population of C. vietnamensis plants with a combination of high and low seed ratios was constructed by crossing a C. vietnamensis female parent. Bulked segregant RNA analysis and full-length transcriptome sequencing were performed to determine the molecular mechanisms underlying a high seed ratio. (3) Results: Seed ratio is a complex quantitative trait with a normal distribution, which is significantly associated with four other traits of fruit (seed weight, seed number, fruit diameter, and pericarp thickness). Two candidate regions related to high seed ratio (HSR) were predicted. One spanned 140.8-148.4 Mb of chromosome 2 and was associated with 97 seed-yield-related candidate genes ranging in length from 278 to 16,628 bp. The other spanned 35.3-37.3 Mb on chromosome 15 and was associated with 38 genes ranging in length from 221 to 16,928 bp. Using the full-length transcript as a template, a total of 115 candidate transcripts were obtained, and 78 transcripts were predicted to be functionally annotated. The DEGs from two set pairs of cDNA sequencing bulks were enriched to cytochrome p450 CYP76F14 (KOG0156; GO:0055114, HSR4, HSR7), the gibberellin phytohormone pathway (GO:0016787, HSR5), the calcium signaling pathway (GO:0005509, HSR6), the polyubiquitin-PPAR signaling pathway (GO:0005515, HSR2, HSR3), and several main transcription factors (bZIP transcription factor, HSR1) in C. vietnamensis.

Novel circRNA-miRNA-mRNA networks regulated by maternal exercise in fetal hearts of pregestational diabetes

BackgroundMaternal exercise lowers the incidence of congenital heart defects (CHDs) induced by pregestational diabetes. However, the molecular mechanisms underlying the beneficial effects of maternal exercise remain unclear. The present study aimed to identify circular RNA (circRNA), microRNA (miRNA) and mRNA networks that are regulated by maternal exercise in fetal hearts of pregestational diabetes. MethodsPregestational diabetes was induced in adult C57BL/6 female mice by streptozotocin. The expression profiles of circRNAs, miRNAs and mRNAs in E10.5 fetal hearts of offspring of control and diabetic mothers with or without exercise were analyzed using next generation sequencing. circRNA-miRNA-mRNA networks in fetal hearts were mapped and key candidate transcripts were verified by qPCR analysis. ResultsPregestational diabetes dysregulated the expression of 206 circRNAs, 66 miRNAs and 391 mRNAs in fetal hearts. Maternal exercise differentially regulated 188 circRNAs, 57 miRNAs and 506 mRNAs in fetal hearts of offspring of pregestational diabetes. A total of 5 circRNAs, 12 miRNAs, and 28 mRNAs were incorporated into a final maternal exercise-associated regulatory network in fetal hearts of offspring of maternal diabetes. Notably, maternal exercise normalized the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs in fetal hearts of pregestational diabetes. ConclusionMaternal exercise reverses the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs, and partially normalizes circRNA, miRNA, and mRNA expression profiles in fetal hearts of pregestational diabetes. These findings shed new light on the potential mechanisms of the beneficial effects of maternal exercise on the developing heart in diabetic pregnancies.

Transcriptome diversity assessment of Gossypium arboreum (FDH228) leaves under control, drought and whitefly infestation using PacBio long reads

Alternative splicing (AS) and alternative polyadenylation (APA) are common mechanisms in eukaryotes to increase the complexity of transcriptomes and subsequently proteomes. Analysis of long reads transcriptomics data can result in the discovery of novel transcripts, splice sites, AS or APA events. Gossypium arboreum is an important cultivated cotton species and a putative contributor of the A sub-genome to the modern tetraploid cotton; and inherently tolerant to several biotic and abiotic stresses. Specifically, its variety ‘FDH228′ is considered to be an important resistance source. In this study, we sequenced the G. arboreum (var. FDH228) transcriptome using PacBio IsoSeq and illumina short read sequencing under three different conditions i.e. untreated/healthy, treated with biotic stress through whitefly infestation, and treated with abiotic stress via water deprivation, for the discovery and surveying of canonical and non-canonical AS, APA and transcript fusion events. We were able to obtain 15,419 unique transcripts from all samples representing 11,343 genes, out of which 10,832 were annotated and 520 were novel with respect to the published reference genome. These transcripts were grouped into different structural categories including 60 Antisense, 11,959 having a full-splice match, 999 with incomplete-splice match, 30 fusion transcripts, 177 genic, 479 intergenic, 771 novels in the catalog, and 944 Novel but not found in the catalog. Subsequently, randomly selected candidate transcripts were experimentally validated using qRT-PCR. Our comprehensive identification of canonical and non-canonical splicing events, and novel and fusion transcripts aids in the understanding of the resistance mechanisms for this specific germplasm.

Meta-Analysis of the Public RNA-Seq Data of the Western Honeybee Apis mellifera to Construct Reference Transcriptome Data.

Simple SummaryThe honeybee is an economically and ecologically essential insect species and a critical social behavior model. It is vital to honey production and pollination. Therefore, it has been studied extensively in entomological research. The Western honeybee (Apis mellifera) is a representative species of honeybees. Reference transcriptome data are required to perform the meta-analyses using public RNA-Seq data and transcriptome analysis using newly sequenced RNA-Seq data. The reference transcriptome sequences were constructed, which consisted of 149,685 transcripts, and 194,174 predicted amino acid sequences. Half the predicted protein sequences were functionally annotated using protein sequence datasets for several model and insect species. We performed meta-analysis to search for novel immune-response-related transcripts. We identified 3–20 transcripts that were upregulated or downregulated in response to both viral and bacterial infections. However, the immune-related transcripts were not included among them. Autophagy-related protein 3 was downregulated by viral infections. The results suggested that autophagy was implicated in the viral immune responses, and autophagy was not regulated by the canonical immune pathways in A. mellifera. The results of the meta-analysis suggested that the reference transcriptome data could be used for transcriptome analysis in A. mellifera.The Western honeybee (Apis mellifera) is valuable in biological research and agriculture. Its genome sequence was published before those for other insect species. RNA-Seq data for A. mellifera have been applied in several recently published studies. Nevertheless, these data have not been prepared for use in subsequent meta-analyses. To promote A. mellifera transcriptome analysis, we constructed reference transcriptome data using the reference genome sequence and RNA-Seq data curated from about 1,000 runs of public databases. The new reference transcriptome data construct comprised 149,685 transcripts, and 194,174 protein sequences were predicted. Approximately 50–60% of the predicted protein sequences were functionally annotated using the protein sequence data for several model and insect species. Novel candidate immune-related transcripts were searched by meta-analysis using immune-response-related RNA-Seq and reference transcriptome data. Three to twenty candidate transcripts including autophagy-related protein 3 were upregulated or downregulated in response to both viral and bacterial infections. The constructed reference transcriptome data may facilitate future transcriptome analyses of A. mellifera.