Stage-specific Expression Patterns Research Articles

Integrated phenotypic, transcriptomics and metabolomics: growth status and metabolite accumulation pattern of medicinal materials at different harvest periods of Astragalus Membranaceus Mongholicus

BackgroundAstragalus membranaceus var. mongholicus (Astragalus), acknowledged as a pivotal “One Root of Medicine and Food”, boasts dual applications in both culinary and medicinal domains. The growth and metabolite accumulation of medicinal roots during the harvest period is intricately regulated by a transcriptional regulatory network. One key challenge is to accurately pinpoint the harvest date during the transition from conventional yield content of medicinal materials to high and to identify the core regulators governing such a critical transition. To solve this problem, we performed a correlation analysis of phenotypic, transcriptome, and metabolome dynamics during the harvesting of Astragalus roots.ResultsFirst, our analysis identified stage-specific expression patterns for a significant proportion of the Astragalus root genes and unraveled the chronology of events that happen at the early and later stages of root harvest. Then, the results showed that different root developmental stages can be depicted by co-expressed genes of Astragalus. Moreover, we identified the key components and transcriptional regulation processes that determine root development during harvest. Furthermore, through correlating phenotypes, transcriptomes, and metabolomes at different harvesting periods, period D (Nov.6) was identified as the critical period of yield and flavonoid content increase, which is consistent with morphological and metabolic changes. In particular, we identified a flavonoid biosynthesis metabolite, isoliquiritigenin, as a core regulator of the synthesis of associated secondary metabolites in Astragalus. Further analyses and experiments showed that HMGCR, 4CL, CHS, and SQLE, along with its associated differentially expressed genes, induced conversion of metabolism processes, including the biosynthesis of isoflavones and triterpenoid saponins substances, thus leading to the transition to higher medicinal materials yield and active ingredient content.ConclusionsThe findings of this work will clarify the differences in the biosynthetic mechanism of astragaloside IV and calycosin 7-O-β-D-glucopyranoside accumulation between the four harvesting periods, which will guide the harvesting and production of Astragalus.

Xbp1 promotes odontoblastic differentiation through modulating mitochondrial homeostasis.

Odontoblast differentiation depends on the orderly recruitment of transcriptional factors (TFs) in the transcriptional regulatory network. The depletion of crucial TFs disturbs dynamic alteration of the chromatin landscape and gene expression profile, leading to developmental defects. Our previous studies have revealed that the basic leucine zipper (bZIP) TF family is crucial in odontoblastic differentiation, but the function of bZIP TF family member XBP1 is still unknown. Here, we showed the stage-specific expression patterns of the spliced form Xbp1s during tooth development. Elevated Xbp1 expression and nuclear translocation of XBP1S in mesenchymal stem cells (MSCs) were induced by differentiation medium invitro. Diminution of Xbp1 expression impaired the odontogenic differentiation potential of MSCs. The further integration of ATAC-seq and RNA-seq identified Hspa9 as a direct downstream target, an essential mitochondrial chaperonin gene that modulated mitochondrial homeostasis. The amelioration of mitochondrial dysfunction rescued the impaired odontogenic differentiation potential of MSCs caused by the diminution of Xbp1. Furthermore, the overexpression of Hspa9 rescued Xbp1-deficient defects in odontoblastic differentiation. Our study illustrates the crucial role of Xbp1 in odontoblastic differentiation via modulating mitochondrial homeostasis and brings evidence to the therapy of mitochondrial diseases caused by genetic defects.

Characterization of the DUF868 gene family in Nicotiana and functional analysis of NtDUF868-E5 involved in pigment metabolism

Domains of unknown function (DUF) proteins represent a large group of uncharacterized protein families. The DUF868 gene family in Nicotiana has not yet been described. In the present study, we identified 12, 11, and 25 DUF868 family members in the genome of Nicotiana sylvestris, N. tomentosiformis, and N. tabacum, respectively. Based on phylogenetic analysis, these were categorized into five groups (A-E). Within each group, the gene structures, motifs, and tertiary structures showed high similarity. NtDUF868 family expansion during evolution was mainly driven by segmental duplication events. MicroRNA (miRNA) target site prediction identified 12 miRNA members that target 16 NtDUF868 family genes. The promoters of these genes contain cis-regulatory elements responsive to light, phytohormones, and abiotic stresses. Expression profiling revealed their tissue- and stage-specific expression patterns. RNA-sequencing and quantitative reverse transcription PCR revealed that the NtDUF868 family genes are potentially involved in the response to abiotic and biotic stresses, particularly drought and hormone stresses, and in the resistance to black shank and bacterial wilt. We generated transformed plants using NtDUF868-E5 overexpression and gene-editing vectors. NtDUF868-E5 overexpression resulted in enhanced tobacco plant growth and development, leading to increased leaf photosynthetic capacity and higher chlorophyll and carotenoid contents. This study provided a comprehensive genome-wide analysis of the DUF868 gene family, shedding light on their potential roles in plant growth and stress responses.

Abstract A056: Lymph and liver metastasis in E2F5 conditional knockout mouse model

Abstract Alterations to normal development in mammary gland biology can result in tumor formation. Mammary development and functional processes are controlled by various transcription factors, including the E2Fs. Commonly known as cell cycle regulators, the E2Fs While the activator E2Fs are well studied, the function of the repressor E2Fs in mammary biology is not well known. This is due in part to the early lethality observed in the knockouts of E2F5. To predict a role for E2F5 in mammary developmental biology we queried scRNAseq data and generated a predictive gene signature. This demonstrated that there were functional stage specific expression patterns for E2F5, including virgin development and involution after lacation. Moreover, when we used the predictive signature for activity, we noted that low levels of E2F5 activity corresponded to a poor prognosis in human breast cancer. Based on these preliminary data we hypothesized that E2F5 played an essential role in mammary development and that loss could contribute to tumor biology. To test the premise that E2F5 had a role in mammary development, we generated a mammary epithelial specific knockout of E2F5. At 4 weeks of age, the outgrowth of the epithelium into the fat bad of the mammary gland of these mice significantly lagged behind the littermate controls. This demonstrated a clear role for E2F5 in development of the mammary gland. We also computationally predicted a role in involution and this was also confirmed through in vivo experiments. We noted accelerated involution with the loss of E2F5 following removal of pups. The second major hypothesis that we tested was that E2F5 had a role in tumor biology, and we planned to test this by interbreeding the E2F5 conditional knockout mice to various other strains. As we aged the E2F5 conditional knockout mice, we were surprised to note that mammary tumors formed after a long latency with the loss of E2F5 alone. These tumors were highly heterogeneous at both a histological and gene expression level, with samples clustering with each of the major human PAM50 subtypes of breast cancer. These tumors were also highly metastatic with lesions developing in the lungs, as many mouse models do. However, unlike most mouse models of breast cancer the E2F5 conditional knockout tumors also metastasized to the liver and lymph nodes. Given the extended latency of the model system, we transplanted the E2F5 conditional knockout tumors into syngeneic recipient mice. We repeatedly observed lymphatic metastases or liver metastases, dependent on the primary tumor. To enrich these tissue trophisms, we repeatedly implanted lymph or liver metastases into the mammary fat pad, resulting in significant enrichment. Preliminary RNAseq and Cut and Run have been completed to examine E2F5 specific gene expression alterations that lead to trophism and metastasis enrichment, with targets that are being currently validated. Since most genetically engineered mouse models have not been characterized to have lymph or liver metastasis, this work represents an important new model. Citation Format: Briana To, Jesus Garcia-Lerena, John Vusich, Carson Broeker, Jonathan Rennhack, Daniel Hollern, Eran Andrechek. Lymph and liver metastasis in E2F5 conditional knockout mouse model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr A056.

Resveratrol facilitates bone formation in high-glucose conditions.

Periodontitis is known to be affected by high-glucose conditions, which poses a challenge to periodontal tissue regeneration, particularly in bone formation. In this study, the potential effects of resveratrol (3,5,4'-trihydroxystilbene, RSV) in facilitating bone formation under high-glucose conditions after periodontitis has been investigated. We focused on the analysis of osteoblasts and periodontal ligament cells, which are essential for bone formation including cell proliferation and differentiation. And we aimed to investigate the impact of RSV on bone healing, employed diabetic mouse model induced by streptozotocin and confirmed through histological observation. High-glucose conditions adversely affected cell proliferation and ALP activity in both MC3T3-E1 and hPDLF in vitro, with more significant impact on MC3T3-E1 cells. RSV under high-glucose conditions had positive effects on both, showing early-stage effects for MC3T3-E1 cells and later-stage effects for hPDLF cells. RSV seemed to have a more pronounced rescuing role in MC3T3-E1 cells. Increased ALP activity was observed and the expression levels of significant genes, such as Col 1, TGF-β1, ALP, and OC, in osteogenic differentiation were exhibited stage-specific expression patterns. Upregulated Col 1 and TGF-β1 were detected in the early stage, and then ALP and OC expressions became more pronounced in the later stages. Similarly, stronger positive reactions against RUNX2 were detected in the RSV-treated group compared to the control. Furthermore, in in vivo experiment, RSV stimulates the growth and differentiation of osteoblasts, thereby promoting bone formation. High-glucose levels have the potential to impair cellular functions and the regenerative capacity to facilitate bone formation with MC3T3-E1 rather than hPDLF cells. Resveratrol appears to facilitate the inherent abilities of MC3T3-E1 cells compared with hPDLF cells, indicating its potential capacity to restore functionality during periodontal regeneration.

Transcriptomic profiles reveal the characteristics of oocytes and cumulus cells at GV, MI, and MII in follicles before ovulation

BackgroundThe oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte relies on communication between the oocyte and the surrounding CCs. However, oocyte evaluation is primarily based on morphological parameters currently, which offer limited insight into the quality and competence of the oocyte. Here, we conducted transcriptomic profiling of oocytes and their CCs from 47 patients undergoing preimplantation genetic testing for aneuploidy (PGT-A). We aimed to investigate the molecular events occurring between oocytes and CCs at different stages of oocyte maturation (germinal vesicle [GV], metaphase I [MI], and metaphase II [MII]). Our goal is to provide new insights into in vitro oocyte maturation (IVM).ResultsOur findings indicate that oocyte maturation is a complex and dynamic process and that MI oocytes can be further classified into two distinct subtypes: GV-like-MI oocytes and MII-like-MI oocytes. Human oocytes and cumulus cells at three different stages of maturation were analyzed using RNA-seq, which revealed unique transcriptional machinery, stage-specific genes and pathways, and transcription factor networks that displayed developmental stage-specific expression patterns. We have also identified that both lipid and cholesterol metabolism in cumulus cells is active during the late stage of oocyte maturation. Lipids may serve as a more efficient energy source for oocytes and even embryogenesis.ConclusionsOverall, our study provides a relatively comprehensive overview of the transcriptional characteristics and potential interactions between human oocytes and cumulus cells at various stages of maturation before ovulation. This study may offer novel perspectives on IVM and provide a reliable reference data set for understanding the transcriptional regulation of follicular maturation.

The trehalose 6-phosphate phosphatase family in plants.

Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signalling metabolite linking plant growth and development to carbon metabolism. While recent work has focused predominantly on the enzymes that produce Tre6P, little is known about the proteins that catalyse its degradation, the trehalose 6-phosphate phosphatases (TPPs). Often occurring in large protein families, TPPs exhibit cell-, tissue- and developmental stage-specific expression patterns, suggesting important regulatory functions in controlling local levels of Tre6P and trehalose as well as Tre6P signalling. Furthermore, growing evidence through gene expression studies and transgenic approaches shows that TPPs play an important role in integrating environmental signals with plant metabolism. This review highlights the large diversity of TPP isoforms in model and crop plants and identifies how modulating Tre6P metabolism in certain cell types, tissues, and at different developmental stages may promote stress tolerance, resilience and increased crop yield.

Comprehensive characterization and expression analysis of enzymatic antioxidant gene families in passion fruit (Passiflora edulis)

Passion fruit, a valuable tropical fruit, faces climate-related growth challenges. Antioxidant enzymes are vital for both stress protection and growth regulation in plants. We first provided systemic analysis of enzymatic antioxidant gene families in passion fruit, identifying 90 members including 11 PeSODs, 45 PeAPXs, 8 PeCATs, 7 PeGPXs, 6 PeMDHARs, 8 PeDHARs, and 5 PeGRs. Gene members in each gene family with same subcellular localization showed closer phylogenetic relationship. Many antioxidant genes exhibited tissue- or developmental stage-specific expression patterns during floral and fruit development, with some widely expressed. Their co-expressed genes were linked to photosynthesis and energy metabolism, suggesting roles in protecting highly proliferating tissues from oxidative damage. Potential genes for enhancing temperature stress resistance were identified. The involvement of diverse regulatory factors including miRNAs, transcription factors, and CREs might contribute to the complex roles of antioxidant genes. This study informs future research on antioxidant genes and passion fruit breeding.

Phylogenomic Analysis of Cytochrome P450 Gene Superfamily and Their Association with Flavonoids Biosynthesis in Peanut (Arachis hypogaea L.).

Cytochrome P450s (CYPs) constitute extensive enzyme superfamilies in the plants, playing pivotal roles in a multitude of biosynthetic and detoxification pathways essential for growth and development, such as the flavonoid biosynthesis pathway. However, CYPs have not yet been systematically studied in the cultivated peanuts (Arachis hypogaea L.), a globally significant cash crop. This study addresses this knowledge deficit through a comprehensive genome-wide analysis, leading to the identification of 589 AhCYP genes in peanuts. Through phylogenetic analysis, all AhCYPs were systematically classified into 9 clans, 43 gene families. The variability in the number of gene family members suggests specialization in biological functions. Intriguingly, both tandem duplication and fragment duplication events have emerged as pivotal drivers in the evolutionary expansion of the AhCYP superfamily. Ka/Ks analysis underscored the substantial influence of strong purifying selection on the evolution of AhCYPs. Furthermore, we selected 21 genes encoding 8 enzymes associated with the flavonoid pathway. The results of quantitative real-time PCR (qRT-PCR) experiments unveiled stage-specific expression patterns during the development of peanut testa, with discernible variations between pink and red testa. Importantly, we identified a direct correlation between gene expression levels and the accumulation of metabolites. These findings offer valuable insights into elucidating the comprehensive functions of AhCYPs and the underlying mechanisms governing the divergent accumulation of flavonoids in testa of different colors.

Genome-wide identification, molecular structures and functional exploration of the membrane attack complex/perforin domain-containing proteins and validation of GmmiRNA169o-GmMACPF-9 module in soybean cold stress

Soybean (Glycine max L.) is a widely cultivated legume and an enriched source of edible oil and protein. Membrane Attack Complex/Perforin Domain-Containing Proteins (MACPFs) played significant roles in plant growth and biotic and abiotic stresses. Here, we performed a genome-wide scanning for the putative GmMACPFs and a total of 20 GmMACPFs were identified in soybean. The conserved domain and phylogenetic analysis of MACPF showed the evolutionary diversification of MACPF genes family among legumes. The GmMACPFs protein characteristics were predicted, including theoretical molecular weight (38.06 to 68.38 kDa), protein isoelectric point (6.61 to 9.41), subcellular localization (nucleus, cytoplasm or mitochondria) and various TFs binding sites. GmMACPFs are located on 12 chromosomes out of a total 20 chromosomes in soybean. The 2 kb promoter analysis revealed that GmMACPFs contains a large number of phytohormones and stress responsive cis-acting elements (CAE) and transcription factor binding sites. We explored the 3D protein structure of GmMACPFs, their posttranscriptional regulators especially abiotic stress responsive miRNAs, and the evolutionary relationship among different legumes and Arabidopsis. GmMACPFs expression pattern was retrieved from RNA-seq libraries for tissue specific and under various abiotic stresses in soybean. According to the results, GmMACPF genes exhibited spatiotemporal, tissue, development stage and abiotic stress specific expression patterns in soybean. Our RT-qPCR analysis indicates the antagonistic expression pattern between GmmiRNA-GmMACPF modules under cold, salt, drought and heat stresses. Transient over expression of GmmiRNA169o-GmMACPF-9 module in soybean hairy root and biochemical analysis showed that GmmiRNA169o-OE plants accumulated less reactive oxygen species (ROS), malondialdehyde (MDA) and accumulated higher superoxide dismutase (SOD) and peroxidase (POD) to enhance cold stress tolerance. These results enriched our knowledge of the functions of GmmiRNA169o and GmMACPF-9 and provided candidate miRNAs-GmMACPFs modules especially GmmiRNA169o for environmental stress resilient soybean breeding through genetic engineering.

Genome-wide identification of GH9 gene family and the assessment of its role during fruit abscission zone formation in Vaccinium ashei.

The genomic location and stage-specific expression pattern of GH9 genes reveal their critical roles during fruit abscission zone formation in Vaccinium ashei. Glycosyl hydrolase family 9 (GH9) cellulases play a crucial role in both cellulose synthesis and hydrolysis during plant growth and development. Despite this importance, there is currently no study on the involvement of GH9-encoding genes, specifically VaGH9s, in abscission zone formation of rabbiteye blueberries (Vaccinium ashei). In this study, we identified a total of 61 VaGH9s in the genome, which can be classified into 3 subclasses based on conserved motifs and domains, gene structures, and phylogenetic analyses. Our synteny analysis revealed that VaGH9s are more closely related to the GH9s of Populus L. than to those of Arabidopsis, Vitis vinifera, and Citrus sinensis. In silico structural analysis predicted that most of VaGH9s are hydrophilic, and localized in cell membrane and/or cell wall, and the variable sets of cis-acting regulatory elements and functional diversity with four categories of stress response, hormone regulation, growth and development, and transcription factor-related elements are present in the promoter sequence of VaGH9s genes. Transcriptomic analysis showed that there were 22 differentially expressed VaGH9s in fruit abscission zone tissue at the veraison stage, and the expression of VaGH9B2 and VaGH9C10 was continuously increased during fruit maturation, which were in parallel with the increasing levels of cellulase activity and oxidative stress indicators, suggesting that they are involved in the separation stage of fruit abscission in Vaccinium ashei. Our work identified 22 VaGH9s potentially involved in different stages of fruit abscission and would aid further investigation into the molecular regulation of abscission in rabbiteye blueberries fruit.

P-221 Single-cell transcriptomic profiles reveal the characteristics of oocytes and CCs at GV, MI, and MII in oocytes before ovulation

Abstract Study question what is the mechanism of the interaction between oocytes and CCs supporting oocyte development during oocyte maturation? Summary answer Cumulus cells are responsible for the accumulation of lipids and fatty acids to ensure oocytes have enough energy available for later embryogenesis. What is known already The oocyte and its surrounding cumulus cells (CCs) exist as an inseparable entity. The maturation of the oocyte is reliant on the materials and energy provided by CCs Although several reports have addressed the transcriptome of oocytes and granulosa cells during human folliculogenesis, many questions remain unanswered regarding the interaction between oocytes and CCs during the final stages of folliculogenesis before ovulation. Therefore, the mechanism of the interaction between oocytes and CCs supporting oocyte development during oocyte maturation is needed to be studied. Study design, size, duration We collected a total of 139 samples from 47 preimplantation genetic testing (PGT) patients for RNA sequencing , including 97 CCs (GV: 35; MI: 30; MII: 32) and 42 oocytes (GV: 22; MI: 11; MII: 9).10 cells were randomly selected from individually cumulus-oocyte complex per CC sample to avoid bias due to the different number of CCs in each follicle. The samples were collected within a time frame of 3 months. Participants/materials, setting, methods The oocytes removed from CGCs were quickly transferred to the operating dish for marking, the maturation stage was assessed and recorded by observing the nucleus of oocytes, the CCs were mechanically isolated. Isolated RNA from oocytes and CCs underwent library preparation using an oligo deoxy-thymidine(dT) priming approach followed by deep sequencing. Data processing and bioinformatics analysis were performed using software, mainly including FASTP, STAR, String Tie, ARCANE and DESeq2 along with functional annotation analysis. Main results and the role of chance We found that oocyte maturation is a dynamic process and MI oocytes can be subdivided into GV-like-MI oocytes and MII-like-MI oocytes. we revealed unique transcriptional machinery, transcription factor networks, and crosstalk, displaying developmental stage-specific expression patterns at the three stages of oocyte maturation. We also identified that both lipid and cholesterol metabolism in cumulus cells are active during the late stage of oocyte maturation, and lipids may serve as a more efficient energy source for oocytes and even embryogenesis. Moreover, we also found that the metabolic profile of MII CCs, particularly those associated with the synthesis pathway of heparan sulfate synthesis, predicts the quality of DAY 3 blastomeres（P = 0.0001）. Limitations, reasons for caution The transcriptional profile of mural granulosa cells during the final stages of follicle maturation has not been fully explored. Wider implications of the findings This study provides a relatively systematic and comprehensive overview of the transcriptional features and interactions between oocytes and CCs at different stages of maturation before ovulation and may provide insights into developing a non-invasive method for the clinical assessment of oocyte quality and prediction of DAY3 blastomere quality in ART. Trial registration number not applicable

Functional characterization of Cinnamate 4-hydroxylase gene family in soybean (Glycine max).

Cinnamate 4-hydroxylase (C4H) is the first key cytochrome P450 monooxygenase (P450) enzyme in the phenylpropanoid pathway. It belongs to the CYP73 family of P450 superfamily, and catalyzes the conversion of trans-cinnamic acid to p-coumaric acid. Since p-coumaric acid serves as the precursor for the synthesis of a wide variety of metabolites involved in plant development and stress resistance, alteration in the expression of soybean C4H genes is expected to affect the downstream metabolite levels, and its ability to respond to stress. In this study, we identified four C4H genes in the soybean genome that are distributed into both class I and class II CYP73 family. GmC4H2, GmC4H14 and GmC4H20 displayed tissue- and developmental stage-specific gene expression patterns with their transcript accumulation at the highest level in root tissues. GmC4H10 appears to be a pseudogene as its transcript was not detected in any soybean tissues. Furthermore, protein homology modelling revealed substrate docking only for GmC4H2, GmC4H14 and GmC4H20. To demonstrate the function of GmC4Hs, we modified a cloning vector for the heterologous expression of P450s in yeast, and used it for microsomal protein production and enzyme assay. Our results confirmed that GmC4H2, GmC4H14 and GmC4H20 contain the ability to hydroxylate trans-cinnamic acid with varying efficiencies.

Genome-Wide Analysis of the BBX Genes in Platanus × acerifolia and Their Relationship with Flowering and/or Dormancy.

The B-BOX (BBX) gene family is widely distributed in animals and plants and is involved in the regulation of their growth and development. In plants, BBX genes play important roles in hormone signaling, biotic and abiotic stress, light-regulated photomorphogenesis, flowering, shade response, and pigment accumulation. However, there has been no systematic analysis of the BBX family in Platanus × acerifolia. In this study, we identified 39 BBX genes from the P. × acerifolia genome, and used TBtools, MEGA, MEME, NCBI CCD, PLANTCARE and other tools for gene collinearity analysis, phylogenetic analysis, gene structure, conserved domain analysis, and promoter cis-element analysis, and used the qRT-PCR and transcriptome data for analyzing expression pattern of the PaBBX genes. Collinearity analysis indicated segmental duplication was the main driver of the BBX family in P. × acerifolia, and phylogenetic analysis showed that the PaBBX family was divided into five subfamilies: I, II, III, IV and V. Gene structure analysis showed that some PaBBX genes contained super-long introns that may regulate their own expression. Moreover, the promoter of PaBBX genes contained a significant number of cis-acting elements that are associated with plant growth and development, as well as hormone and stress responses. The qRT-PCR results and transcriptome data indicated that certain PaBBX genes exhibited tissue-specific and stage-specific expression patterns, suggesting that these genes may have distinct regulatory roles in P. × acerifolia growth and development. In addition, some PaBBX genes were regularly expressed during the annual growth of P. × acerifolia, corresponding to different stages of flower transition, dormancy, and bud break, indicating that these genes may be involved in the regulation of flowering and/or dormancy of P. × acerifolia. This article provided new ideas for the study of dormancy regulation and annual growth patterns in perennial deciduous plants.

Identification and Characterization of Development-Related microRNAs in the Red Flour Beetle, Tribolium castaneum

MicroRNAs (miRNAs) play important roles in insect growth and development, but they were poorly studied in insects. In this study, a total of 883 miRNAs were detected from the early embryo (EE), late larva (LL), early pupa (EP), late pupa (LP), and early adult (EA) of Tribolium castaneum by microarray assay. Further analysis identified 179 differentially expressed unique miRNAs (DEmiRNAs) during these developmental stages. Of the DEmiRNAs, 102 DEmiRNAs exhibited stage-specific expression patterns during development, including 53 specifically highly expressed miRNAs and 20 lowly expressed miRNAs in EE, 19 highly expressed miRNAs in LL, 5 weakly expressed miRNAs in EP, and 5 abundantly expressed miRNAs in EA. These miRNAs were predicted to target 747, 265, 472, 234, and 121 genes, respectively. GO enrichment analysis indicates that the targets were enriched by protein phosphorylation, calcium ion binding, sequence-specific DNA binding transcription factor activity, and cytoplasm. An RNA interference-mediated knockdown of the DEmiRNAs tca-miR-6-3p, tca-miR-9a-3p, tca-miR-9d-3p, tca-miR-11-3p, and tca-miR-13a-3p led to defects in metamorphosis and wing development of T. castaneum. This study has completed the identification and characterization of development-related miRNAs in T. castaneum, and will enable us to investigate their roles in the growth and development of insect.

Improved Annotation of the Peach (Prunus persica) Genome and Identification of Tissue- or Development Stage-Specific Alternative Splicing through the Integration of Iso-Seq and RNA-Seq Data

Alternative splicing (AS) is an important way to generate notable regulatory and proteomic complexity in eukaryotes. However, accurate full-length splicing isoform discovery by second-generation sequencing (SGS) technologies is beset with the precise assembly of multiple isoforms from the same gene loci. In recent years, third-generation sequencing (TGS) technologies have been adopted to gain insight into different aspects of transcriptome complexity, such as complete sequences of mRNA, alternative splicing, fusion transcript, and alternative polyadenylation (APA). Here, we combined PacBio Iso-Seq and Illumina RNA-Seq technologies to decipher the full-length transcriptome of peach. In total, 40,477 nonredundant high-quality consensus transcript sequences were obtained from equally pooled libraries from 10 samples of 6 organs, including leaf, shoot, flower, fruit peel, fruit mesocarp, and fruit stone, of which 18,274 isoforms were novel isoforms of known genes and 546 isoforms were novel gene transcripts. We also discovered 148 fusion transcripts, 15,434 AS events, 508 potential lncRNAs, and 4368 genes with APA events. Of these AS events, the most abundant (62.48%) AS type was intron retention (IR). Moreover, the expression levels of different isoforms identified in this study were quantitatively evaluated, and highly tissue- or development stage-specific expression patterns were observed. The novel transcript isoforms and new characteristics of the peach transcriptome revealed by this study will facilitate the annotation of the peach genome and lay the foundations for functional research in the future.

Genome-wide analysis reveals the crucial role of lncRNAs in regulating the expression of genes controlling pollen development.

The genomic location and stage-specific expression pattern of many long non-coding RNAs reveal their critical role in regulating protein-coding genes crucial in pollen developmental progression and male germ line specification. Long non-coding RNAs (lncRNAs) are transcripts longer than 200bp with no apparent protein-coding potential. Multiple investigations have revealed high expression of lncRNAs in plant reproductive organs in a cell and tissue-specific manner. However, their potential role as essential regulators of molecular processes involved in sexual reproduction remains largely unexplored. We have used developing field mustard (Brassica rapa) pollen as a model system for investigating the potential role of lncRNAs in reproductive development. Reference-based transcriptome assembly performed to update the existing genome annotation identified novel expressed protein-coding genes and long non-coding RNAs (lncRNAs), including 4347 long intergenic non-coding RNAs (lincRNAs, 1058 expressed) and 2,045 lncRNAs overlapping protein-coding genes on the opposite strand (lncNATs, 780 expressed). The analysis of expression profiles reveals that lncRNAs are significant and stage-specific contributors to the gene expression profile of developing pollen. Gene co-expression networks accompanied by genome location analysis identified 38 cis-acting lincRNA, 31 cis-acting lncNAT, 7 trans-acting lincRNA and 14 trans-acting lncNAT to be substantially co-expressed with target protein-coding genes involved in biological processes regulating pollen development and male lineage specification. These findings provide a foundation for future research aiming at developing strategies to employ lncRNAs as regulatory tools for gene expression control during reproductive development.

Postnatal ontogeny of Neuromedin S and its receptors NMUR1 and NMUR2 expression in mouse testis

Neuromedin S (NMS) is a well-known anorexigenic neuropeptide. Despite some reports of the presence of its transcript and precursor protein in testis, the expression and localization of NMS and its receptors during the postnatal development of mammalian testis remains elusive. We investigated the expression patterns and testicular localization of NMS and its receptors NMUR1 and NMUR2, during 5, 10, 20, 30, and 90 days of postnatal development, using real time PCR, immunoblot analysis and immunohistochemistry in mice. NMS and its receptors are present at all age groups at transcript level in mouse testis. At the protein level, NMS and NMUR2 are present in all age groups, whereas NMUR1 is present primarily in 30- and 90-day testis. Immunolocalization study showed that NMS and NMUR2 are expressed in spermatogonia, spermatocytes, Sertoli cells, and Leydig cells, in contrast to NMUR1 which is expressed exclusively in the Leydig cells of 30- and 90-day testis. The results also confirm the intranuclear localization of NMS in spermatogonia and spermatocytes. Although NMS-NMUR2 is expressed in Sertoli cells at all stages of the spermatogenic cycle, they showed a stage-specific expression pattern in spermatogonia and primary spermatocytes. In conclusion, NMS and its receptors NMUR1 and NMUR2 are expressed in the testis and may regulate spermatogenesis, possibly by modulating steroidogenesis and Sertoli cell function in the testis.

Developmental Transcriptome Analysis of Red-Spotted Apollo Butterfly, Parnassius bremeri.

Parnassius bremeri (P. bremeri), a member of the genus Snow Apollo in the swallowtail family (Papilionidae), is a high alpine butterfly that lives in Russia, Korea, and China. It is an endangered wildlife (Class I) in South Korea and is a globally endangered species. The lack of transcriptomic and genomic resources of P. bremeri significantly hinders the study of its population genetics and conservation. The detailed information of the developmental stage-specific gene expression patterns of P. bremeri is of great demand for its conservation. However, the molecular mechanism underlying the metamorphic development of P. bremeri is still unknown. In the present study, the differentially expressed genes (DEGs) across the metamorphic developmental stages were compared using high-throughput transcriptome sequencing. We identified a total of 72,161 DEGs from eight comparisons. GO enrichment analysis showed that a range of DEGs were responsible for cuticle development and the melanin biosynthetic pathway during larval development. Pathway analysis suggested that the signaling pathways, such as the Wnt signaling pathway, hedgehog signaling pathway and Notch signaling pathway, are regulated during the developmental stages of P. bremeri. Furthermore, sensory receptors were also activated, especially during the larval to adult transition stage. Collectively, the results of this study provide a preliminary foundation and understanding of the molecular mechanism in their transcriptomes for further research on the metamorphic development of P. bremeri.

Emerging roles of circular RNAs in tuberculosis.

Tuberculosis (TB) remains a major global health issue, resulting in around 1.5 million people deaths each year. Better diagnostic and therapeutic tools are urgently needed. Circular RNAs (circRNAs) are a new class of noncoding RNAs with a covalently closed structure, and exhibit a tissue-, cell-, and developmental stage-specific expression pattern. Recently, circRNAs were thought to be regulatory molecules implicated in the onset and progression of a series of human diseases including tuberculosis. In tuberculosis, circRNAs have been shown to regulate host anti-TB immune responses, such as decreasing monocyte apoptosis, enhancing autophagy and promoting macrophage polarization. Importantly, circRNAs are physically stable and abundant in several types of body fluids. Therefore they are considered as promising minimally-invasive biomarkers. In this review, we focus on the recent advances in the immune regulatory roles of circRNAs, as well as their potential diagnostic value in TB.