Dynamic transcriptome landscape of Asian domestic honeybee (Apis cerana) embryonic development revealed by high-quality RNA sequencing.
ABSTRACT: BACKGROUND:Honeybee development consists of four stages: embryo, larva, pupa and adult. Embryogenesis, a key process of cell division and differentiation, takes 3 days in honeybees. However, the embryonic transcriptome and the dynamic regulation of embryonic transcription are still largely uncharacterized in honeybees, especially in the Asian honeybee (Apis cerana). Here, we employed high-quality RNA-seq to explore the transcriptome of Asian honeybee embryos at three ages, approximately 24, 48 and 72 h (referred to as Day1, Day2 and Day3, respectively). RESULTS:Nine embryo samples, three from each age, were collected for RNA-seq. According to the staging scheme of honeybee embryos and the morphological features we observed, our Day1, Day2 and Day3 embryos likely corresponded to the late stage four, stage eight and stage ten development stages, respectively. Hierarchical clustering and principal component analysis showed that same-age samples were grouped together, and the Day2 samples had a closer relationship with the Day3 samples than the Day1 samples. Finally, a total of 18,284 genes harboring 55,646 transcripts were detected in the A. cerana embryos, of which 44.5% consisted of the core transcriptome shared by all three ages of embryos. A total of 4088 upregulated and 3046 downregulated genes were identified among the three embryo ages, of which 2010, 3177 and 1528 genes were upregulated and 2088, 2294 and 303 genes were downregulated from Day1 to Day2, from Day1 to Day3 and from Day2 to Day3, respectively. The downregulated genes were mostly involved in cellular, biosynthetic and metabolic processes, gene expression and protein localization, and macromolecule modification; the upregulated genes mainly participated in cell development and differentiation, tissue, organ and system development, and morphogenesis. Interestingly, several biological processes related to the response to and detection of light stimuli were enriched in the first-day A. cerana embryogenesis but not in the Apis mellifera embryogenesis, which was valuable for further investigations. CONCLUSIONS:Our transcriptomic data substantially expand the number of known transcribed elements in the A. cerana genome and provide a high-quality view of the transcriptome dynamics of A. cerana embryonic development.
Project description:Next-generation proteomics of Vero E6 cells infected by Italy-INMI1 SARS-CoV-2 virus for defining the kinetics of whole viral particle antigen production for vaccines. Cells from Day1, Day2, Day3, Day4, Day7 post-infection at two multiplicities of infection.
Project description:Since exposure therapy for anxiety disorders incorporates extinction of contextual anxiety, relapses may be due to reinstatement processes. Animal research demonstrated more stable extinction memory and less anxiety relapse due to vagus nerve stimulation (VNS). We report a valid human three-day context conditioning, extinction and return of anxiety protocol, which we used to examine effects of transcutaneous VNS (tVNS). Seventy-five healthy participants received electric stimuli (unconditioned stimuli, US) during acquisition (Day1) when guided through one virtual office (anxiety context, CTX+) but never in another (safety context, CTX-). During extinction (Day2), participants received tVNS, sham, or no stimulation and revisited both contexts without US delivery. On Day3, participants received three USs for reinstatement followed by a test phase. Successful acquisition, i.e. startle potentiation, lower valence, higher arousal, anxiety and contingency ratings in CTX+ versus CTX-, the disappearance of these effects during extinction, and successful reinstatement indicate validity of this paradigm. Interestingly, we found generalized reinstatement in startle responses and differential reinstatement in valence ratings. Altogether, our protocol serves as valid conditioning paradigm. Reinstatement effects indicate different anxiety networks underlying physiological versus verbal responses. However, tVNS did neither affect extinction nor reinstatement, which asks for validation and improvement of the stimulation protocol.
Project description:BACKGROUND: Transcriptome analysis during embryogenesis usually requires pooling of embryos to obtain sufficient RNA. Hence, the measured levels of gene-expression represent the average mRNA levels of pooled samples and the biological variation among individuals is confounded. This can irreversibly reduce the robustness, resolution, or expressiveness of the experiment. Therefore, we developed a robust method to isolate abundant high-quality RNA from individual embryos to perform single embryo transcriptome analyses using zebrafish as a model organism. Available methods for embryonic zebrafish RNA isolation minimally utilize ten embryos. Further downscaling of these methods to one embryo is practically not feasible. FINDINGS: We developed a single embryo RNA extraction method based on sample homogenization in liquid nitrogen, RNA extraction with phenol and column purification. Evaluation of this method showed that: the quality of the RNA was very good with an average RIN value of 8.3-8.9; the yield was always >/= 200 ng RNA per embryo; the method was applicable to all stages of zebrafish embryogenesis; the success rate was almost 100%; and the extracted RNA performed excellent in microarray experiments in that the technical variation was much lower than the biological variation. CONCLUSIONS: Presented is a high-quality, robust RNA isolation method. Obtaining sufficient RNA from single embryos eliminates the necessity of sample pooling and its associated drawbacks. Although our RNA isolation method has been setup for transcriptome analysis in zebrafish, it can also be used for other model systems and other applications like (q)PCR and transcriptome sequencing.
Project description:Somatic embryogenesis is a notable illustration of cell totipotency, by which somatic cells undergo dedifferentiation and then differentiate into somatic embryos. Our previous work demonstrated that pretreatment of immature zygotic embryos with 0.5 M sucrose solution for 72 h efficiently induced somatic embryo initiation in camphor tree. To better understand the molecular basis of somatic embryogenesis induced by osmotic stress, de novo transcriptome sequencing of three tissues of camphor tree (immature zygotic embryos, sucrose-pretreated immature zygotic embryos, and somatic embryos induced from sucrose-pretreated zygotic embryos) were conducted using Illumina Hiseq 2000 platform.A total of 30.70 G high quality clean reads were obtained from cDNA libraries of the three samples. The overall de novo assembly of cDNA sequence data generated 205592 transcripts, with an average length of 998 bp. 114229 unigenes (55.56 % of all transcripts) with an average length of 680 bp were annotated with gene descriptions, gene ontology terms or metabolic pathways based on Blastx search against Nr, Nt, Swissprot, GO, COG/KOG, and KEGG databases. CEGMA software identified 237 out of 248 ultra-conserved core proteins as 'complete' in the transcriptome assembly, showing a completeness of 95.6 %. A total of 897 genes previously annotated to be potentially involved in somatic embryogenesis were identified. Comparative transcriptome analysis showed that a total of 3335 genes were differentially expressed in the three samples. The differentially expressed genes were divided into six groups based on K-means clustering. Expression level analysis of 52 somatic embryogenesis-related genes indicated a high correlation between RNA-seq and qRT-PCR data. Gene enrichment analysis showed significantly differential expression of genes responding to stress and stimulus.The present work reported a de novo transcriptome assembly and global analysis focused on gene expression changes during initiation and formation of somatic embryos in camphor tree. Differential expression of somatic embryogenesis-related genes indicates that sucrose induced somatic embryogenesis may share or partly share the mechanisms of somatic embryogenesis induced by plant hormones. This study provides comprehensive transcript information and gene expression data for camphor tree. It could also serve as an important platform resource for further functional studies in plant embryogenesis.
Project description:Learning new information is crucial in daily activities and occurs continuously during a subject's lifetime. Retention of learned material is required for later recall and reuse, although learning capacity is limited and interference between consecutively learned information may occur. Learning processes are impaired in Parkinson's disease (PD); however, little is known about the processes related to retention and interference. The aim of this study is to investigate the retention and anterograde interference using a declarative sequence learning task in drug-naive patients in the disease's early stages. Eleven patients with PD and eleven age-matched controls learned a visuomotor sequence, SEQ1, during Day1; the following day, retention of SEQ1 was assessed and, immediately after, a new sequence of comparable complexity, SEQ2, was learned. The comparison of the learning rates of SEQ1 on Day1 and SEQ2 on Day2 assessed the anterograde interference of SEQ1 on SEQ2. We found that SEQ1 performance improved in both patients and controls on Day2. Surprisingly, controls learned SEQ2 better than SEQ1, suggesting the absence of anterograde interference and the occurrence of learning optimization, a process that we defined as "learning how to learn." Patients with PD lacked such improvement, suggesting defective performance optimization processes.
Project description:Varroa destructor (Vd) is a honeybee ectoparasite. Its original host is the Asian honeybee, Apis cerana, but it has also become a severe, global threat to the European honeybee, Apis mellifera. Previous studies have shown that Varroa can mimic a host's cuticular hydrocarbons (HC), enabling the parasite to escape the hygienic behaviour of the host honeybees. By transferring mites between the two honeybee species, we further demonstrate that Vd is able to mimic the cuticular HC of a novel host species when artificially transferred to this new host. Mites originally from A. cerana are more efficient than mites from A. mellifera in mimicking HC of both A. cerana and A. mellifera. This remarkable adaptability may explain their relatively recent host-shift from A. cerana to A. mellifera.
Project description:MicroRNAs (miRNAs) are key regulators of developmental processes, such as cell fate determination and differentiation. Previous studies showed Dicer knockdown in honeybee embryos disrupt the processing of functional mature miRNAs and impairs embryo patterning. Here we investigated the expression profiles of miRNAs in honeybee embryogenesis and the role of the highly conserved miR-34-5p in the regulation of genes involved in insect segmentation. A total of 221 miRNAs were expressed in honey bee embryogenesis among which 97 mature miRNA sequences have not been observed before. Interestingly, we observed a switch in dominance between the 5-prime and 3-prime arm of some miRNAs in different embryonic stages; however, most miRNAs present one dominant arm across all stages of embryogenesis. Our genome-wide analysis of putative miRNA-target networks and functional pathways indicates miR-34-5p is one of the most conserved and connected miRNAs associated with the regulation of genes involved in embryonic patterning and development. In addition, we experimentally validated that miR-34-5p directly interacts to regulatory elements in the 3'-untranslated regions of pair-rule (even-skipped, hairy, fushi-tarazu transcription factor 1) and cytoskeleton (actin5C) genes. Our study suggests that miR-34-5p may regulate the expression of pair-rule and cytoskeleton genes during early development and control insect segmentation.
Project description:No standard chemotherapy is used as neoadjuvant therapy in triple negative breast cancer (TNBC). This study has compared carboplatin plus paclitaxel with commonly used epirubicin plus paclitaxel as neoadjuvant chemotherapy (NAC) in TNBC.91 patients with a median age of 47 years (PC 47 patients, EP 44 patients) were enrolled. 65% of the patients were premenopausal. While the objective response rate was similar in the PC and EP arm (89.4% vs. 79.5%, P = 0.195), the pCR rate in the PC arm was significantly higher (38.6% vs. 14.0%, P = 0.014). The median follow-up time was 55.0 months. 5-year RFS were 77.6% and 56.2%, significantly higher in the PC arm, P = 0.043. No significant difference in OS was observed between the two arms (P = 0.350). Adverse events were similar, except for more thrombocytopenia in the PC arm (P = 0.001).Patients with stage II/III TNBC were randomized to receive either paclitaxel (175 mg/m2, day1) plus carboplatin (Area Under the Curve = 5, day2) (PC) or epirubicin (75mg/m2, day1) plus paclitaxel (175 mg/m2, day2) (EP) as NAC every three weeks for 4-6 cycles. The primary endpoint was rate of pathologic complete response (pCR).The secondary endpoints included relapse-free survival (RFS), overall survival (OS) and safety.This study suggested that the addition of carboplatin to paclitaxel was superior to the regimen of epirubicin plus paclitaxel as NAC for TNBC in terms of improving pCR rate and RFS. Further phase 3 study has already started.
Project description:Rice (Oryza sativa) is an excellent model monocot with a known genome sequence for studying embryogenesis. Here we report the transcriptome profiling analysis of rice developing embryos using RNA-Seq as an attempt to gain insight into the molecular and cellular events associated with rice embryogenesis. RNA-Seq analysis generated 17,755,890 sequence reads aligned with 27,190 genes, which provided abundant data for the analysis of rice embryogenesis. A total of 23,971, 23,732, and 23,592 genes were identified from embryos at three developmental stages (3-5, 7, and 14 DAP), while an analysis between stages allowed the identification of a subset of stage-specific genes. The number of genes expressed stage-specifically was 1,131, 1,443, and 1,223, respectively. In addition, we investigated transcriptomic changes during rice embryogenesis based on our RNA-Seq data. A total of 1,011 differentially expressed genes (DEGs) (log(2)Ratio ? 1, FDR ? 0.001) were identified; thus, the transcriptome of the developing rice embryos changed considerably. A total of 672 genes with significant changes in expression were detected between 3-5 and 7 DAP; 504 DEGs were identified between 7 and 14 DAP. A large number of genes related to metabolism, transcriptional regulation, nucleic acid replication/processing, and signal transduction were expressed predominantly in the early and middle stages of embryogenesis. Protein biosynthesis-related genes accumulated predominantly in embryos at the middle stage. Genes for starch/sucrose metabolism and protein modification were highly expressed in the middle and late stages of embryogenesis. In addition, we found that many transcription factor families may play important roles at different developmental stages, not only in embryo initiation but also in other developmental processes. These results will expand our understanding of the complex molecular and cellular events in rice embryogenesis and provide a foundation for future studies on embryo development in rice and other cereal crops.
Project description:The Asian honeybee Apis cerana is one of two bee species that have been commercially kept with immense economic value. Here we present the analysis of genomic sequence and transcriptomic exploration for A. cerana as well as the comparative genomic analysis of the Asian honeybee and the European honeybee A. mellifera. The genome and RNA-seq data yield new insights into the behavioral and physiological resistance to the parasitic mite Varroa the evolution of antimicrobial peptides, and the genetic basis for labor division in A. cerana. Comparison of genes between the two sister species revealed genes specific to A. cerana, 54.5% of which have no homology to any known proteins. The observation that A. cerana displayed significantly more vigilant grooming behaviors to the presence of Varroa than A. mellifera in conjunction with gene expression analysis suggests that parasite-defensive grooming in A. cerana is likely triggered not only by exogenous stimuli through visual and olfactory detection of the parasite, but also by genetically endogenous processes that periodically activates a bout of grooming to remove the ectoparasite. This information provides a valuable platform to facilitate the traits unique to A. cerana as well as those shared with other social bees for health improvement.