Chicken blastoderms and primordial germ cells possess a higher expression of DNA repair genes and lower expression of apoptosis genes to preserve their genome stability

DNA is susceptible to damage by various sources. When the DNA is damaged, the cell repairs the damage through an appropriate DNA repair pathway. When the cell fails to repair DNA damage, apoptosis is initiated. Although several genes are involved in five major DNA repair pathways and two major apoptosis pathways, a comprehensive understanding of those gene expression is not well-understood in chicken tissues. We performed whole-transcriptome sequencing (WTS) analysis in the chicken embryonic fibroblasts (CEFs), stage X blastoderms, and primordial germ cells (PGCs) to uncover this deficiency. Stage X blastoderms mostly consist of undifferentiated progenitor (pluripotent) cells that have the potency to differentiate into all cell types. PGCs are also undifferentiated progenitor cells that later differentiate into male and female germ cells. CEFs are differentiated and abundant somatic cells. Through WTS analysis, we identified that the DNA repair pathway genes were expressed more highly in blastoderms and high in PGCs than CEFs. Besides, the apoptosis pathway genes were expressed low in blastoderms and PGCs than CEFs. We have also examined the WTS-based expression profiling of candidate pluripotency regulating genes due to the conserved properties of blastoderms and PGCs. In the results, a limited number of pluripotency genes, especially the core transcriptional network, were detected higher in both blastoderms and PGCs than CEFs. Next, we treated the CEFs, blastoderm cells, and PGCs with hydrogen peroxide (H2O2) for 1 h to induce DNA damage. Then, the H2O2 treated cells were incubated in fresh media for 3–12 h to observe DNA repair. Subsequent analyses in treated cells found that blastoderm cells and PGCs were more likely to undergo apoptosis along with the loss of pluripotency and less likely to undergo DNA repair, contrasting with CEFs. These properties of blastoderms and PGCs should be necessary to preserve genome stability during the development of early embryos and germ cells, respectively.

Integrated Genomic and Bioinformatics Approaches to Identify Molecular Links between Endocrine Disruptors and Adverse Outcomes

Exposure to Endocrine Disrupting Chemicals (EDC) has been linked with several adverse outcomes. In this review, we examine EDCs that are pervasive in the environment and are of concern in the context of human, animal, and environmental health. We explore the consequences of EDC exposure on aquatic life, terrestrial animals, and humans. We focus on the exploitation of genomics technologies and in particular whole transcriptome sequencing. Genome-wide analyses using RNAseq provides snap shots of cellular, tissue and whole organism transcriptomes under normal physiological and EDC perturbed conditions. A global view of gene expression provides highly valuable information as it uncovers gene families or more specifically, pathways that are affected by EDC exposures, but also reveals those that are unaffected. Hypotheses about genes with unknown functions can also be formed by comparison of their expression levels with genes of known function. Risk assessment strategies leveraging genomic technologies and the development of toxicology databases are explored. Finally, we review how the Adverse Outcome Pathway (AOP) has exploited this high throughput data to provide a framework for toxicology studies.

Integrative Analyses of Whole-Transcriptome Sequencing Reveals CeRNA Regulatory Network of mRNAs, lncRNAs, miRNAs and circRNAs in Pulmonary Hypertension Treated with FGF21

Noncoding RNAs have been shown to play important roles in hypoxic pulmonary hypertension (HPH). Our preliminary data showed that HPH is attenuated by fibroblast growth factor 21 (FGF21) administration. Therefore, we further investigated the whole transcriptome RNA expression patterns and interactions in a mice HPH model treated with FGF21. By whole-transcriptome sequencing, differentially expressed mRNA, miRNA, lncRNA, and circRNA were successfully identified in normoxia (Nx) vs. hypoxia (Hx) and Hx vs. hypoxia + FGF21 (Hx + F21). Through intersection and predictive analysis, differentially co-expressed mRNA, miRNA, lncRNA, and circRNA were selected, followed by functional enrichment analysis. MAPK signaling pathway and epigenetic modification were enriched and may play fundamental roles in the therapeutic effects of FGF21. A ceRNA regulatory network was constructed with miR-7a-5p, miR-449c-5p, miR-676-3p and miR-674-3p as the core. Then the quantitative real time-PCR validation results were consistent with the results of whole-transcriptome sequencing. This study may provide potential biomarkers, pathway and ceRNA regulatory network in HPH treated with FGF21.

Triptolide’s anti-inflammatory effects on ARDS by down-regulating miR-9-5p and up-regulating LRG1 and CLDN5

Background: Acute respiratory distress syndrome (ARDS) is a life-threatening respiratory disease and its treatment is not fully established. Triptolide, one of Tripterygium wilfordii’s main active components, has been proved to alleviate Lipopolysaccharide (LPS)-induced ARDS. Imbalance of MicroRNAs (miRNAs) is recognized as the pathogenic mechanism of various diseases, including ARDS. However, the specific miRNAs that play a key regulatory role in the anti-inflammatory effect of triptolide in ARDS remain elusive.Methods: In this study, we administered triptolide in a mouse model of ARDS, and whole transcriptome sequencing was applied to identify meaningful miRNAs and validate them in vitro. Results: The results showed that triptolide may reduce the inflammatory response in ARDS by regulating miR-9-5p. The data further proved that LRG1 and CLDN5 expression are regulated by miR-9-5p, and triptolide can down-regulate the expression of miR-9-5p by regulating negatively the expression of LRG1 and CLDN5.Conclusion: Our study revealed that miR-9-5p was the specific miRNAs that plays key role in triptolide’s alleviation of ARDS inflammation by regulating target genes, and its inhibitory effect on LRG1 and CLDN5 expression was verified.

Integration of Genomic Profiling and Organoid Development in Precision Oncology

Precision oncology involves an innovative personalized treatment strategy for each cancer patient that provides strategies and options for cancer treatment. Currently, personalized cancer medicine is primarily based on molecular matching. Next-generation sequencing and related technologies, such as single-cell whole-transcriptome sequencing, enable the accurate elucidation of the genetic landscape in individual cancer patients and consequently provide clinical benefits. Furthermore, advances in cancer organoid models that represent genetic variations and mutations in individual cancer patients have direct and important clinical implications in precision oncology. This review aimed to discuss recent advances, clinical potential, and limitations of genomic profiling and the use of organoids in breast and ovarian cancer. We also discuss the integration of genomic profiling and organoid models for applications in cancer precision medicine.

Three-Dimensional Culture Decreases the Angiogenic Ability of Mouse Macrophages

Macrophages play important roles in angiogenesis; however, previous studies on macrophage angiogenesis have focused on traditional 2D cultures. In this study, we established a 3D culture system for macrophages using collagen microcarriers and assessed the effect of 3D culture on their angiogenic capabilities. Macrophages grown in 3D culture displayed a significantly different morphology and arrangement under electron microscopy compared to those grown in 2D culture. Tube formation assays and chick embryo chorioallantoic membrane assays further revealed that 3D-cultured macrophages were less angiogenic than those in 2D culture. Whole-transcriptome sequencing showed that nearly 40% of genes were significantly differently expressed, including nine important angiogenic factors of which seven had been downregulated. In addition, the expression of almost all genes related to two important angiogenic pathways was decreased in 3D-cultured macrophages, including the two key angiogenic factors, VEGFA and ANG2. Together, the findings of our study improve our understanding of angiogenesis and 3D macrophage culture in tissues, and provide new avenues and methods for future research on macrophages.

Genetic Basis of Follicle Development in Dazu Black Goat by Whole-Transcriptome Sequencing

The follicle development (FD) is an important factor determining litter size in animals. Recent studies have found that noncoding RNAs (ncRNAs) play an important role in FD. In particular, the role of the regulatory mechanism of competing endogenous RNAs (ceRNAs) that drive FD has attracted increasing attention. Therefore, this study explored the genetic basis of goat FD by obtaining the complete follicular transcriptome of Dazu black goats at different developmental stages. Results revealed that 128 messenger RNAs (mRNAs), 4 long noncoding RNAs (lncRNAs), 49 microRNAs (miRNAs), and 290 circular RNAs (circRNAs) were significantly differentially expressed (DE) between large and small follicles. Moreover, DEmRNAs were enriched in many signaling pathways related to FD, as well as GO terms related to molecular binding and enzyme activity. Based on the analysis of the ceRNA network (CRN), 34 nodes (1 DElncRNAs, 10 DEcircRNAs, 14 DEmiRNAs, and 9 DEmRNAs) and 35 interactions (17 DEcircRNAs–DEmRNAs, 2 DElncRNAs–DEmiRNAs, and 16 DEmRNA–DEmiRNAs) implied that the CRN could be involved in the FD of goats. In conclusion, we described gene regulation by DERNAs and lncRNA/circRNA–miRNA–mRNA CRNs in the FD of goats. This study provided insights into the genetic basis of FD in precise transcriptional regulation.

TRAWLING: a Transcriptome Reference Aware of spLIciNG events

Alternative splicing is critical for human gene expression regulation and plays an important role in multiple human diseases. In this context, RNA sequencing has emerged as powerful approach to detect alternative splicing events.In parallel, fast alignment-free methods have emerged as a viable alternative to quantify gene and transcript level abundance from RNAseq data. However, the ability to detect differential splicing events is dependent on the annotation of the transcript reference provided by the user.Here, we introduce a new reference transcriptome aware of splicing events, TRAWLING, which simplifies the detection of aberrant splicing events in a fast and simple way. In addition, we evaluate the performances and the benefits of aligning transcriptome data to TRAWLING using three different RNA sequencing datasets: whole transcriptome sequencing, single cell RNA sequencing and Digital RNA with pertUrbation of Genes.Collectively, our comprehensive evaluation underlines the value of using TRAWLING in transcriptomic data analysis.Availability and implementationOur code is available at https://github.com/Novartis/TRAWLING

Pre-Operative Physical Function Predicts Post-Operative Skeletal Muscle Gene Expression in Old Mice

In older adults, pre-operative physical function predicts post-operative outcomes. The biological mechanisms underlying vulnerability to physical decline remain poorly understood. Using a mouse model of laparotomy, we sought to identify biological correlates of post-operative function. 24-month-old male C57BL/6N mice were categorized as high functioning (HF) or low functioning (LF) based on pre-operative performance on the accelerating rotarod. On post-operative days (POD) 2 and 4, LF mice had lower rotarod latency to fall times than HF mice did. Forelimb grip strength was reduced after laparotomy in both HF and LF groups on POD 1 and 3 and did not differ significantly between these groups. Whole transcriptome sequencing analysis (RNAseq) of soleus muscles collected on POD 5 showed 224 and 228 differentially expressed genes (DEGs) for HF and LF, respectively, compared to their respective controls. Only 21 DEGs were observed in both groups, including Pparα, Fst and Pla2g15. Such changes may be hallmarks of the post-surgical response in aging. Pathway analysis of DEGs using Ingenuity Pathway Analysis software (Qiagen) revealed one pathway common to HF and LF (osteoarthritis) whereas activation of GP6 signaling and apoptosis signaling was observed in HF and inhibition of PPARα/RXR activation and PPARα signaling was noted in LF. We conclude that pre-operative performance on the accelerating rotarod correlates with differences in skeletal muscle gene expression, which may contribute to the differences in functional outcomes post-operatively in HF and LF mice. Further studies are needed to delineate the roles of these signaling pathways in physical resilience to surgery.

Gene expression signatures as candidate biomarkers of response to PD-1 blockade in non-small cell lung cancers

Although anti-PD-1/PD-L1 monotherapy has achieved clinical success in non-small cell lung cancer (NSCLC), definitive predictive biomarkers remain to be elucidated. In this study, we performed whole-transcriptome sequencing of pretreatment tumor tissue samples and pretreatment and on-treatment whole blood samples (WB) samples obtained from a clinically annotated cohort of NSCLC patients (n = 40) treated with nivolumab (anti-PD-1) monotherapy. Using a single-sample gene set enrichment scoring method, we found that the tumors of responders with lung adenocarcinoma (LUAD, n = 20) are inherently immunogenic to promote antitumor immunity, whereas those with lung squamous cell carcinoma (LUSC, n = 18) have a less immunosuppressive tumor microenvironment. These findings suggested that nivolumab may function as a molecular targeted agent in LUAD and as an immunomodulating agent in LUSC. In addition, our study explains why the reliability of PD-L1 expression on tumor cells as a predictive biomarker for the response to nivolumab monotherapy is quite different between LUAD and LUSC.