Transcriptomic analysis is crucial for understanding the functional elements of the genome, with the classic method consisting of screening transcriptomics datasets for differentially expressed genes (DEGs). Additionally, since 2005, weighted gene co-expression network analysis (WGCNA) has emerged as a powerful method to explore relationships between genes. However, an approach combining both methods, i.e., filtering the transcriptome dataset by DEGs or other criteria, followed by WGCNA (DEGs + WGCNA), has become common. This is of concern because such approach can affect the resulting underlying architecture of the network under analysis and lead to wrong conclusions. Here, we explore a plot twist to transcriptome data analysis: applying WGCNA to exploit entire datasets without affecting the topology of the network, followed with the strength and relative simplicity of DEG analysis (WGCNA + DEGs). We tested WGCNA + DEGs against DEGs + WGCNA to publicly available transcriptomics data in one of the most transcriptomically complex tissues and delicate processes: vertebrate gonads undergoing sex differentiation. We further validate the general applicability of our approach through analysis of datasets from three distinct model systems: European sea bass, mouse, and human.In all cases, WGCNA + DEGs clearly outperformed DEGs + WGCNA. First, the network model fit and node connectivity measures and other network statistics improved. The gene lists filtered by each method were different, the number of modules associated with the trait of interest and key genes retained increased, and GO terms of biological processes provided a more nuanced representation of the biological question under consideration. Lastly, WGCNA + DEGs facilitated biomarker discovery.We propose that building a co-expression network from an entire dataset, and only thereafter filtering by DEGs, should be the method to use in transcriptomic studies, regardless of biological system, species, or question being considered. 

/pdf/improved-biomarker-discovery-through-a-plot-twist-in-1egffmmz.pdf

Improved biomarker discovery through a plot twist in transcriptomic data analysis

Background: Asthma was a chronic inflammatory illness driven by complicated genetic regulation and environmental exposure. The complex pathophysiology of asthma has not been fully understood. Ferroptosis was involved in inflammation and infection. However, the effect of ferroptosis on asthma was still unclear. The study was designed to identify ferroptosis-related genes in asthma, providing potential therapeutic targets. Methods: We conducted a comprehensive analysis combined with WGCNA, PPI, GO, KEGG, and CIBERSORT methods to identify ferroptosis-related genes that were associated with asthma and regulated the immune microenvironment in GSE147878 from the GEO. The results of this study were validated in GSE143303 and GSE27066, and the hub genes related to ferroptosis were further verified by immunofluorescence and RT-qPCR in the OVA asthma model. Results: 60 asthmatics and 13 healthy controls were extracted for WGCNA. We found that genes in the black module (r = −0.47, p < 0.05) and magenta module (r = 0.51, p < 0.05) were associated with asthma. CAMKK2 and CISD1 were discovered to be ferroptosis-related hub genes in the black and magenta module, separately. We found that CAMKK2 and CISD1 were mainly involved in the CAMKK-AMPK signaling cascade, the adipocytokine signaling pathway, the metal cluster binding, iron-sulfur cluster binding, and 2 iron, 2 sulfur cluster binding in the enrichment analysis, which was strongly correlated with the development of ferroptosis. We found more infiltration of M2 macrophages and less Tregs infiltration in the asthma group compared to healthy controls. In addition, the expression levels of CISD1 and Tregs were negatively correlated. Through validation, we found that CAMKK2 and CISD1 expression were upregulated in the asthma group compared to the control group and would inhibit the occurrence of ferroptosis. Conclusion: CAMKK2 and CISD1 might inhibit ferroptosis and specifically regulate asthma. Moreover, CISD1 might be tied to the immunological microenvironment. Our results could be useful to provide potential immunotherapy targets and prognostic markers for asthma.

/pdf/ferroptosis-related-genes-are-involved-in-asthma-and-2pbqt3m4.pdf

Ferroptosis-related genes are involved in asthma and regulate the immune microenvironment

Transportation is an inevitable phase for the cattle industry, and its effect on the respiratory system of transported cattle remains controversial. To reveal cattle’s nasopharyngeal microbiota dynamics, we tracked a batch of beef calves purchased from an auction market, transported to a farm by vehicle within three days and adaptively fed for seven days. Before and after the transport and after the placement, a total of 18 nasopharyngeal mucosal samples were collected, and microbial profiles were obtained using a metagenomic shot-gun sequencing approach. The diversity, composition, structure, and function of the microbiota were collected at each time point, and their difference was analyzed. The results showed that, before the transportation, there were a great abundance of potential bovine respiratory diseases (BRD) related pathogens, and the transportation did not significantly change their abundance. After the transportation, seven days of placement significantly decreased the risk of BRD by decreasing the abundance of potential BRD related pathogens even if the diversity was decreased. We also discussed the controversial results of transportation’s effect in previous works and the decrease in diversity induced by placement. Our work provided more accurate information about the effect of transportation and the followed placement on the calf nasopharyngeal microbial community, indicated the importance of adaptive placement after long-distance transport, and is helpful to prevent BRD induced by transportation stress.

Metagenomics Reveals That Proper Placement After Long-Distance Transportation Significantly Affects Calf Nasopharyngeal Microbiota and Is Critical for the Prevention of Respiratory Diseases.

The beef cattle industry in China has advanced remarkably since its reform and opening up; consequently, China has become the world’s third-largest beef cattle producer. China is also one of the countries with the most substantial research input and output in the field of beef cattle feed and nutrition. The progress and innovation by China in the research field of beef cattle feed and nutrition have undoubtedly promoted the development of the domestic beef cattle industry. This review summarizes recent advances in feed resource development, nutrient requirements, and nutritional regulation of beef cattle in China. Limitations in current research and perspectives on future work are also discussed. 

Recent advances in feed and nutrition of beef cattle in China — A review

Transportation stress causes significant changes in physiological responses in goats; however, studies exploring the transcriptome of stress are very limited. The objective of this study was to determine the differential gene expressions and related pathways in the blood samples using RNA-seq procedure in Spanish goats subjected to different durations of transportation stress. Fifty-four male Spanish goats (8-mo old; BW = 29.7 ± 2.03 kg) were randomly subjected to one of three treatments (TRT; n = 18 goats/treatment): (1) transported for 180 min, (2) transported for 30 min, or (3) held in pens (control). Blood samples were collected before and after treatment for stress hormone, metabolite, and transcriptomic analysis. RNA-seq technology was used to obtain the transcriptome profiles of blood. Analysis of physiological data using SAS showed that plasma cortisol concentrations were higher (P < 0.01) in 180 min and 30 min groups compared to the control group. Enrichment analysis of DEGs related to transportation stress through Gene Ontology and KEGG databases revealed that the differentially expressed genes related to inflammatory pathways, caspases, and apoptosis such as IL1R2, CASP14, CD14, TLR4, and MAPK14 were highly enriched in the transported group of goats compared to non-transported goats. Stress in goats leads to a sequence of events at cellular and molecular levels that causes inflammation and apoptosis. 

/pdf/differential-gene-expression-analysis-using-rna-seq-in-the-2kr4gxt5.pdf

Differential gene expression analysis using RNA-seq in the blood of goats exposed to transportation stress

The transportation is a crucial phase in beef cattle industry, and the annual losses caused by beef cattle transport stress are substantial. Several studies have described the effect of long distance transportation stress on animal health, such as disorder in nervous, endocrine, immune, and metabolic system. However, molecular mechanisms underlying short distance transportation stress is still poorly understood. Present study aims to investigate the effect of short distance transportation by measuring the hematological indices and transcriptomic analysis. In this study, a total 10 Qinchuan cattle were used to compare the molecular characteristics of blood before and after transportation. We have found that a stress-related marker "white blood cell count (WBC)" increased significantly after transportation. The decrease in triglyceride (TG), cholestenone (CHO), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) showed that energy expenditure was increased after transportation, but not enough to activate fatty decomposition. Intriguingly, the decrease of malondialdehyde (MDA) showed that cattle were more resilience to oxidative stress. The RNA-seq showed that 1,092 differentially expressed genes (DEGs) were found (329 up-regulated and 763 down-regulated) between group before and group after. The GO and KEGG enrichment showed that the metabolic pathway and B cell function related pathways were enriched. Furthermore, median absolute deviation (MAD) top 5,000 genes were used to construct a co-expression network by weighted correlation network analysis (WGCNA), and 11 independent modules were identified. Combing with protein-protein interaction (PPI) analysis, the verification of quantitative real-time PCR (qPCR) and the correlation of B cell function, structural maintenance of chromosomes 3 (SMC3), jun proto-oncogene (JUN), and C-X-C motif chemokine ligand 10 (CXCL10) were suggested as potential molecular markers in identification of short distance transportation. Collectively, the blood RNA-seq analysis and WGCNA indicated that the disorder of B cell differentiation, proliferation, survival, and apoptosis were the potential molecular mechanism in short distance transportation stress. In conclusion, our results provide the novel insight about potential biomarkers for short distance transportation stress, which may serve as for diagnosing and preventing this condition in beef industry.

/pdf/transcriptome-characterization-of-short-distance-transport-u5dcjmimls.pdf

Transcriptome Characterization of Short Distance Transport Stress in Beef Cattle Blood.

MicroRNAs (miRNAs) have been determined to participate in the process of oestradiol production. Generally, there are two pathways by which oestradiol levels change, one being the state of cells (i.e. the status of enzymes involved in the synthesis of hormones such as oestradiol) and the other being the number of cells that secrete oestradiol. It is known that oestrogens are the main steroids produced by granulosa cells (GCs) of mature ovarian follicles. In this study we explored the function of miR-18b in rabbit GCs by overexpressing or inhibiting its activity. We found that miR-18b silencing promoted the secretion of oestradiol by significantly affecting the expression of steroidogenesis-related genes. Thus, miR-18b may act as a negative regulator of the production of enzymes related to oestradiol synthesis and affect oestradiol production. Furthermore, the effects of miR-18b on the proliferation, cell cycle and apoptosis of GCs were investigated using a cell counting kit (CCK-8) proliferation assay, detection of annexin V–fluorescein isothiocyanate apoptosis, flow cytometry and quantitative polymerase chain reaction. The results showed that miR-18b upregulated GC apoptosis (miR-18b overexpression decreases cell growth and stimulates apoptosis). These findings suggest that miR-18b and the oestrogen receptor 1 (ESR1) gene may be attractive targets to further explore the molecular regulation of GCs. The miR-18b may also explain, in part, the abnormal folliculogenesis in mammals caused by conditions such as polycystic ovary syndrome, primary ovarian insufficiency, and others.

miR-18b regulates the function of rabbit ovary granulosa cells.

The invention discloses a detection method for 19-bp duplication and deletion polymorphism of a Chinese yellow cattle PLAG1 (Pleomorphic Adenoma Gene 1) gene and application of the detection method. The method takes Chinese yellow cattle whole genome DNA (Deoxyribonucleic Acid) as a template, and a primer pair P1 is used for treating a partial segment of the Chinese yellow cattle PLAG1 gene through PCR (Polymerase Chain Reaction) amplification; then the segment obtained by the PCR amplification is subjected to agarose gel electrophoresis; a gene type of an insertion/deletion polymorphism siteon the Chinese yellow cattle PLAG1 gene is identified according to an electrophoresis result; different gene types of the duplication and deletion polymorphism of the gene are remarkably related withgrowth properties including body height, body length, chest circumference, chest width, chest depth, waist height and the like four types of Chinese yellow cattle including Qiachuan cattle, Nanyang cattle, Jiaxian cattle and Luxi cattle. The method disclosed by the invention can be applied to marker-assisted selection breeding of growth characters of beef cattle, and a good genetic resource groupwith good growth performance is easily and rapidly established.

Detection method for 19-bp duplication and deletion polymorphism of Chinese yellow cattle PLAG1 (Pleomorphic Adenoma Gene 1) gene and application of detection method

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Papers

Transcriptome Characterization of Short Distance Transport Stress in Beef Cattle Blood.

miR-18b regulates the function of rabbit ovary granulosa cells.

Detection method for 19-bp duplication and deletion polymorphism of Chinese yellow cattle PLAG1 (Pleomorphic Adenoma Gene 1) gene and application of detection method