Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs

Fat deposition is an economically important trait in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Backfat tissue (three samples for each stage) was initially collected from different developmental stages (60, 120, 180, 240, 300, and 360 days after birth), and histological analysis and RNA sequencing (RNA-seq) were then conducted. Fragments per kilobase of transcript per million (FPKM) method was used to qualify gene expressions, and differentially expressed genes (DEGs) were identified. Furthermore, strongly co-expressed genes in modules, which were named by color, were clustered by Weighted gene co-expression network analysis (WGCNA) based on dynamic tree cutting algorithm. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment were subsequently implemented, and hub genes were described in each module. Finally, QPCR analysis was employed to validate RNA-seq data. The results showed that adipocyte area increased and adipocyte number decreased with development of backfat tissue. A total of 1,024 DEGs were identified in five comparison groups (120 days vs. 60 days, 180 days vs. 120 days, 240 days vs. 180 days, 300 days vs. 240 days, and 360 days vs. 300 days). The turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had the highest correlation coefficient with 60, 120, 180, 240, 300, and 360 days developmental stage, while the tan, black and turquoise module had strong relationship with backfat thickness, adipocyte area, and adipocyte number, respectively. Thirteen hub genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized. ACSL1 and ACOX1 might perform function in the early developmental stage of backfat tissue (60 days), and FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position in backfat tissue around 120 days developmental stage. Besides, hub genes SELP and DNM1 in modules significantly associated with backfat thickness and adipocyte area might be involved in the process of backfat tissue development. These findings contribute to understand the integrated mechanism underlying backfat tissue development and promote the progress of genetic improvement in Ningxiang pigs

Prolonged administration of total glucosides of paeony improves intestinal immune imbalance and epithelial barrier damage in collagen-induced arthritis rats based on metabolomics-network pharmacology integrated analysis

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and joint damage with complex pathological mechanisms. In recent years, many studies have shown that the dysregulation of intestinal mucosal immunity and the damage of the epithelial barrier are closely related to the occurrence of RA. Total glucosides of paeony (TGP) have been used clinically for the treatment of RA in China for decades, while the pharmacological mechanism is still uncertain. The purpose of this study was to investigate the regulatory effect and mechanism of TGP on intestinal immunity and epithelial barrier in RA model rats. The results showed that TGP alleviated immune hyperfunction by regulating the ratio of CD3+, CD4+ and CD8+ in different lymphocyte synthesis sites of the small intestine, including Peyer’s patches (PPs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Specially, TGP first exhibited immunomodulatory effects on sites close to the intestinal lumen (IELs and LPLs), and then on PPs far away from the intestinal lumen as the administration time prolonged. Meanwhile, TGP restores the intestinal epithelial barrier by upregulating the ratio of villi height (V)/crypt depth (C) and expression of tight junction proteins (ZO-1, occludin). Finally, the integrated analysis of metabolomics-network pharmacology was also used to explore the possible regulation mechanism of TGP on the intestinal tract. Metabolomics analysis revealed that TGP reversed the intestinal metabolic profile disturbance in CIA rats, and identified 32 biomarkers and 163 corresponding targets; network pharmacology analysis identified 111 potential targets for TGP to treat RA. By intersecting the results of the two, three key targets such as ADA, PNP and TYR were determined. Pharmacological verification experiments showed that the levels of ADA and PNP in the small intestine of CIA rats were significantly increased, while TGP significantly decreased their ADA and PNP levels. In conclusion, purine metabolism may play an important role in the process of TGP improving RA-induced intestinal immune imbalance and impaired epithelial barrier

Use of Inverse PCR for Analysis of Class 1 Integrons Carrying an Unusual 3′ Conserved Segment Structure▿

By using inverse PCR and DNA sequencing, 13 sul3-associated mutational integrons, 2 defective class 1 integrons, and 1 qnrB2-associated complex sul1-type class 1 integrons were identified in Salmonella enterica serovar Choleraesuis, Pseudomonas aeruginosa, and Enterobacter cloacae, respectively. In addition, conjugation and Southern hybridization demonstrated that unusual class 1 integrons were located on plasmids or integrated into chromosomal DNA. Thus, an inverse PCR assay can be a valuable tool for the analysis of unusual structures of the 3′ conserved region of class 1 integrons

Identifying EGFR-Expressed Cells and Detecting EGFR Multi-Mutations at Single-Cell Level by Microfluidic Chip

Abstract EGFR mutations companion diagnostics have been proved to be crucial for the efficacy of tyrosine kinase inhibitor targeted cancer therapies. To uncover multiple mutations occurred in minority of EGFR-mutated cells, which may be covered by the noises from majority of un-mutated cells, is currently becoming an urgent clinical requirement. Here we present the validation of a microfluidic-chip-based method for detecting EGFR multi-mutations at single-cell level. By trapping and immunofluorescently imaging single cells in specifically designed silicon microwells, the EGFR-expressed cells were easily identified. By in situ lysing single cells, the cell lysates of EGFR-expressed cells were retrieved without cross-contamination. Benefited from excluding the noise from cells without EGFR expression, the simple and cost-effective Sanger’s sequencing, but not the expensive deep sequencing of the whole cell population, was used to discover multi-mutations. We verified the new method with precisely discovering three most important EGFR drug-related mutations from a sample in which EGFR-mutated cells only account for a small percentage of whole cell population. The microfluidic chip is capable of discovering not only the existence of specific EGFR multi-mutations, but also other valuable single-cell-level information: on which specific cells the mutations occurred, or whether different mutations coexist on the same cells. This microfluidic chip constitutes a promising method to promote simple and cost-effective Sanger’s sequencing to be a routine test before performing targeted cancer therapy

Table5_Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs.XLSX

Fat deposition is an economically important trait in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Backfat tissue (three samples for each stage) was initially collected from different developmental stages (60, 120, 180, 240, 300, and 360 days after birth), and histological analysis and RNA sequencing (RNA-seq) were then conducted. Fragments per kilobase of transcript per million (FPKM) method was used to qualify gene expressions, and differentially expressed genes (DEGs) were identified. Furthermore, strongly co-expressed genes in modules, which were named by color, were clustered by Weighted gene co-expression network analysis (WGCNA) based on dynamic tree cutting algorithm. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment were subsequently implemented, and hub genes were described in each module. Finally, QPCR analysis was employed to validate RNA-seq data. The results showed that adipocyte area increased and adipocyte number decreased with development of backfat tissue. A total of 1,024 DEGs were identified in five comparison groups (120 days vs. 60 days, 180 days vs. 120 days, 240 days vs. 180 days, 300 days vs. 240 days, and 360 days vs. 300 days). The turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had the highest correlation coefficient with 60, 120, 180, 240, 300, and 360 days developmental stage, while the tan, black and turquoise module had strong relationship with backfat thickness, adipocyte area, and adipocyte number, respectively. Thirteen hub genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized. ACSL1 and ACOX1 might perform function in the early developmental stage of backfat tissue (60 days), and FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position in backfat tissue around 120 days developmental stage. Besides, hub genes SELP and DNM1 in modules significantly associated with backfat thickness and adipocyte area might be involved in the process of backfat tissue development. These findings contribute to understand the integrated mechanism underlying backfat tissue development and promote the progress of genetic improvement in Ningxiang pigs.</p

Table2_Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs.DOCX

Fat deposition is an economically important trait in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Backfat tissue (three samples for each stage) was initially collected from different developmental stages (60, 120, 180, 240, 300, and 360 days after birth), and histological analysis and RNA sequencing (RNA-seq) were then conducted. Fragments per kilobase of transcript per million (FPKM) method was used to qualify gene expressions, and differentially expressed genes (DEGs) were identified. Furthermore, strongly co-expressed genes in modules, which were named by color, were clustered by Weighted gene co-expression network analysis (WGCNA) based on dynamic tree cutting algorithm. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment were subsequently implemented, and hub genes were described in each module. Finally, QPCR analysis was employed to validate RNA-seq data. The results showed that adipocyte area increased and adipocyte number decreased with development of backfat tissue. A total of 1,024 DEGs were identified in five comparison groups (120 days vs. 60 days, 180 days vs. 120 days, 240 days vs. 180 days, 300 days vs. 240 days, and 360 days vs. 300 days). The turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had the highest correlation coefficient with 60, 120, 180, 240, 300, and 360 days developmental stage, while the tan, black and turquoise module had strong relationship with backfat thickness, adipocyte area, and adipocyte number, respectively. Thirteen hub genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized. ACSL1 and ACOX1 might perform function in the early developmental stage of backfat tissue (60 days), and FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position in backfat tissue around 120 days developmental stage. Besides, hub genes SELP and DNM1 in modules significantly associated with backfat thickness and adipocyte area might be involved in the process of backfat tissue development. These findings contribute to understand the integrated mechanism underlying backfat tissue development and promote the progress of genetic improvement in Ningxiang pigs.</p

Table3_Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs.XLSX

Fat deposition is an economically important trait in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Backfat tissue (three samples for each stage) was initially collected from different developmental stages (60, 120, 180, 240, 300, and 360 days after birth), and histological analysis and RNA sequencing (RNA-seq) were then conducted. Fragments per kilobase of transcript per million (FPKM) method was used to qualify gene expressions, and differentially expressed genes (DEGs) were identified. Furthermore, strongly co-expressed genes in modules, which were named by color, were clustered by Weighted gene co-expression network analysis (WGCNA) based on dynamic tree cutting algorithm. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment were subsequently implemented, and hub genes were described in each module. Finally, QPCR analysis was employed to validate RNA-seq data. The results showed that adipocyte area increased and adipocyte number decreased with development of backfat tissue. A total of 1,024 DEGs were identified in five comparison groups (120 days vs. 60 days, 180 days vs. 120 days, 240 days vs. 180 days, 300 days vs. 240 days, and 360 days vs. 300 days). The turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had the highest correlation coefficient with 60, 120, 180, 240, 300, and 360 days developmental stage, while the tan, black and turquoise module had strong relationship with backfat thickness, adipocyte area, and adipocyte number, respectively. Thirteen hub genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized. ACSL1 and ACOX1 might perform function in the early developmental stage of backfat tissue (60 days), and FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position in backfat tissue around 120 days developmental stage. Besides, hub genes SELP and DNM1 in modules significantly associated with backfat thickness and adipocyte area might be involved in the process of backfat tissue development. These findings contribute to understand the integrated mechanism underlying backfat tissue development and promote the progress of genetic improvement in Ningxiang pigs.</p

Table6_Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs.XLSX

Fat deposition is an economically important trait in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Backfat tissue (three samples for each stage) was initially collected from different developmental stages (60, 120, 180, 240, 300, and 360 days after birth), and histological analysis and RNA sequencing (RNA-seq) were then conducted. Fragments per kilobase of transcript per million (FPKM) method was used to qualify gene expressions, and differentially expressed genes (DEGs) were identified. Furthermore, strongly co-expressed genes in modules, which were named by color, were clustered by Weighted gene co-expression network analysis (WGCNA) based on dynamic tree cutting algorithm. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment were subsequently implemented, and hub genes were described in each module. Finally, QPCR analysis was employed to validate RNA-seq data. The results showed that adipocyte area increased and adipocyte number decreased with development of backfat tissue. A total of 1,024 DEGs were identified in five comparison groups (120 days vs. 60 days, 180 days vs. 120 days, 240 days vs. 180 days, 300 days vs. 240 days, and 360 days vs. 300 days). The turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had the highest correlation coefficient with 60, 120, 180, 240, 300, and 360 days developmental stage, while the tan, black and turquoise module had strong relationship with backfat thickness, adipocyte area, and adipocyte number, respectively. Thirteen hub genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized. ACSL1 and ACOX1 might perform function in the early developmental stage of backfat tissue (60 days), and FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position in backfat tissue around 120 days developmental stage. Besides, hub genes SELP and DNM1 in modules significantly associated with backfat thickness and adipocyte area might be involved in the process of backfat tissue development. These findings contribute to understand the integrated mechanism underlying backfat tissue development and promote the progress of genetic improvement in Ningxiang pigs.</p

Table8_Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs.XLSX

Fat deposition is an economically important trait in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Backfat tissue (three samples for each stage) was initially collected from different developmental stages (60, 120, 180, 240, 300, and 360 days after birth), and histological analysis and RNA sequencing (RNA-seq) were then conducted. Fragments per kilobase of transcript per million (FPKM) method was used to qualify gene expressions, and differentially expressed genes (DEGs) were identified. Furthermore, strongly co-expressed genes in modules, which were named by color, were clustered by Weighted gene co-expression network analysis (WGCNA) based on dynamic tree cutting algorithm. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment were subsequently implemented, and hub genes were described in each module. Finally, QPCR analysis was employed to validate RNA-seq data. The results showed that adipocyte area increased and adipocyte number decreased with development of backfat tissue. A total of 1,024 DEGs were identified in five comparison groups (120 days vs. 60 days, 180 days vs. 120 days, 240 days vs. 180 days, 300 days vs. 240 days, and 360 days vs. 300 days). The turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had the highest correlation coefficient with 60, 120, 180, 240, 300, and 360 days developmental stage, while the tan, black and turquoise module had strong relationship with backfat thickness, adipocyte area, and adipocyte number, respectively. Thirteen hub genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized. ACSL1 and ACOX1 might perform function in the early developmental stage of backfat tissue (60 days), and FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position in backfat tissue around 120 days developmental stage. Besides, hub genes SELP and DNM1 in modules significantly associated with backfat thickness and adipocyte area might be involved in the process of backfat tissue development. These findings contribute to understand the integrated mechanism underlying backfat tissue development and promote the progress of genetic improvement in Ningxiang pigs.</p

Image1_Identification of crucial modules and genes associated with backfat tissue development by WGCNA in Ningxiang pigs.TIF

Fat deposition is an economically important trait in pigs. Ningxiang pig, one of the four famous indigenous breeds in China, is characterized by high fat content. The underlying gene expression pattern in different developmental periods of backfat tissue remains unclear, and the purpose of this investigation is to explore the potential molecular regulators of backfat tissue development in Ningxiang pigs. Backfat tissue (three samples for each stage) was initially collected from different developmental stages (60, 120, 180, 240, 300, and 360 days after birth), and histological analysis and RNA sequencing (RNA-seq) were then conducted. Fragments per kilobase of transcript per million (FPKM) method was used to qualify gene expressions, and differentially expressed genes (DEGs) were identified. Furthermore, strongly co-expressed genes in modules, which were named by color, were clustered by Weighted gene co-expression network analysis (WGCNA) based on dynamic tree cutting algorithm. Gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment were subsequently implemented, and hub genes were described in each module. Finally, QPCR analysis was employed to validate RNA-seq data. The results showed that adipocyte area increased and adipocyte number decreased with development of backfat tissue. A total of 1,024 DEGs were identified in five comparison groups (120 days vs. 60 days, 180 days vs. 120 days, 240 days vs. 180 days, 300 days vs. 240 days, and 360 days vs. 300 days). The turquoise, red, pink, paleturquoise, darkorange, and darkgreen module had the highest correlation coefficient with 60, 120, 180, 240, 300, and 360 days developmental stage, while the tan, black and turquoise module had strong relationship with backfat thickness, adipocyte area, and adipocyte number, respectively. Thirteen hub genes (ACSL1, ACOX1, FN1, DCN, CHST13, COL1A1, COL1A2, COL6A3, COL5A1, COL14A1, OAZ3, DNM1, and SELP) were recognized. ACSL1 and ACOX1 might perform function in the early developmental stage of backfat tissue (60 days), and FN1, DCN, COL1A1, COL1A2, COL5A1, COL6A3, and COL14A1 have unignorable position in backfat tissue around 120 days developmental stage. Besides, hub genes SELP and DNM1 in modules significantly associated with backfat thickness and adipocyte area might be involved in the process of backfat tissue development. These findings contribute to understand the integrated mechanism underlying backfat tissue development and promote the progress of genetic improvement in Ningxiang pigs.</p