Glyceraldehyde-3-phosphate Dehydrogenase Expression Research Articles

A Pan-Cancer Analysis of GAPDH as a Common Biomarker for Various Cancers

Objective: To investigate the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and explore its prognostic value across 24 different human cancers. This investigation was conducted using comprehensive bioinformatics and in vitro approaches, incorporating multiple layers of analysis. Methods: GAPDH expression and methylation levels were assessed using bioinformatics tools and validated in cell lines through RNA-seq and targeted bisulfite-seq analyses. The potential prognostic significance of GAPDH was evaluated using the KM plotter. Additionally, cBioPortal was employed to investigate genetic alterations associated with this gene. Pathway analysis was conducted using DAVID. Furthermore, a correlation analysis between GAPDH expression and CD8+ T immune cells was performed using TIMER and CDT. Finally, a gene-drug interaction network analysis was conducted using Cytoscape to examine the relationship between GAPDH and various drugs. Results: GAPDH was found to be commonly upregulated in 24 types of human cancers, with its upregulation significantly correlated with poor relapse-free survival (RFS) and overall survival (OS) in BLCA, CESC, HNSC, KIRP, LIHC, and LUAD. This suggests that GAPDH plays a significant role in the development of these cancers. GAPDH upregulation was also associated with various clinicopathological features in patients with BLCA, CESC, HNSC, KIRP, LIHC, and LUAD. Pathway analysis revealed GAPDH’s involvement in diverse pathways. Additionally, notable correlations were observed between GAPDH expression and its promoter methylation level, genetic alterations, and CD8+ T immune cell levels. Moreover, several regulatory drugs targeting GAPDH were identified, with the potential to modulate its expression and potentially prevent conditions such as BLCA, CESC, HNSC, KIRP, LIHC, and LUAD. Conclusion: Based on our findings, GAPDH emerges as a promising diagnostic and prognostic biomarker for BLCA, CESC, HNSC, KIRP, LIHC, and LUAD.

Availability of individual proteins for quantitative analysis in postmortem brains preserved in two different brain banks.

Postmortem brain research is necessary for elucidating the pathology of schizophrenia; an increasing number of studies require a combination of suitable tissue samples preserved at multiple brain banks. In this study, we examined whether a comparative study of protein expression levels can be conducted using postmortem brain samples preserved in different facilities. We compared the demographic factors of postmortem brain samples preserved in two institutions and measured and compared the expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glial fibrillary acidic protein (GFAP) in the prefrontal cortex and superior temporal gyrus. GAPDH is generally used as a loading control for western blotting, and GFAP is considered as an astrocyte marker in the brain. We found significant differences between the two institutions in postmortem interval, age at death, and preservation time. To reduce the effects of these differences on our measurements, the parameters were set as covariates in our analyses of covariance. Subsequently, no differences in GAPDH and GFAP expression were found between institutions. When studies are conducted using brain samples preserved in different brain banks, differences in demographic factors should be carefully considered and taken into account by statistical methods to minimize their impact as much as possible. Since there was no significant difference in the protein expression levels of GAPDH and GFAP in either region between the two institutions that preserved the postmortem brains, we concluded that it is possible to perform protein quantitative analysis assuming that there is no effect of difference between two institutions.

Preliminary study on the effect of Echinococcus multilocaris on phenotypic transformations of glucose metabolism and polarization types in macrophages

To investigate the effects of Echinococcus multilocularis on the phenotypic transformations of glucose metabolism, polarization types and inflammatory responses in macrophages, so as to provide insights into elucidation of echinococcosis pathogenesis. Bone marrow cells were isolated from C57BL/6J mice at ages of 6 to 8 weeks, and induced into bone marrow-derived macrophages (BMDMs) with mouse macrophage colony-stimulating factor (M-CSF), which served as controls (BMDMs-M0). BMDMs-M0 induced M2 macrophages by interleukin-4 for 24 hours served as the IL-4 induction group, and BMDMs-M0 co-cultured with 2.4 ng/mL E. multilocularis cystic fluid (CF) served as the BMDM-CF co-culture group, while BMDMs-M0 co-cultured with E. multilocularis protoscolex (PSC) at a ratio of 500:1 served as the BMDM-PSC co-culture group. The types of polarization of BMDMs co-cultured with E. multilocularis CF and PSC were analyzed using flow cytometry, and the expression of macrophage markers, inflammatory factors, and glucose metabolism-related enzymes was quantified using fluorescent quantitative real-time PCR (qPCR) and Western blotting assays. There were significant differences among the four groups in terms of Arginase-1 (Arg1) (F = 1 457.00, P < 0.000 1), macrophages-derived C-C motif chemokine 22 (Ccl22) (F = 22 203.00, P < 0.000 1), resistin-like α (Retnla) (F = 151.90, P < 0.000 1), inducible nitric oxide synthase (iNOS) (F = 107.80, P < 0.001), hexokinase (HK) (F = 9 389.00, P < 0.000 1), pyruvate kinase (PK) (F = 641.40, P < 0.001), phosphofructokinase 1 (PFK1) (F = 43.97, P < 0.01), glucokinase (GK) (F = 432.50, P < 0.000 1), pyruvate dehydrogenase kinases1 (PDK1) (F = 737.30, P < 0.000 1), lactic dehydrogenase (LDH) (F = 3 632.00, P < 0.000 1), glucose transporter 1 (GLUT1) (F = 532.40, P < 0.000 1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (F = 460.00, P < 0.000 1), citrate synthase (CS) (F = 5 642.00, P < 0.01), glycogen synthase1 (GYS1) (F = 273.30, P < 0.000 1), IL-6 (F = 1 823.00, P < 0.000 1), IL-10 (F = 291.70, P < 0.000 1), IL-1β (F = 986.60, P < 0.000 1), and tumor necrosis factor (TNF)-α (F = 334.80, P < 0.000 1) and transforming growth factor (TGF)-β mRNA expression (F = 163.30, P < 0.001). The proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-PSC co-culture group [(22.87% ±1.48%) vs. (1.70% ±0.17%); t = 24.61, P < 0.001], and the proportion of M2 macrophages was significantly higher than that of M1 macrophages in the BMDM-CF co-culture group [(20.07% ±0.64%) vs. (1.93% ±0.25%); t = 45.73, P < 0.001]. The mRNA expression of M2 macrophages markers Arg1, Ccl22 and Retnla was significantly higher in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01), and no significant difference was seen in the mRNA expression of the M1 macrophage marker iNOS among the three groups (P > 0.05), while qPCR assay quantified higher mRNA expression of key glycolytic enzymes HK, PK and PFK, as well as inflammatory factors IL-10, IL-1β, TNF-α and TGF-β in the BMDM-CF and BMDM-PSC co-culture groups than in the control group (all P values < 0.01). Western blotting assay determined higher HK, PK and PFK protein expression in the BMDM-PSC co-culture group than in the control group (all P values < 0.05), and qPCR quantified higher GLUT1, GAPDH and IL-6 mRNA expression in the BMDM-CF co-culture group than in the control group (all P values < 0.05), while higher HK, PK and PFK protein and mRNA expression (all P values < 0.01), as well as lower IL-6 and TNF-α and higher TGF-β mRNA expression (both P values < 0.05) was detected in the IL-4 induction group than in the control group. Glycolytic stress test showed no significant difference in the extracellular acidification rate (ECAR) of mouse BMDM among the control group, IL-4 induction group and BMDM-PSC co-culture group (F = 124.4, P < 0.05), and a higher ECAR was seen in the BMDM-PSC co-culture group and a lower ECAR was found in the IL-4 induction group than in the control group (both P values < 0.05). Treatment of E. multilocularis CF or PSC mainly causes polarization of BMDM into M2 macrophages, and phenotypic transformation of glucose metabolism into high-energy and high-glycolytic metabolism, and affects inflammatory responses in BMDM.

Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

The variations in gene expression of GAPDH in Ocimum basilicum cultivars under drought-induced stress conditions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) holds a pivotal role within the glycolytic pathway of higher plants. It has garnered attention as a significant target protein in instances of oxidative stress, where it can engage in thiolation reactions within its active site. Numerous genes encoding cytosolic iterations of GAPDH have been identified and analyzed in specific plant species. This investigation was conducted to gain insights into GAPDH's function amidst drought-induced stress. Within this framework, the basil plant (Ocimum basilicum) was chosen for focused exploration, encompassing the cloning of the comprehensive cDNA of basil GAPDH (ObGAPDH) and scrutinizing its patterns of expression. The complete sequence of Ob-GAPDH spanned 1315 base pairs. The resultant protein derived from this sequence comprised 399 amino acids, projecting a molecular weight of approximately 42.54kDa and an isoelectric point (pI) of 6.01. An examination of the evolutionary connections among various GAPDH proteins unveiled ObGAPDH's shared lineage with GAPDH proteins sourced from other plants, such as Salvia splendens and Sesamum indicum. Furthermore, computational methodologies were harnessed to predict the potential oxidative role of ObGAPDH in response to external signals. Molecular docking simulations illuminated the interaction between ObGAPDH and hydrogen peroxide (H2O2) as a ligand. Scrutinizing the expression patterns of the ObGAPDH gene under conditions of water scarcity stress brought to light diverse levels of transcriptional activity. Collectively, these findings underscore the notion that the regulation of ObGAPDH expression is contingent upon both the specific plant cultivar and the presence of stress stemming from drought conditions.

Comparison of Multiple Strategies for Precision Transgene Knock-In in Gallus gallus Genome via Microhomology-Mediated End Joining.

Precision exogenous gene knock-in is an attractive field for transgenic Gallus gallus (chicken) generation. In this article, we constructed multiple Precise Integration into Target Chromosome (PITCh) plasmid systems mediated by microhomology-mediated end-joining (MMEJ) for large-fragment integration in DF-1 cells and further assess the possibility of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a genomic safe harbor for chickens. We designed three targeted sgRNAs for the all-in-one plasmid at the 3'UTR of GAPDH near the stop codon. The donor-plasmid-carrying microhomology arms correspond to sgRNA and EGFP fragments in the forward and reverse directions. MMEJ-mediated EGFP insertion can be efficiently expressed in DF-1 cells. Moreover, the differences between the forward and reverse fragments indicated that promoter interference does affect the transfection efficiency of plasmids and cell proliferation. The comparison of the 20 bp and 40 bp microhomology arms declared that the short one has higher knock-in efficiency. Even though all three different transgene insertion sites in GAPDH could be used to integrate the foreign gene, we noticed that the G2-20R-EGFP cell reduced the expression of GAPDH, and the G3-20R-EGFP cell exhibited significant growth retardation. Taken together, G1, located at the 3'UTR of GAPDH on the outer side of the last base of the terminator, can be a candidate genomic safe harbor (GSH) loci for the chicken genome. In addition, deleted-in-azoospermia-like (DAZL) and actin beta (ACTB) site-specific gene knock-in indicated that MMEJ has broad applicability and high-precision knock-in efficiency for genetically engineered chickens.

Elevated FBXW10 drives hepatocellular carcinoma tumorigenesis via AR-VRK2 phosphorylation-dependent GAPDH ubiquitination in male transgenic mice

Hepatocellular carcinoma (HCC), the most common liver cancer, occurs mainly in men, but the underlying mechanism remains to be further explored. Here, we report that ubiquitinated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is responsible for HCC tumorigenesis in males. Mechanistically, FBXW10 promotes GAPDH polyubiquitination and activation; VRK2-dependent phosphorylation of GAPDH Ser151 residue is critical for GAPDH ubiquitination and activation. Activated GAPDH interacts with TRAF2, leading to upregulation of the canonical and noncanonical NF-κB pathways, and increases PD-L1 and AR-VRK2 expression, followed by induction of immune evasion, HCC tumorigenesis, and metastasis. Notably, the GAPDH inhibitor koningic acid (KA) activates immune response and protects against FBXW10-driven HCC invivo. In HCC clinical samples, the expression of active GAPDH is positively correlated with that of FBXW10 and VRK2. We propose that the FBXW10/AR/VRK2/GAPDH/NF-κB axis is critical for HCC tumorigenesis in males. Targeting this axis with KA is a potential therapeutic strategy for male HCC patients.

A Systematic Study of Traditional Chinese Medicine for the Treatment of Lung Adenocarcinoma Using a Reverse Network of Key Targets Based on Bioinformatics and Molecular Docking: Curcumin and Trans-Resveratrol as Potential Drug Candidates for Lung Adenocarcinoma

Background Lung adenocarcinoma (LUAD) is a dominant tumor with high morbidity and mortality. In spite of innovations in surgery, chemotherapy, and targeted therapies, the prognosis for patients with LUAD remains unsatisfactory. Therefore, it is particularly important to study the biological markers of the occurrence, development, and prognosis of this disease. Methods In this study, 3 datasets were selected from the Gene Expression Omnibus (GEO) database to screen differentially expressed genes (DEGs). Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using Database Annotation, Visualization and Integrated Discovery (DAVID). In addition, a protein-protein interaction network was performed and 10 key genes were analyzed. Gene and protein expression were analyzed using The Cancer Genome Atlas (TCGA) and The Human Protein Atlas (HPA) databases, respectively. Then, overall survival (OS) rate was analyzed using Kaplan-Meier plotter database. The chemical components were downloaded from the HERB database. Finally, the chemical constituents were docked with 10 targets by molecular docking. By using in vitro studies, we evaluated the effects of curcumin and trans-resveratrol on the proliferation of LUAD cells. Results A total of 122 upregulated and 434 downregulated genes were identified. Then, 10 key genes (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), interleukin-1 beta (IL1B), von Willebrand factor (VWF), matrix metalloproteinase-9 (MMP9), CD44 antigen (CD44), transcription factor Jun (JUN), platelet endothelial cell adhesion molecule (PECAM1), hematopoietic progenitor cell antigen CD34 (CD34), peroxisome proliferator-activated receptor gamma (PPARG), and collagen alpha-1(I) chain (COL1A1)) were obtained. TCGA results showed that GAPDH, MMP9, and COL1A1 were upregulated, while IL-1B, VWF, CD44, JUN, PECAM1, CD34, and PPARG were downregulated in cancer tissues. Kaplan-Meier plotter analysis also showed that high expression of GAPDH, PPARG, and COL1A1, and low expression of VWF, PECAM1, CD34, PPARG, and COL1A1 were associated with poor prognosis in LUAD. Combined with gene expression, staging, immunohistochemistry, and OS prognostic analysis, the results showed that COL1A1 was a potential target for the occurrence and prognosis of LUAD. In addition, 2 key components (curcumin and trans-resveratrol) had good docking effect with COL1A1. We found that curcumin and trans-resveratrol significantly inhibited the proliferation of LUAD cells and significantly decreased the expression of COL1A1 and MMP9. Conclusion In conclusion, our study identified the potential target of LUAD and the main traditional Chinese medicine components (curcumin and trans-resveratrol) for LUAD treatment. This study provides potential therapeutic targets and treatment strategies for the treatment of LUAD.

Research on the oncogenic role of the house-keeping gene GAPDH in human tumors.

As an internal reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in glycolysis. While increasing evidence suggests that GAPDH plays a crucial role in tumorigenesis of some cancers, no systematic analysis of GAPDH has been conducted. Here, we sought to analyze the expression of GAPDH and its oncogenic processes in pan-cancer. GAPDH was investigated in The Cancer Genome Atlas (TCGA) tumor types using several bioinformatic tools including Tumor IMmune Estimation Resource (TIMER), Gene Expression Profiling Interactive Analysis (GEPIA), University of ALabama at Birmingham CANcer (UALCAN), cBio Cancer Genomics Portal (cBioPortal), and Search Tool for Recurring Instances of Neighbouring Genes (STRING) for the expression and relationships with prognosis and immune infiltration separately. Through our analysis, we measured the higher expression of GAPDH across the majority of TCGA tumors. GAPDH overexpression predicts poor survival in patients with tumors expressing a high level of GAPDH. Moreover, the genetic changes in GAPDH contributed to an increased mRNA expression. Additionally, GAPDH expression was negatively correlated with immune infiltration involving cancer-associated fibroblasts, neutrophil cell and endothelial. The house-keeping gene GAPDH might be a promising biomarker for pan-cancer prognosis. And GAPDH is not suitable as an internal reference gene for most cancer research, whether RNA or protein analyses.

GAPDH: A common housekeeping gene with an oncogenic role in pan-cancer

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is one of the most prominent housekeeping proteins and is widely used as an internal control in some semi-quantitative assays. In addition to glycolysis, GAPDH is involved in several cancer-related biological processes and has been reported to be commonly dysregulated in multiple cancer types. Therefore, its role in the physiological process of cancer needs to be urgently elucidated. Pan-cancer analysis indicated that GAPDH is ubiquitously highly expressed in most cancer types, and that patients with a high GAPDH expression of in tumor tissues have a poor prognosis. The concordance of GAPDH expression in tumors with the infiltration of immune cells and immune checkpoints implies a certain association between GAPDH and the tumor microenvironment as well as tumor development. Gene Set Enrichment Analysis revealed that GAPDH may contribute to multiple important cancer-related pathways and biological processes. Multi-omics analysis and in vitro cell experiments revealed that GAPDH overexpression is regulated by DNA copy number amplification and promoter methylation modification. Importantly, a transcription factor, forkhead box M1 (FOXM1), which is capable of regulating GAPDH expression, was also identified and was confirmed to be an oncogene and ubiquitously highly expressed in multiple cancer types. Semi-quantitative chromatin immunoprecipitation, quantitative PCR, and dual-luciferase assays showed that FOXM1 mainly binds to the promoter region of GAPDH in two cancer cell lines. The present findings revealed the implication of GAPDH in tumor development, thus bringing attention to this important molecule and casting doubts on its role as an internal reference gene in cancer studies.

Suppressing of Src-Hic-5-JNK-AKT Signaling Reduced GAPDH Expression for Preventing the Progression of HuCCT1 Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a malignant neoplasm of the bile ducts, being the second most common type of cancer in the liver, and most patients are diagnosed at a late stage with poor prognosis. Targeted therapy aiming at receptors tyrosine kinases (RTKs) such as c-Met or EGFR have been developed but with unsatisfactory outcomes. In our recent report, we found several oncogenic molecules downstream of RTKs, including hydrogen peroxide clone-5 (Hic-5), Src, AKT and JNK, were elevated in tissues of a significant portion of metastatic CCAs. By inhibitor studies and a knockdown approach, these molecules were found to be within the same signal cascade responsible for the migration of HuCCT1 cells, a conventionally used CCA cell line. Herein, we also found Src inhibitor dasatinib and Hic-5 siRNA corporately suppressed HuCCT1 cell invasion. Moreover, dasatinib inhibited the progression of the HuCCT1 tumor on SCID mice skin coupled with decreasing the expression of Hic-5 and EGFR and the activities of Src, AKT and JNK. In addition, we found a glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and several cytoskeletal molecules such as tubulin and cofilin were dramatically decreased after a long-term treatment of the HuCCT1 tumor with a high dose of dasatinib. Specifically, GAPDH was shown to be a downstream effector of the Hic-5/Src/AKT cascade involved in HuCCT1 cell migration. On the other hand, TFK1, another CCA cell line without Hic-5 expression, exhibited very low motility, whereas an ectopic Hic-5 expression enhanced the activation of Src and AKT and marginally increased TFK1 migration. In the future, it is tempting to investigate whether cotargeting Src, Hic-5 and/or GAPDH is efficient for preventing CCA progression in future clinical trials.

GAPDH mediates plant reovirus-induced incomplete autophagy for persistent viral infection in leafhopper vector

ABSTRACT Macroautophagy/autophagy is a conserved mechanism launched by host organisms to fight against virus infection. Double-membraned autophagosomes in arthropod vectors can be remodeled by arboviruses to accommodate virions and facilitate persistent viral propagation, but the underlying mechanism is unknown. Rice gall dwarf virus (RGDV), a plant nonenveloped double-stranded RNA virus, induces the formation of virus-containing double-membraned autophagosomes to benefit persistent viral propagation in leafhopper vectors. In this study, it was found that the capsid protein P2 of RGDV alone induced autophagy. P2 specifically interacted with GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and ATG4B both in vitro and in vivo. Furthermore, the GAPDH-ATG4B complex could be recruited to virus-induced autophagosomes. Silencing of GAPDH or ATG4B expression suppressed ATG8 lipidation, autophagosome formation, and efficient viral propagation. Thus, P2 could directly recruit the GAPDH-ATG4B complex to induce the formation of initial autophagosomes. Furthermore, such autophagosomes were modified to evade fusion with lysosomes for degradation, and thus could be persistently exploited by viruses to facilitate efficient propagation. GAPDH bound to ATG14 and inhibited the interaction of ATG14 with SNAP29, thereby preventing ATG14-SNARE proteins from mediating autophagosome-lysosome fusion. Taken together, these results highlight how RGDV activates GAPDH to initiate autophagosome formation and block autophagosome degradation, finally facilitating persistent viral propagation in insect vectors. The findings reveal a positive regulation of immune response in insect vectors during viral infection.

Selection of suitable candidate genes for mRNA expression normalization in bulbil development of Pinellia ternata

Pinellia ternata (Thunb.) Breit. (Abbreviated as P. ternata). It is a commonly prescribed Chinese traditional medicinal herb for the treatment of phlegm, cough, and morning sick. Bulbil reproduction is one of the main reproductive methods of P. ternata. The accurate quantification of gene expression patterns associated with bulbil development might be helpful to explore the molecular mechanism involved in P. ternata reproduction. Quantitative real-time PCR was the most preferred method for expression profile and function analysis of mRNA. However, the reference genes in different tissues of P. ternata in different periods of bulbil development have not been studied in detail. In present study, the expression stability of eight candidate reference genes were determined with programs: geNorm, NormFinder, BestKeeper, and refFinder. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as the top- rated reference gene in all samples of P. ternata, while different combinations of reference gene proved to be the most stable depending on development stage and tissue type. Furthermore, the reliability of GAPDH expression was verified by six P. ternata related genes in hormone and nutrient biosynthesis pathways, and the expression profiles of these genes were agreed with the results of RNA-seq digital gene expression analysis. These results can contribute to studies of gene expression patterns and functional analysis of P. ternata involved in bulbil development.

Comparison of endogenous development, invasion ability and apoptotic features between diclazuril resistant and sensitive strains of Eimeria tenella

Diclazuril (DIC) is widely used in the poultry industry to control coccidiosis. However, drug resistance makes it less effective, and the underlying mechanism remains unclear. One DIC-resistant E. tenella (RE) isolate and one sensitive E. tenella (SE) isolate were used to compare the differences in their endogenous development, pathogenicity, invasion-related gene expression and apoptotic characteristics. Chickens were allocated into four groups to receive RE or SE strain and their corresponding DIC treatment or not. Caeca tissues were sampled at 96 h, 120 h and 144 h post-infection (PI) for pathological analysis. Meanwhile, second-generation merozoites (Mz2) were separated at 120 h PI to detect alterations in mitochondrial membrane potential (MMP), apoptotic rate and caspase-3 activity and mRNA expression of protein phosphatase 5 (PP5), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), actin depolymerizing factor (ADF) and microneme proteins (MICs). Haematoxylin and eosin staining revealed that DIC treatment strictly blocked the development of the SE strain but slightly affected the RE strain. Meanwhile, the number of SE Mz2 and their MMP decreased at the same time the apoptotic rate increased after DIC treatment. Real-time quantitative PCR and caspase-3 activity studies demonstrated that Mz2 from the RE strain had higher mRNA expression of ADF and MICs along with no significant changes in GAPDH and caspase-3 activity under DIC pressure compared to its control; in contrast, the mRNA expression of ADF, MICs and PP5 was markedly suppressed in Mz2 from SE with upregulated caspase-3 activity and GAPDH transcription. In addition, the mRNA expression of GAPDH and PP5 in Mz2 from RE was remarkably higher than that of SE. Taken together, the higher mRNA expression of invasion-related genes and almost unaffected endogenous development provide a better understanding of coccidian resistance to DIC.

Type 2 Diabetes Induced by Changes in Proteomic Profiling of Zebrafish Chronically Exposed to a Mixture of Organochlorine Pesticides at Low Concentrations.

Effect of organochlorine pesticides (OCPs) mixtures on development of type 2 diabetes mellitus (T2DM) and the underlying mechanism, especially at protein levels, are largely unknown. We exposed a mixture of five OCPs to zebrafish at concentrations of 0, 0.05, 0.25, 2.5, and 25 μg/L for 12 weeks. Differentially expressed proteins (DEPs) were quantitatively identified in female zebrafish livers, and its functional study was conducted. The significantly high glucose and low insulin levels were observed only at 0.05 μg/L, linking to the different pattern of DEPs than other concentrations. A total of 1082 proteins was quantified, of which 321 proteins formed 6 clusters in protein dynamics analysis. The enriched pathways in cluster 3 showing distinct pattern of DEPs could explain the nonlinear response at 0.05 μg/L, indicating that OCP mixtures adversely affected proteins associated with mitochondrial function and energy metabolism. We proposed a feasible mechanism that decrease in expression of aldehyde dehydrogenase led to abnormal accumulation of aldehydes, reducing expression of glyceraldehyde 3-phosphate dehydrogenase, and resulting in disruption of glucose homeostasis. Our findings help to better understand the causality of T2DM by exposure to OCP mixtures and to identify biomarkers in the protein expression level.

Expression stability of ACTB, 18S, and GAPDH in human placental tissues from subjects with PCOS and controls: GAPDH expression is increased in PCOS.

The aim was to assess the expression stability of three commonly used reference genes, namely, β-actin (ACTB), 18S ribosomal RNA (18S), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in placental tissue obtained from pregnant women with polycystic ovary syndrome (PCOS) and healthy controls. mRNA was isolated after delivery from the placentae of 10 PCOS and 10 control women with term, uncomplicated, singleton pregnancies. The expression of ACTB, 18S, and GAPDH was analyzed using real-time polymerase chain reaction (RT-PCR). Gene expression stability was evaluated with the RefFinder, GeNorm, Normfinder, BestKeeper, and Delta-Ct tools. ACTB was ranked as the most stably expressed gene, followed by 18S. The expression of GAPDH varied considerably in both studied groups, while it was increased in PCOS versus controls (5.3-fold, p < 0.05). ACTB is an appropriate reference gene for placental gene expression studies in women with PCOS, whereas GAPDH is unfit for such a role, as its placental expression is increased in PCOS.

Optimization of Adipose Tissue Cryopreservation Techniques in a Murine Model.

Background:The aim of this study was to develop an adipose tissue (AT) cryopreservation protocol that is effective, simple, and maintains the functionality and viability of AT after thawing and transplantation.Methods:Two cryopreservation temperatures (T°), −20°C and −80°C, and two cryoprotective agents (CPAs), trehalose and hydroxyethyl starch (HES), were compared first in an experimental study, using a slowfreezing protocol. The five experimental groups were the following: (a) Fresh AT (control group), (b) T = −20°C, 10%HES, (c) T = −80°C, 10%HES, (d) T = −20°C, 0.35M trehalose, (e) T = −80°C, 0.35M trehalose. We evaluated the morphology (histological studies) and tissue viability by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genic expression. Based on the results of the preliminary study, an in vivo study was performed, choosing as cryopreservation T° −20°C. HES and trehalose were compared as cryoprotective agents and with a control group (fresh AT). AT grafts were transplanted into immunodeficient mice. After 1 month of inoculation, animals were euthanized and samples were recovered. Samples were weighted and processed for histological study, viability study (GAPDH genic expression), and vascularization study (VEGF genic expression).Results:The initial histological study demonstrated that all AT cryopreserved group samples showed typical histological features of AT, similar to that of the control group. Statistically significant differences were not observed (P > 0.05) in GAPDH expression between different groups related to temperature or CPA. Referring to the in vivo studies, cryopreserved groups showed good take of the graft and normal AT architectural preservation, as well as a clear vascular network. Statistically significant differences were not found (P > 0.05) with regard to graft take (%), GAPDH, or VEGF expression.Conclusion:Slow freezing at −20°C using trehalose, and −20°C using HES as cryoprotective agents are both straightforward and easy AT cryopreservation procedures, with results similar to those of fresh AT in terms of tissue viability and morphohistological characteristics.

The Protective Effect of Panax notoginseng Mixture on Hepatic Ischemia/Reperfusion Injury in Mice via Regulating NR3C2, SRC, and GAPDH.

Panax notoginseng mixture (PNM) has the characteristics of multicomponent, multitarget, and multieffect, which can cope with the multidirectional and multidimensional complex pathological process caused by hepatic ischemia/reperfusion injury (HIRI). Our animal experiments showed that PNM composed of notoginseng, dogwood, and white peony root could significantly reduce the level of aspartate transaminase and alanine aminotransferase in the blood of mice with HIRI, indicating that this preparation had a protective effect on HIRI in mice. Therefore, on this basis, the molecular mechanism of PNM intervention in HIRI was further explored by network pharmacology. First, target genes corresponding to active components and HIRI were obtained through databases such as TCMSP, Pharm Mapper, Swiss Target Prediction, GeneCards, and so on. All target genes were standardized by Uniprot database, and a total of 291 target genes with their intersection were obtained. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological processes (BPs) of 291 target genes were obtained through the online public platform of DAVID. A total of 177 KEGG pathways and 337 BPs were obtained by setting p < 0.01 and false discovery rate <0.05. The network mapping map of components and disease targets was drawn by Cytoscape, and the top 10 Hub target genes related to HIRI were obtained. At the same time, the String database was used to obtain the protein–protein interaction dataset, which was imported into Cytoscape, and the first 10 Hub target genes were obtained. The Hub target genes obtained by the above two methods were molecular docking with their corresponding small molecule compounds through DockThor online tool. The results showed that the docking of paeoniflorin with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), paeoniflorin and loganin with SRC, ginsenoside Rb1 with NR3C2, ursolic acid and oleanolic acid with IL-6, paeoniflorin docking VEGFA, and MMP9. Finally, NR3C2, SRC, and GAPDH were identified as target genes in this study by referring to relevant literature reports. After verification by immunohistochemical experiments, compared with the sham group, the above three target genes were highly expressed in the HIRI group (p < 0.01). Compared with the HIRI group, the expression of three target genes in the PNM + HIRI group was significantly decreased (p < 0.01). The results showed that PNM could protect mouse HIRI by decreasing the expression of NR3C2, SRC, and GAPDH.

Glyceraldehyde-3-Phosphate Dehydrogenase Facilitates Macroautophagic Degradation of Mutant Huntingtin Protein Aggregates

Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector-transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

Effect of MG‐132 on myofibrillogenesis and the ubiquitination of GAPDH in quail myotubes

In the three-step myofibrillogenesis model, mature myofibrils are formed through two intermediate structures: premyofibrils and nascent myofibrils. We have recently reported that several inhibitors of the Ubiquitin Proteosome System, for example, MG-132, and DBeQ, reversibly block progression of nascent myofibrils to mature myofibrils. In this investigation, we studied the effects of MG132 and DBeQ on the expression of various myofibrillar proteins including actin, myosin light and heavy chains, tropomyosin, myomesin, and myosin binding protein-C in cultured embryonic quail myotubes by western blotting using two loading controls-α-tubulin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Surprisingly, we found that MG-132 affected the level of expression of GAPDH but DBeQ did not. Reverse transcription polymerase chain reaction (RT-PCR) and quantitative reverse transcription-PCR (qRT-PCR) showed no significant effect of MG-132 on GAPDH transcription. Two-dimensional (2D) western blot analyses with extracts of control and MG-132-treated cells using anti-ubiquitin antibody indicated that MG132-treated myotubes show a stronger emitter-coupled logic signal. However, Spot% and Spot volume calculations for all spots from both western blot film signals and matched Coomassie-stained 2D polyacrylamide gel electrophoresis showed that the intensity of staining in a spot of ~39 kDa protein is 3.5-fold lower in the gel of MG-132-treated extracts. Mass spectrometry analyses identified the ~39 kDa protein as quail GAPDH. Immunohistochemical analysis of fixed MG-132-treated myotubes with anti-GAPDH antibody showed extensive clump formation, which may be analogous to granule formation by stress response factors in MG132-treated cells. This is the first report on in vivo ubiquitination of GAPDH. This may be essential for the moonlighting (Jeffery, 1999) activity of GAPDH for tailoring stress in myotubes.