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Soil salinity stress causes osmotic/ionic imbalances and induces oxidative stress that causes cellular structure damage, perturbs metabolism, antioxidant system (comprising enzymatic and non-enzymatic components) and hence inhibits plant growth performance. In this study, we used genome-wide association scan (GWAS) in 174 diverse spring barley accessions which were exposed to salt stress under field conditions at the vegetative stage to uncover the genetic basis of antioxidant components and agronomic traits. High activities of enzymatic and content of non-enzymatic antioxidants were observed under salt stress compared to control conditions. Under salt stress, all the agronomic and yield-related traits were significantly reduced. Six genomic regions were associated with antioxidants and agronomic traits under salt stress conditions which were found to be linked with candidate genes. Several significant associations were physically located inside or near genes which are potentially involved in antioxidants. Two candidate genes at 2H (40,659,364 bp) and 7H (416,743,127 bp) were found to be involved in Dihydroflavonol 4-reductase/flavanone protein and Glyceraldehyde-3-phosphate dehydrogenase, respectively. The allelic variation at SNP of BK_07 at 7H inside the GAPDH gene demonstrates a negative selection of accessions carrying A allele. This allele appears in cultivars with lower activity of enzymatic antioxidants e.g. superoxide dismutase and catalases under salt stress conditions. These accessions are predominantly two-rowed, cultivars, originated from Europe, and carrying photoperiod sensitive alleles. The detected associated molecular markers in this work are considered as an important source for selection of increased amount of antioxidant compounds in barley under stress conditions.

To understand the fundamental mechanisms on genes in plant cells can be performed by gene expression studies. The study needs reference genes that act as internal control to avoid expression data bias. Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene is one of reference genes that has been validated in plants. This study aims to analyze the DNA sequences encoding GAPDH in cocor bebek (Kalanchoe x laetivirens). Methods included total DNA extraction from fresh leaf, polymerase chain reaction (PCR), electrophoresis at 1% agarose and DNA sequence data analysis. The DNA sequence of K. x laetivirens GAPDH gene obtained were 1107 bp and predicted to consist of 5 exons and 5 introns. This sequence had about 68.96%-73.81% similarity to the DNA sequence of the GAPDH gene in several plant species. The amino acid sequence of K. x laetivirens GAPDH had 5 conserved regions and residues of cysteine (C) and histidine. The sequence obtained in this study was the first to be reported from the genus Kalanchoe. This sequence can be used for analysis of gene expression in K. x laetivirens and can be as reference for isolating the GAPDH gene for other plant species in the genus Kalanchoe.

Molecular markers are fast, reliable and robust for early detection of pathogens. They need unique stable regions specific to pathogens for developing good diagnostics. The multiple sequence alignment and phylogenetic studies of conserved loci such as Internal Transcribed Spacer (ITS), Translation elongation factor 1α (TEF-1α), β-tubulin gene and glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of wheat pathogens identified intergeneric (5–30%) and intrageneric (2–24%) variability with multiple sequence polymorphism (5–250 bp) for selection of pathogen-specific regions. ITS region precisely showed specificity for Tilletia indica and storage pathogens (Aspergillus spp., Penicillium spp. and Fusarium spp.), while TEF-1α gene distinctly differentiated 5 Fusarium species with high potential (10%) and found highly suitable for developing qPCR or LAMP assays for detection of Fusaruim head blight disease. β-tubulin gene showed, enormous variabilities among species of Puccinia (15%) and Aspergillus (23%) for developing their diagnostics. GAPDH gene with the highest intergenic sequence variability (> 30%) found suitable for developing markers at generic level. Further, it has differentiated different species of Aspergilli and Bipolaris. The variability among different pathogens enabled the selection and development of appropriate diagnostic marker such as, PCR, qPCR, RPA, LAMP assay, macro- or micro-array. As a proof of concept, a PCR-based diagnostics (MN754026) for Tilletia indica was developed, which amplified a band of 148 bp only in Tilletia indica and detected as little as 10 pg of DNA. Developed maker has tremendous potential for timely management of this quarantined pathogen.

Aims Acidithiobacillus caldus is an important sulphur-oxidizing bacterium that plays crucial roles in the bioleaching industry. This study aims to analyse the optimal reference gene for real-time quantitative PCR (RT-qPCR) under different conditions and investigate the transcription levels of the sigma factor genes in the stress response. Methods and results We selected six housekeeping genes and analysed them via RT-qPCR using two energy resources, under four stress conditions. Three statistical approaches BestKeeper, geNorm, and NormFinder were utilized to determine transcription stability of these reference genes. The gapdH gene was the best internal control gene using elemental sulphur as an energy resource and under heat stress, map was the best internal control gene under pH and osmotic stress, era was the best internal control gene for the K2 S4 O6 energy resource, and rpoC was the best internal control gene under Cu2+ stress. Furthermore, the expressional levels of 11 sigma factors were analysed by RT-qPCR in the stress response. Conclusions Stable internal control genes for RT-qPCR analysis of A. caldus were determined, and the expression patterns of sigma factor genes of A. caldus were investigated. Significance and impact of the study The identification of the optimal reference gene and analysis of transcription levels of sigma factors in A. caldus can provide clues for reference gene selection and the study of sigma factor function.

Many recent studies have been focused on prevalence and impact of two helminth parasites, eyeworm Oxyspirura petrowi and caecal worm Aulonocephalus pennula, in the northern bobwhite quail (Colinus virginianus). However, few studies have attempted to examine the effect of these parasites on the bobwhite immune system. This is likely due to the lack of proper reference genes for relative gene expression studies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme that is often utilized as a reference gene, and in this preliminary study, we evaluated the similarity of bobwhite GAPDH to GAPDH in other avian species to evaluate its potential as a reference gene in bobwhite. GAPDH was identified in the bobwhite full genome sequence and multiple sets of PCR primers were designed to generate overlapping PCR products. These products were then sequenced and then aligned to generate the sequence for the full-length open reading frame (ORF) of bobwhite GAPDH. Utilizing this sequence, phylogenetic analyses and comparative analysis of the exon–intron pattern were conducted that revealed high similarity of GAPDH encoding sequences among bobwhite and other Galliformes. Additionally, This ORF sequence was also used to predict the encoded protein and its three-dimensional structure which like the phylogenetic analyses reveal that bobwhite GAPDH is similar to GAPDH in other Galliformes. Finally, GAPDH qPCR primers were designed, standardized, and tested with bobwhite both uninfected and infected with O. petrowi, and this preliminary test showed no statistical difference in expression of GAPDH between the two groups. These analyses are the first to investigate GAPDH in bobwhite. These efforts in phylogeny, sequence analysis, and protein structure suggest that there is > 97% conservation of GADPH among Galliformes. Furthermore, the results of these in silico tests and the preliminary qPCR indicate that GAPDH is a prospective candidate for use in gene expression analyses in bobwhite.

In this study, transgenic Arabidopsis lines expressing a potato gene (D43), encoding Glyceraldehyde 3-phosphate dehydrogenase, were studied. The D43 plants exhibited improved morphological parameters and accumulation of photosynthetic pigments compared to wild-type (WT) plants under salinity stress conditions. In addition, the D43 transgenic plants showed significantly reduced electrolyte leakage, higher stomatal conductance, lower malondialdehyde (MDA) content, and higher proline content than the WT plants under salinity stress. The gene expression analysis showed that the D43 plants accumulated 1.7-fold, 2.2-fold, and 1.3-fold higher mRNA transcripts of genes encoding the antioxidant enzymes ascorbate peroxidase (APX), superoxide dismutase (SOD), and catalase (CAT), respectively under salt-stress conditions. Furthermore, they significantly altered the expression of seven major stress-responsive genes, which indicated that overexpression of the potato D43 gene gave salinity stress resistance to Arabidopsis. Chlorophyll-a fluorescence kinetics confirmed the efficient photon absorption, electron transport, and overall PSII efficiency that led to improved photosynthesis in the D43 plants subjected to NaCl-induced salinity stress. Overall, our findings have suggested that potato D43 is a potential candidate gene for developing salinity stress resistance in higher plants.

Gen glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) merupakan salah satu gen referensi yang sering bertindak sebagai kontrol internal pada analisis ekspresi gen di beberapa spesies tumbuhan. Penelitian ini bertujuan menganalisis sekuen gen GAPDH parsial pada sirsak ( Annona muricata L.). Metode meliputi persiapan sampel tanaman, isolasi DNA total menggunakan Genomic DNA mini kit Plant ( Geneaid ), amplifikasi gen GAPDH dengan teknik polymerase chain reaction (PCR), elektroforesis pada 1% gel agarose dan analisis data sekuen DNA. Studi ini telah memperoleh sekuen DNA dari gen GAPDH parsial sirsak sepanjang 961 pb. Sekuen tersebut memiliki kemiripan sekitar 68,93–84,35% dengan sekuen mRNA gen GAPDH pada beberapa spesies tumbuhan. Sekuen ini diprediksi terdiri dari 5 ekson dan 4 intron. Total ekson diprediksi terdiri dari 429 pb. Sekuen ini adalah yang pertama kali dilaporkan dari genus Annona dan juga dari famili Annonaceae . Sekuen ini dapat dimanfaatkan untuk analisis ekspresi gen pada sirsak dan dapat menjadi dasar untuk mengisolasi gen GAPDH spesies lain di dalam genus Annona dan famili Annonaceae . Abstract GAPDH ( glyceraldehyde-3-phosphate dehydrogenase) gene is one of reference genes that is frequently became an internal control in any plant species. This study reports a DNA sequence of parsial GAPDH gene on soursop ( Annona muricata L. ). Methods included sample preparation, total DNA isolation using Genomic DNA mini kit Plant ( Geneaid ) , amplification of GAPDH gene using PCR (polymerase chain reaction) technique, electrophoresis using 1% agarose gel and data analysis. This study had been obtained the DNA sequence of soursop partial GAPDH gene sizing 961 bp. The sequence had 68.93 – 84.35% similarity to GAPDH mRNA of some plants species. The soursop partial GAPDH gene was predicted consisting of 5 exons and 4 introns. The total exons length was 429 bp. The sequence is the first reported from Annona genus and also Annonaceae family . The sequence can be used for gene expression in soursop and also can be used to isolate GAPDH gene of other species in Annona genus and Annonaceae family .

Silver nanoparticles (AgNPs) are widely used in medicine, however, the underlying mechanisms of their action on cellular signaling have not been completely determined, and fundamental studies are required to clarify them. We aimed to investigate AgNPs effects on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as both the internal control gene and the redox-sensitive enzyme involved in apoptosis-related pathways and the formation of amyloid aggregates. To achieve this purpose, MCF-7 cells were treated with different concentrations (0, 3, 22, and 200 μg/ml) of AgNPs and then cell viability, generation of reactive oxygen species (ROS), induction of apoptosis, expression of GAPDH gene, the formation of amyloid aggregates, and GAPDH activity were assessed. The results indicated that treatment with AgNPs significantly reduced cell viability and increased apoptosis in a dose-dependent manner. The ROS levels increased at lower concentrations of AgNPs (up to 22 μg/ml) and during short-term exposure (30 min). The level of GAPDH gene expression was significantly upregulated by 1.22, 1.47, and 1.56 fold, in the concentrations of 3, 22, and 200 μg/ml, respectively. The amount of amyloid aggregates was significantly increased in a dose-dependent manner. The results of enzyme activity showed that AgNPs were affected on the activity of GAPDH protein, however, it has fluctuated that could not be interpreted by our limited data. In conclusion, our results suggested that AgNPs could affect the GAPDH gene expression and enzyme activity, therefore the selection of GAPDH as a gene and protein internal control in the (AgNPs)-related studies requires careful consideration. Additionally, AgNPs may cause apoptosis due to the increase in the production of amyloid aggregates.

Bipolaris species are important plant pathogens with a worldwide distribution in tropical and temperate environments. Species recognition in Bipolaris has been problematic due to a lack of molecular data from ex-type cultures, the use of few gene regions for species resolution and overlapping morphological characters. In this study, we evaluate the efficiency of different DNA barcodes in species delimitation in Bipolaris by phylogenetic analyses, Automatic Barcode Gap Discovery and Objective Clustering. GAPDH is determined to be the best single marker for the genus. These approaches are used to clarify the taxonomic placement of all sequences currently named as Bipolaris in GenBank based on ITS and GAPDH gene sequence data. In checking various publications, we found that the majority of new host records of fungal species published in the Plant Disease journal from 2010 to 2019 were based on BLAST searches of the ITS sequences and up to 82% of those records could be erroneous. Therefore, relying on BLAST searches from GenBank to name species is not recommended. Editorial boards of journals and reviewers of new record papers should be aware of this problem. In naming Bipolaris species, whether new or known, it is recommended to perform phylogenetic analyses based on GAPDH using the correct taxon sampling for accurate results and the species relationship should have reliable statistical support. At least two new species are represented by molecular data in GenBank and we provide an updated taxonomic revision of Bipolaris. We accept 45 species in Bipolaris and notes are provided for all the species including hosts and geographic distribution.

Anaplasma marginale, A. ovis, and A. phagocytophilum are the causative agents of bovine anaplasmosis, ovine anaplasmosis, and granulocytic anaplasmosis, respectively. The gold standard for diagnosis of post-acute and long-term persistent infections is the serological cELISA, which does not discriminate between Anaplasma species and requires highly equipped laboratories and trained personnel. This study addresses the development of a rapid, isothermal, sensitive, species-specific RPA assays to detect three Anaplasma species in blood and cELISA A. marginale-positive serum samples. Three RPA primer and probe sets were designed targeting msp4 genes of each Anaplasma species and the internal control (GAPDH gene) for each assay. The limit of detection of gel-based or RPA-basic assays is 8.99 × 104 copies/µl = A. marginale, 5.04 × 106 copies/µl = A. ovis, and 4.58 × 103 copies/µl = A. phagocytophilum, and for each multiplex lateral flow or RPA-nfo assays is 8.99 × 103 copies/µl of A. marginale, 5.04 × 103 copies/µl of A. ovis, 4.58 × 103 copies/µl of A. phagocytophilum, and 5.51 × 103 copies/µl of internal control (GAPDH). Although none of the 80 blood samples collected from Oklahoma cattle were positive, the RPA-nfo assays detected all A. marginale cattle blood samples with varying prevalence rates of infection, 83% of the 24 cELISA A. marginale-positive serum samples, and all A. phagocytophilum cell culture samples. Overall, although early detection of three Anaplasma species was not specifically addressed, the described RPA technique represents an improvement for detection of three Anaplasma in regions where access to laboratory equipment is limited.

Heptelidic acid (HA), a sesquiterpene lactone, is a known inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Recently, we found that HA was produced by Aspergillus oryzae RIB40 and acted as the growth inhibitor of the salt-tolerant lactic acid bacteria in soy sauce brewing. Although several decades have passed since the discovery of HA, the genes involved in its biosynthesis and biosynthetic pathway have not yet been fully identified. In this study, we identified the HA biosynthetic gene cluster (HA cluster) using gene disruption and expression analysis. We also revealed that two transcription regulatory genes adjacent to the HA cluster were responsible for the expression of HA biosynthetic genes in A. oryzae. Interestingly, the HA cluster contained a gene encoding GAPDH (gpdB), which showed much higher resistance to HA than the GAPDH gene (gpdA) located at the other locus, but which did not seem to act as a self-resistant gene.

We evaluated the effect of the Schistosoma japonicum antigen of glyceraldehyde-3-phosphate dehydrogenase (SjGAPDH) on the growth of schistosomula. Quantitative real-time PCR and immunohistochemical analysis were performed to evaluate the level and immunolocalization of SjGAPDH mRNA and protein, respectively. RNA inference was conducted to further study the role of SjGAPDH on the schistosomula growth of S. japonicum. The results demonstrated that SjGAPDH mRNA was expressed during all stages of S. japonicum development, during which it gradually increased over time. SjGAPDH protein was mainly distributed on the surface and in some parenchyma of S. japonicum. Double-stranded RNA-mediated GAPDH gene knockdown reduced SjGAPDH mRNA transcription by more than 90%, and western blot analysis showed that SjGAPDH protein expression was decreased by more than 60%. Light microscopy observation revealed that the size of schistosomula including the length, width, volume, and area of schistosomula in the SjGAPDH interference group was significantly lower than those in the enhanced green fluorescent protein control group. Thus, SjGAPDH may be involved in the growth of S. japonicum schistosomula and a useful target for treating schistosomiasis.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

The results of past research studies show that platelets are one of the main sources of reactive oxygen species (ROS) and reactive nitrogen species (RNS) to be found in the course of many pathological states. The aim of this study was to determine the level of oxidative/nitrative stress biomarkers in blood platelets obtained from multiple sclerosis (MS) patients (n=110) and to verify their correlation with the clinical parameters of the psychophysical disability of patients. The mitochondrial metabolism of platelets was assessed by measuring the intracellular production of ROS using the fluorescence method with DCFH-DA dye and by identification of changes in the mitochondrial membrane potential of platelets using the JC-1 dye. Moreover, we measured the mRNA expression for the gene encoding the cytochrome c oxidase subunit I (MTCO-1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in platelets and megakaryocytes using the RT-qPCR method, as well as the concentration of NADPH oxidase (NOX-1) by the ELISA method. Our results proved an increased level of oxidative/nitrative damage of proteins (carbonyl groups, 3-nitrotyrosine) (p<0.0001) and decreased level of -SH in MS (p<0.0001) and also a pronounced correlation between these biomarkers and parameters assessed by the Expanded Disability Status Scale and the Beck’s Depression Inventory. The application of fluorescence methods showed mitochondrial membrane potential disruption (p<0.001) and higher production of ROS in platelets from MS compared to control (p<0.0001). Our research has also confirmed the impairment of red-ox metabolism in MS, which was achieved by increasing the relative mRNA expression in platelets for the genes studied (2-fold increase for theMTCO-1gene and 1.5-fold increase inGAPDHgene,p<0.05), as well as the augmented concentration of NOX-1 compared to control (p<0.0001). Our results indicate that the oxidative/nitrative damage of platelets is implicated in the pathophysiology of MS, which reflects the status of the disease.

Schistosomes are intravascular blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic analysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNA interference significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to ∼8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in Escherichia coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite's hosts, and has a Michaelis–Menten constant for glyceraldehyde-3-phosphate (GAP) of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thrombolysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade blood clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo. This article has an associated First Person interview with the first author of the paper., Summary: This work provides evidence of a novel function for a blood fluke's glycolytic enzyme (GAPDH) activating plasmin to potentially degrade blood clots, thus aiding parasite survival in the host vasculature.

Haemaphysalis flava (Acari: Ixodidae) harbors pathogenic microorganisms and transfers these to hosts during blood feeding. Proteomic analysis in the midgut contents of H. flava detected glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and contig 1683 was retrieved as a GAPDH gene fragment by searching our previous transcriptomic library. In the study, the 5' and 3' ends of contig 1683 were cloned by rapid amplification of cDNA ends (RACE) and a full length, 1340 bp cDNA of Hf-GAPDH was obtained. The open-reading frame had 999 bp and coded for 333 amino acids. Hf-GAPDH was predicted to have an N-terminal NAD binding domain and a C-terminal glyceraldehyde dehydrogenase catalytic domain. The molecular structure of Hf-GAPDH was analyzed and the evolutionary relationship also established. The GAPDH protein sequence was conserved among ticks. The expression pattern of Hf-GAPDH, analyzed by real-time PCR, significantly differed among life phases, feeding stages, and tissues. As the ticks grew, the expression level of Hf-GAPDH was up-regulated. The expression levels of Hf-GAPDH in salivary glands and midguts from half-engorged ticks were lower than the same tissues from engorged ticks. This study will provide reference data for the follow-up verification of the GAPDH-related function and the feasibility as a potential anti-tick vaccine.

The housekeeping gene encoding β-actin appears to be the most widely-used internal reference for gene expression studies in experimental animals or their cell lines. However, the effectiveness of β-actin to normalize mRNA levels expression in many crustacean species is still object of debate. To date, it is still unclear if β-actin is suitable to be utilized as the internal reference in qualitative real-time gene expression study in crab species. To address this concern, we evaluated 5 candidate reference genes encoding β-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A, elongation factor 1-α (EF1-α), and 18 S ribosomal RNA (18 S rRNA) in the swimming crabs (Portunus trituberculatus) models. Our data showed that the β-actin gene expression varied significantly across individual swimming crab individuals in gills or hemocytes and the expression of 18 S rRNA, EF1-α, cyclophilin or GAPDH gene were relatively stable compared to that of β-actin. Moreover, the expression stability of the reference genes among different tissues in normal crabs or after WSSV challenge was also tested by geNorm and NormFinder software. Among tissues, 18 S rRNA was most stably expressed in different tissues, followed by cyclophilin A and EF1-α, compared to β-actin and GAPDH. Upon to viral simulation, GAPDH was found to be the most stable internal control gene in gills and cyclophilin A was ranked as the most stable gene in hemocytes.

Background Development of Parkinson’s disease (PD) is attributed to both genetic and environmental factors. Furthermore,GAPDH may play a key role in the development of neurodegenerative disease. Examination of genetic polymorphism in patients with sporadic PD will help uncover the mechanisms of PD pathogenesis and provide new insights into the treatment of PD. Methods and results The SNaPshot method was applied to determine the gene sequences in 265 patients with idiopathic PD and 269 control cases (sex- and age-matched). The rs1136666 polymorphism of GAPDH was determined to be closely associated with PD. Subsequently, the CC genotype of the rs1136666 fragment was transfected into SH-SY5Y cells via a plasmid. The genetic expression of rs1136666 CC could induce SH-SY5Y cell injury and apoptosis via regulation of the oxidant–antioxidant and apoptosis–antiapoptosis balance. rs1136666 CC of the GAPDH had a pro-apoptotic effect similar to that of rotenone, and combination of the rs1136666 CC genetic variation and the rotenone neurotoxic effect could aggravate oxidative stress, cell injury, and apoptosis better than either single treatment alone. Conclusion This study confirmed that the rs1136666 CC allele of theGAPDH increased the risk of PD, particularly in older male patients.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the sixth step of glycolysis, and is also known to perform other (moonlighting) activities in animal cells. We have earlier identified an additional GAPDH gene in Trichoderma virens genome. This gene is consistently associated with the vir cluster responsible for biosynthesis of a range of volatile sesquiterpenes in Trichoderma virens. This gene is also associated with an orthologous gene cluster in Aspergillus spp. Both glycolytic GAPDH and the vir cluster-associated GAPDH show more than 80% similarity with essentially conserved NAD+ cofactor- and substrate-binding sites. However, a conserved indel is consistently present only in GAPDH associated with the vir cluster, both in T. virens and Aspergillus spp. Using gene knockout, we demonstrate here that the vir cluster-associated GAPDH is involved in biosynthesis of volatile sesquiterpenes in T. virens. We thus, for the first time, elucidate the non-glycolytic role of a GAPDH in a fungal system, and also prove for the first time that a GAPDH, a primary metabolism protein, is involved in secondary metabolism.

Reproducible and stable transgene expression is an important goal in both basic research and biotechnology, with each application demanding a range of transgene expression. Problems in achieving stable transgene expression include multi-copy transgene silencing, chromosome-position effects, and loss of expression during long-term culture, induced cell quiescence, and/or cell differentiation. Previously, we described the “BAC TG-EMBED” method for copy-number dependent, chromosome position-independent expression of embedded transgenes within a BAC containing ~170 kb of the mouse Dhfr locus. Here we demonstrate wider applicability of the method by identifying a BAC and promoter combination that drives reproducible, copy-number dependent, position-independent transgene expression even after induced quiescence and/or cell differentiation into multiple cell types. Using a GAPDH BAC containing ~200 kb of the human GAPDH gene locus and a 1.2 kb human UBC promoter, we achieved stable GFP-ZeoR reporter expression in mouse NIH 3T3 cells after low-serum induced cell cycle arrest or differentiation into adipocytes. More notably, GFP-ZeoR expression remained stable and copy-number dependent even after differentiation of mouse ESCs into several distinct lineages. These results highlight the potential use of BAC TG-EMBED as an expression platform for high-level but stable, long-term expression of transgene independent of cell proliferative or differentiated state.

DNA-based vaccination or genetic immunization is one of the most promising, effective and prophylactic measures to control aquatic animal diseases. This immunization strategy involves administration of eukaryotic antigen expression vectors (DNA vaccine) into the host that encode for an antigen under the control of a eukaryotic promoter which resulted into elicitation of strong cellular and humoral immune responses. In the present study, a bicistronic DNA vaccine (designated as pGPD+IFN) was constructed which contains an additional immune adjuvant gene (Interferon gamma gene of Labeo rohita) along with a regular antigenic gene (glyceraldehyde-3-phosphate dehydrogenase gene of Edwardsiella tarda) with the purpose to maximize the protective efficacy of the vaccine against E. tarda infection. After construction of the vaccine a pilot study was orchestrated in vitro to ascertain the positive co-expression of the dual genes in vaccine-transfected SSN-1 cell line. Successful co-expression of GAPDH and IFN-γ genes in the transfected cell were confirmed by Western blot and RT-PCR respectively. Further, an in vivo vaccine trial was conducted in which rohu (L. rohita) fingerlings were intramuscularly (I/M) injected (initial and booster immunised) with two DNA constructs one group with pGPD+IFN and the other with pGPD (containing GAPDH gene only) and challenged with E. tarda (1 × 105 CFU/fish) at 35 day post-initial vaccination. The protective immune responses were determined in terms of relative percentage survival (RPS), specific antibody production, non-specific immune response and expression kinetics of immune-related iNOS gene. Evaluation of RPS analysis revealed that pGPD+IFN group recorded highest RPS of 63.16% while the pGPD vaccinated group showed 47.37% when compared with 63.33% cumulative mortality of control group. The results regarding respiratory burst activity, myeloperoxidase activity as well as antibody titre also showed pGPD+IFN group with highest activities at all the time points. Furthermore, the current study displayed pGPD+IFN group having significant (p

In molecular biology, real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) has been commonly used for the evaluation of gene expression in human cells and tissues. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene is commonly used as a reference gene for the analysis of RT-qPCR experiments. However, recent studies have recognized several variants of GAPDH gene transcripts in human tissues. Thus, the application of the GAPDH gene in RT-qPCR amplification was also established with false interpretations of differential gene expression analysis. Thereof, a consistent amplification of reference gene transcript is essentially required from various tissues or cells treated under differential conditions. In this study, we designed a set of oligo primers for consistent amplification of the human GAPDH transcripts as a reference for human gene amplification. The designed primers for the GAPDH gene showed equalized amplification of GAPDH transcripts from human fibroblasts cells for differential gene expression analysis under various experimental conditions.

Background Estimation of the postmortem interval (PMI) is a critical issue in forensic science. Various approaches have been used to determine the PMI including physical, biochemical and entomological methods. Most of these methods have practical limitations or provide insufficient results in certain conditions. Postmortem degradation of RNA may be a useful tool for PMI estimation if there is a correlation between the quantity of residual RNA and the elapsed time. This study aimed to evaluate the use of GAPDH mRNA quantity in the brain as a possible indicator for PMI in different environmental conditions. Methods Seventy-eight adult female albino rats were sacrificed by cervical dislocation. Rats were divided into five groups, the control group and 4 studied groups left after sacrificing in different conditions (ambient air at 30 °C and at 6 °C, buried in sand and submerged under water). Brain samples were obtained at different intervals (0, 24, 48 and 96 h postmortem). The mRNA of GAPDH gene of rats’ brain was quantitatively detected by qRT-PCR. Results The decrease of GAPDH mRNA levels with increasing PMI were observed in all study groups. There were significant negative correlations between brain GAPDH mRNA and Time intervals in rats left in air at 30 °C, buried in sand and recovered from the water. Conclusion GAPDH mRNA in rat ’s brain could be a useful marker for PMI estimation in several environmental conditions.

This study has been able to determine the physiochemical properties, secondary and tertiary structure, and phylogenetic analysis of GAPDH among domestic animals under in silico platform. Eighteen nucleotide and protein sequence of GAPDH gene of different mammalian species were retrieved from National Centre for Biotechnology information (NCBI). The percentage of identity and similarity was done by Basic Local Alignment Search Tool (BLAST), physiochemical properties were analyzed by ExPASy”s ProtParam tool, the secondary and 3-D structure was predicted by GOR IV and Swiss modeling respectively. Phylogenetic analysis among the animals was done by Molecular Evolutionary Genetics Analysis. It was found that the percentage of identity and similarity among all animals were almost more than 90%. The physiochemical analysis showed this protein is very stable, hydrophilic and intracytoplasmic in nature. The secondary structure analysis showed that GAPDH has more number of random coil (49.85%) Extended strand (27.93%), alpha helix (22.22%) of the protein. The QMEAN Z score was found 0.33 under protein modeling which interfered that this protein is of comparable quality. The phylogenetic analysis of this gene showed that the highest time of divergence occurred between sheep and common chimpanzee but least time of divergence observed between killer whale and dolphin. So it can be concluded that the GAPDH gene is highly conserved along all animal species.

Objective: To evaluate the effect of imatinib mesylate on cell viability, anti cancer effect through modulation of KAI1/CD82 gene expression in breast cancer MCF-7 cell line. Methods: The effects of imatinib mesylate on cell viability in MCF-7 cell line were assessed using MTT assay and IC50 value was determined. GAPDH and KAI1/CD82 were selected as reference and target genes, respectively. Quantitative real time PCR technique was applied for investigation of KAI1/CD82 gene expression in human breast cancer MCF-7 cells. Subsequently, the quantity of KAI1 compared to GAPDH gene expressions were analyzed using the formula; 2−ΔΔCt. Results: Imatinib was showed to have a dose-dependent inhibitory effect on the viability of MCF-7 cells. CD82/GAPDH gene expression ratios were 1.322 ± 0.030 (P > 0.05), 2.052 ± 0.200 (P

Breast cancer has been increasing in developing countries; this cancer is becoming a fundamental health problem for these countries. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classical glycolytic enzyme correlated with the hormonal receptor concentrations, cell proliferation, tumor grade and stage, drug resistance, and outcome of breast cancer cells. Furthermore, genetic variations in miRNA binding sites could be played an essential role in breast cancer risk and survival. In this study, the SNP rs1803622 at the 3′-UTR of GAPDH was chosen as a candidate SNP for investigation based on bioinformatics studies. This polymorphism was genotyped in an Iranian population with 123 breast cancer patients and 117 matched healthy controls using the Allele-Specific Primer (ASP)-PCR method. The functional impact of this genetic variation on the post-transcriptional regulation of GAPDH gene were bioinformatically studied. The genotyping data, for the first time, successfully identified frequencies of GG, GT, and TT to be 7%, 61%, and 32% in breast cancer patients, respectively. In particular, the assessed frequencies of allele G was 38% while risk allele T was 62% in these population. Results revealed that compared with the GG genotype of rs1803622, T allele was related to a significantly increased risk of breast cancer in a dominant genetic model Significant difference in allele and genotype distributions in the SNP rs1803622 was found between the high and low expression levels of estrogen, progesterone, and HER2 receptors. Furthermore, allele and genotype frequencies in the SNP rs1803622 were also related to the stage and grade of breast cancer. Surprisingly, Genotype and allele frequencies in this SNP were also significantly different in metastatic and non-metastatic breast cancer samples. Finally, the bioinformatics data suggested that the SNP rs1803622 might be involved in transcriptional regulation of the GAPDH gene by hsa-miR-548g.

Patchouli (Pogostemon cablin) is an economically important aromatic plant and is often vulnerable to nematode attack (Meloidogyne incognita). The application of PGPR has tremendously changing disease management approach in plants and a systematic understanding the molecular mechanism behind it is needed to control over the disease more precisely. Since, qRT-PCR is the most frequently used molecular tools in studying molecular mechanism but is greatly dependent upon suitable reference or housekeeping genes. This study is aim to select suitable reference genes for qRT-PCR experiments in patchouli under the condition of nematode attack and PGPR treatments. Expression pattern of six reference genes (Actin, EF1, ACP, COX3, GAPDH and Pat18S) were statistically analyzed and validated using different algorithms viz., ΔCt, BestKeeper, GeNorm, NormFinder and RefFinder. The result showed that the Actin and ACP are the most suitable reference genes and the combination of ACP & GAPDH gene pairs can also be used whenever the experiment needed.

Fasciola gigantica is an important food-borne trematode responsible for the hepatobiliary disease, commonly known as fascioliasis. In F. gigantica, the glyceraldehyde 3-phosphate dehydrogenase (FgGAPDH) is a key enzyme of the glycolytic pathway and catalyzes the reversible oxidative phosphorylation of d-glyceraldehyde-3-phosphate (G-3-P) to 1,3-bisphosphoglycerate (1,3-BPG), with the simultaneous reduction of NAD+ to NADH. In the present study, we analyzed the sequence of FgGAPDH and investigated its structural, binding, and catalytic properties. Sequence alignment of FgGAPDH showed 100% identity with the sister fluke Fasciola hepatica GAPDH. The gapdh gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified. The purified FgGAPDH exists as a homo-tetramer, composed of a ~ 37-kDa subunit under non-dissociating conditions at 300 mM salt concentration indicating that higher salt stabilizes the tetrameric state. The binding of the cofactor NAD+ caused a conformational rearrangement in the enzyme structure, leading to the stabilization of the enzyme. A homology model of FgGAPDH was constructed, the cofactor (NAD+) and substrate (G-3-P) were docked, and the binding sites were identified in a single chain. The inter-subunit cleft of GAPDH that has been exploited for structure-based drug design in certain protozoan parasites is closed in the case of FgGAPDH, similar to the human GAPDH. Thus, the conformation of FgGAPDH in this region is similar to the human enzyme. Therefore, GAPDH may not be a suitable target for drug discovery against fascioliasis. Still, the analysis of the structural and functional attributes of GAPDH will be significant in understanding the various roles of this enzyme in the parasite as well as provide new insights into the biochemistry of flukes.

We assessed if leptin, a cytokine hormone known to enhance energy expenditure by promoting lipid and carbohydrate catabolism in response to physiologic stress, might directly regulate cellular glycolysis. A transcriptomic analysis of prolactin cells in the tilapia (Oreochromis mossambicus) pituitary rostral pars distalis (RPD) revealed that recombinant leptin (rtLep) differentially regulates 1,995 genes, in vitro. Machine learning algorithms and clustering analyses show leptin influences numerous cellular gene networks including metabolism; protein processing, transport, and metabolism; cell cycle and the hypoxia response. Leptin stimulates transcript abundance of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (gapdh) in a covariate manner to the hypoxic stress gene network. Orthogonal tests confirm that rtLepA dose-dependently increases gapdh gene expression in the RPD along with transcript abundance of 6-phosphofructo-1-kinase (pfk1), the rate limiting glycolytic enzyme. Functional testing demonstrated that leptin stimulates PFK activity and glycolytic output, while Stattic (a STAT3 blocker) was sufficient to suppress these responses, indicating leptin stimulates glycolysis through a STAT3-dependent mechanism. Leptin also stimulated pfk1 gene expression and lactate production in primary hepatocyte incubations in a similar manner to those shown for the pituitary RPD. This work characterizes a critical metabolic action of leptin to directly stimulate glycolysis across tissue types in a teleost model system, and suggest that leptin may promote energy expenditure, in part, by stimulating glycolysis. These data in a teleost fish, suggest that one of leptin's ancient, highly-conserved functions among vertebrates may be stimulation of glycolysis to facilitate the energetic needs associated with various stressors.

Background: CRISPR/Cas9 system is becoming the dominant genome editing tool in a variety of organisms. CRISPR/Cas9 mediated knock out has been demonstrated both in chicken cell lines and in chicken germ cells that served to generate genetically modified birds. However, there is limited data about CRISPR/Cas9 dependent homology directed repair (HDR) for avian, even in cell culture. Few attempts have been made with integrations in safe harbor loci of chicken genome that induces constitutive expression of the inserted gene. Gene expression under an endogenous promoter would be more valuable than under a constitutive exogenous promoter, as it allows the gene expression to be tissue-specific. Methods: Three gRNAs were chosen to target chicken 3’-untranslated region of GAPDH gene. Cas9-mediated activity in the targeted locus for the gRNAs in DF-1 cells was estimated by T7E1 assay. To edit the locus, the HDR cassette was added along with CRISPR/Cas9. The inserted sequence contained eGFP in frame with a GAPDH coding sequence via P2A and Neomycin resistance gene (neoR) under cytomegalovirus promoter. Correct integration of the cassette was confirmed with fluorescent microscopy, PCR analysis and sequencing. Enrichment of modified cells was done by G418 selection. Efficiency of integration was assessed with fluorescence activated cell sorting (FACS). Results: We have established a CRISPR/Cas9 system to target an endogenous locus and precisely insert a gene under endogenous control. In our system, we used positive and negative selection to enrich modified cells and remove cells with undesirable insertions. The efficiency of CRISPR/Cas9-mediated HDR was increased up to 90% via G418 enrichment. We have successfully inserted eGFP under control of the chicken GAPDH promoter. Conclusions: The approach can be used further to insert genes of interest under control of tissue-specific promoters in primordial germ cells in order to produce genetically modified birds with useful for biotechnological purposes features.

We describe a novel technology for detecting nucleic acids: Probe Alteration Link Self-Assembly Reactions (PALSAR). PALSAR comprises DNA self-assembly of pairs of short DNA probes formed by alternate hybridization of three complementary regions in a pair of honeycomb probes (HCPs). Self-assembly occurs at designated salt concentrations and reaction temperatures and requires no enzymes. We prepared pairs of HCPs to detect mRNAs encoded by the GAPDH gene β-actin (BA) gene, CD3D gene, CD4 gene, major vault protein (MV) gene and the signalling lymphocytic activation molecule-associated protein (SAP) gene, and succeeded in quantitatively detecting these mRNAs. PALSAR could detect mRNA directly without synthesizing cDNA. Moreover, multiple mRNAs could be detected simultaneously in a single reaction tube and there was a good correlation between the results obtained PALSAR and those by real-time PCR.

Background Clostridium thermocellum is a promising microorganism for conversion of cellulosic biomass to biofuel, without added enzymes; however, the low ethanol titer produced by strains developed thus far is an obstacle to industrial application. Results Here, we analyzed changes in the relative concentration of intracellular metabolites in response to gradual addition of ethanol to growing cultures. For C. thermocellum, we observed that ethanol tolerance, in experiments with gradual ethanol addition, was twofold higher than previously observed in response to a stepwise increase in the ethanol concentration, and appears to be due to a mechanism other than mutation. As ethanol concentrations increased, we found accumulation of metabolites upstream of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) reaction and depletion of metabolites downstream of that reaction. This pattern was not observed in the more ethanol-tolerant organism Thermoanaerobacterium saccharolyticum. We hypothesize that the Gapdh enzyme may have different properties in the two organisms. Our hypothesis is supported by enzyme assays showing greater sensitivity of the C. thermocellum enzyme to high levels of NADH, and by the increase in ethanol tolerance and production when the T. saccharolyticum gapdh was expressed in C. thermocellum. Conclusions We have demonstrated that a metabolic bottleneck occurs at the GAPDH reaction when the growth of C. thermocellum is inhibited by high levels of ethanol. We then showed that this bottleneck could be relieved by expression of the gapdh gene from T. saccharolyticum. This enzyme is a promising target for future metabolic engineering work. Electronic supplementary material The online version of this article (10.1186/s13068-017-0961-3) contains supplementary material, which is available to authorized users.

MeGAPCs were identified as negative regulators of plant disease resistance, and the interaction of MeGAPCs and MeATG8s was highlighted in plant defense response. As an important enzyme of glycolysis metabolic pathway, glyceraldehyde-3-P dehydrogenase (GAPDH) plays important roles in plant development, abiotic stress and immune responses. Cassava (Manihot esculenta) is most important tropical crop and one of the major food crops, however, no information is available about GAPDH gene family in cassava. In this study, 14 MeGAPDHs including 6 cytosol GAPDHs (MeGAPCs) were identified from cassava, and the transcripts of 14 MeGAPDHs in response to Xanthomonas axonopodis pv manihotis (Xam) indicated their possible involvement in immune responses. Further investigation showed that MeGAPCs are negative regulators of disease resistance against Xam. Through transient expression in Nicotiana benthamiana, we found that overexpression of MeGAPCs led to decreased disease resistance against Xam. On the contrary, MeGAPCs-silenced cassava plants through virus-induced gene silencing (VIGS) conferred improved disease resistance. Notably, MeGAPCs physically interacted with autophagy-related protein 8b (MeATG8b) and MeATG8e and inhibited autophagic activity. Moreover, MeATG8b and MeATG8e negatively regulated the activities of NAD-dependent MeGAPDHs, and are involved in MeGAPCs-mediated disease resistance. Taken together, this study highlights the involvement of MeGAPCs in plant disease resistance, through interacting with MeATG8b and MeATG8e.

Baylisascaris schroederi, a roundworm parasite of giant pandas, badly affects the health of its hosts. Diagnosis of this disease currently depends mainly on sedimentation floatation and Polymerase Chain Reaction (PCR) methods to detect the eggs. However, neither of these methods is suitable for diagnosis of early-stage panda baylisascariasis and no information on early diagnosis of this disease is available so far. Therefore, to develop an effective serologic diagnostic method, this study produced recombinant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and thioredoxin peroxidase (Tpx) proteins from B. schroederi using a prokaryotic expression system. We determined the immunological characteristics of these proteins and their location in the parasite. Indirect enzyme-linked immunosorbent assays (ELISAs) were established to detect B. schroederi infection in giant pandas based on GAPDH and Tpx respectively. The open reading frame of the GAPDH gene (1083 bp) encoded a 39 kDa protein, while the predicted molecular weight of Tpx (588 bp) was 21.6 kDa. Western-blotting analysis revealed that both recombinant proteins could be recognized with positive serum of pandas infected with B. schroederi. Immunohistochemical staining showed that the endogenous GAPDH of B. schroederi was widely distributed in the worm while Tpx was mainly localized in the muscle, eggs, gut wall, uterus wall and hypodermis. Serological tests showed that the GAPDH-based indirect ELISA had a sensitivity of 95.83% and specificity of 100%, while the test using Tpx as the antigen had sensitivity of 75% and specificity of 91.7%. Thus, B. schroederi Tpx is unsuitable as a diagnostic antigen for baylisascariasis, but B. schroederi GAPDH is a good candidate diagnostic antigen for B. schroederi in pandas.

During the purification of human recombinant erythropoietin (rEPO) from host cells, residual DNA may remain in final products. This contamination is a risk factor for patients and may result in the inactivation of some tumor suppressor genes or activation of oncogenes if its concentration is more than the standard defined by WHO. Based on WHO’s criteria, acceptable level of residual DNA in biopharmaceuticals is less than 10–100 pg/dose. In this study, we have designed a sensitive and specific quantitative real-time polymerase chain reaction (PCR) assay for the detection of residual DNA in human rEPO products. All reported sequences of CHO’s GAPDH gene were retrieved from GenBank, and a multiple alignment was performed using Mega 6 software to find conserved regions of the gene. Primers and probe were designed by AlleleID7 software for the highly conserved region. Quantitative real-time PCR showed an R2 value more than 0.99 and the efficiency equal to 101% indicating a highly accurate and efficienc...

Ischemia represents an existential threat not only to organ and cell viability but also to life itself. For that reason, organisms respond with specific changes in cell structure and function, which may be organ specific. These include modulation of GAPDH gene expression, its protein– protein interactions, changes in subcellular localization, and its posttranslational modification. In particular, GAPDH mRNA synthesis is diminished, coordinating with increases in inducible nitric oxide (NO) synthase during neuronal ischemia. GAPDH is required for glutamate excitotoxicity during neuronal ischemia; for poly (ADP) ribose polymerase inhibition of glycolysis during renal ischemia; and NO-induced changes in gene expression and cell function in leucocytes as well as in retinal cells; and, lastly, differential age-related responses to ischemia in skeletal muscle.

Diabetes presents as a consequence of the hyperglycemic stress resulting from either a deficiency in the synthesis of insulin or an alteration in its basic mechanisms of cell regulation. Although insulin regulates GAPDH gene expression in normal cells, recent studies indicate fundamental roles for moonlighting GAPDH in the cellular mechanisms, which underlie diabetic pathogenesis. These include hyperglycemic perturbations as a consequence of GAPDH enzyme inhibition following its structural modification by oxidative stress-induced poly (ADP-ribose) polymerase 1, a notable role for moonlighting GAPDH in the development of diabetic retinopathy, as well as its distinctive function in diabetes-related apoptosis. As such, these studies indicate the significance of moonlighting GAPDH in the clinical and cellular presentation of this human disease.

While involved in a functional genomics program, we found that the overexpression of potato (Solanum tuberosum) glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene in yeast improves its water-deficit stress (drought) tolerance. But, the effect of altered (under and over) expression of GAPDH on water-deficit stress tolerance of higher plants is not yet studied. In this study, we used a versatile reverse genetics tool called virus-induced gene silencing and down-regulated the expression of GAPDH gene in tobacco (Nicotiana benthamiana) to examine the relevance (effect of underexpression) of GAPDH on drought tolerance of higher plants. Leaf discs made from silenced and nonsilenced tobacco plants were subjected to water-deficit stress. Measure of cell viability and the content of chlorophyll in stressed and nonstressed leaf discs were determined to quantify the effect of stress. Leaf discs made from the gene-silenced plants were found to be more severely affected by the stress than the leaf discs made from nonsilenced plants, implying the importance of GAPDH gene in drought tolerance of plants. Furthermore, to reiterate the involvement of GAPDH in drought tolerance of plants, potato transgenic plants constitutively overexpressing the GAPDH gene were generated and their performance under drought condition was analyzed. Transgenic potato plants showed improved drought tolerance when compared to wild-type potato. On the whole, our results confirm that the GAPDH gene plays an important role in drought tolerance of higher plants, and its constitutive overexpression by genetic engineering can be used to improve drought tolerance of crop plants like potato.

Escherichia coli (E. coli) consists of commensal (ComEC) and diarrhoeagenic (DEC) groups. ComEC are detected using traditional culture methods. Conformational steps are performed after culturing if it is required to test for the presence of DEC, increasing cost and time in obtaining the results. The aim of this study was to develop a single-step multiplex polymerase chain reaction (m-PCR) that can simultaneously amplify genes associated with DEC and ComEC, with the inclusion of controls to monitor inhibition. A total of 701 samples, taken from clinical and environmental water sources in South Africa, were analysed with the optimised m-PCR which targeted the eaeA, stx1, stx2, lt, st, ial, eagg, astA and bfp virulence genes. The mdh and gapdh genes were included as an internal and external control, respectively. The presence of the external control gapdh gene in all samples excluded any possible PCR inhibition. The internal control mdh gene was detected in 100 % of the environmental and 85 % of the clinical isolates, confirming the classification of isolates as E. coli PCR positive samples. All DEC types were detected in varying degrees from the mdh positive environmental and clinical isolates. Important gene code combinations were detected for clinical isolates of 0.4 % lt and eagg. However, 2.3 % of eaeA and ial, and 8.7 % of eaeA and eagg were reported for environmental water samples. The E. coli astA toxin was detected as positive at 35 and 17 % in environmental isolates and clinical isolates, respectively. Interestingly, 25 % of the E. coli astA toxin detected in environmental isolates and 17 % in clinical isolates did not contain any of the other virulence genes tested. In conclusion, the optimised single-step 11-gene m-PCR reactions could be successfully used for the identification of pathogenic and non-pathogenic E. coli types. The m-PCR was also successful in showing monitoring for PCR inhibition to ensure correct reporting of the results.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that plays important roles in multiple cellular processes including phytohormone signaling, plant development, and transcriptional regulation. Although GAPDH genes have been well characterized in various plant species such as Arabidopsis, tobacco, wheat, rice, and watermelon, comprehensive analysis has yet to be completed at the whole genome level in sweet orange (Citrus sinensis). In this study, six GAPDH genes distributed across four chromosomes were identified within the sweet orange genome. Their gene structures, conserved subunits, and subcellular localization were also characterized. Cis-element analysis of CsGAPDHs’ promoter regions and the results of dark treatments indicate that CsGAPDH may be involved in photosynthesis. CsGAPDH genes expressed either in a tissue-specific manner or constitutively were ultimately identified along with their expression response to phosphorus deficiency treatments. In addition, a dual-luciferase transient assay was performed to reveal the transcriptional activation of CsGAPDH proteins. Gene Ontology (GO) analysis for proteins interacting with CsGAPDHs helped to uncover the roles these CsGAPDHs play in other plant processes such as citrus seed germination. This study provides a systematic analysis of the CsGAPDH gene family in the sweet orange genome, which can serve as a strong foundation for further research into the biochemical properties and physiological functions of CsGAPDHs.

Neuroblastoma (NB), originating from neural crest cells, is the most common extracranial tumor of childhood. Retinoic acid (RA) which is the biological active form of vitamin A regulates differentiation of NB cells, and RA derivatives have been used for NB treatment. PPARα (peroxisome proliferator-activated receptor) plays an important role in the oxidation of fatty acids, carcinogenesis, and differentiation. URG4/URGCP gene is a proto-oncogene and that overexpression of URG4/URGCP is associated with metastasis and tumor recurrence in osteosarcoma. It has been known that URG4/URGCP gene is an overexpressed gene in hepatocellular carcinoma and gastric cancers. This study aims to detect gene expression patterns of PPARα and URG4/URGCP genes in SH-SY5Y NB cell line after RA treatment. Expressions levels of PPARα and URG4/URGCP genes were analyzed after RA treatment for reducing differentiation in SH-SY5Y NB cell line. To induce differentiation, the cells were treated with 10 μM RA in the dark for 3-10 days. Gene expression of URG4/URGCP and PPARα genes were presented as the yield of polymerase chain reaction (PCR) products from target genes compared with the yield of PCR products from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. SH-SY5Y cells possess small processes in an undifferentiated state, and after treatment with RA, the cells developed long neurites, resembling a neuronal phenotype. PPARα gene expression increased in RA-treated groups; URG4/URGCP gene expression decreased in SH-SY5Y cells after RA treatment compared with that in the control cells. NB cell differentiation might associate with PPARα and URG4/URGCP gene expression profile after RA treatment. © 2013 International Society of Oncology and BioMarkers (ISOBM).

A protocol for the rapid detection and semi-quantification of human enteric adenovirus based on the quantification of viral mRNA during cell culture infectivity assay was developed. Infectivity assays for adenovirus incorporated cell culture and reverse transcription real-time PCR, where viral mRNA detection was used to monitor the progress of adenovirus infection (CC/mRNA qPCR). The cell line used was G293. This specific infectivity assay was calibrated against different initial concentrations of human adenovirus 41. In addition, the expression of the host's housekeeping (HK) gene, GAPDH, served as internal control for the mRNA assays for quality assurance of the mRNA extraction and reverse transcription steps. The concentrations of infectious human adenoviruses in different sewage samples were estimated semi-quantitatively using the CC/mRNA qPCR assay and calibration obtained for adenovirus 41. A linear relationship between concentrations of viral mRNA (hexon gene) and infectious units was observed between 10 7 to 10 1 infectious units per assay ( R 2 = 0.97) in samples analyzed 3–5 days post infection. The expressions of host cell GAPDH gene were not significantly affected by infections with different concentrations of human adenovirus 41, and between virus positive and negative cell cultures ( p > 0.1). The estimated concentrations of human adenoviruses in sewage samples ranged between 10 2 to 10 3 mRNA-IU/L. Most of the viruses detected in sewage samples were from human adenovirus species F. The CC/mRNA qPCR assay can be used for quantifying infectious human adenovirus 41, estimating the levels of human adenoviruses in sewage samples, and applied to other sample settings. The CC/mRNA qPCR protocol described here represents an improvement in the detection of human enteric adenoviruses by reducing incubation time (5 days); whereas the conventional cell culture method requires longer incubation periods (10–20 days). More importantly, this protocol can be used to more rapidly and semi-quantitatively estimate the levels of infectious human adenoviruses in environmental samples.

BACKGROUND:The PCR assays usually employed for Leishmania diagnosis does not simultaneously detect a constitutive gene that would certify the viability of the DNA sample. We present a multiplex PCR approach for the simultaneous diagnosis of the Leishmania sp. kDNA fragment and a catalytic domain segment of a conserved region of the mammalian gapdh gene. METHODOLOGY:The proposed multiplex protocol was designed through in silico PCR. The annealing temperature, concentration of primer pairs, number of cycles, distinct polymerase enzymes and premix kit were defined to achieve an optimal reaction. The DNA detection sensitivity was tested with different concentrations of known L. tropica DNA, and the reproducibility of the assay was confirmed using samples from 106 wild mammals that were previously subject to Leishmania sp. kDNA analysis through singleplex reactions. PRINCIPAL FINDINGS:The following optimal conditions were established: 95°C for 1 min followed by 30 cycles of 95°C for 30 s, 61°C for 30 s, and 72°C for 30 s and a final extension at 72°C for 1 min. The multiplex PCR system was capable of detecting 0.1 ng of L. tropica diluted in 100 ng of mammalian DNA. Of 51 kDNA samples that were previously found to be positive, 45 (88.2%) were positive for both targets, two were positive only for kDNA and four were negative for both. Of 55 kDNA samples that were previously identified as negative, 38 (69.1%) were positive for gapdh whereas the other 17 were negative for both targets. CONCLUSIONS/SIGNIFICANCE:The proposed multiplex PCR system allows the simultaneous detection of the gapdh gene and Leishmania sp. kDNA in tissue samples derived from distinct wild mammal species. The amplification of the gapdh mammalian gene in the same reaction ensures the quality and viability of the DNA in the sample, eliminating the possibility of false-negative results that would impair an accurate description of the infection rates in a given population.

Development of nonviral vectors for the successful application of gene therapy through siRNA/DNA transfection of cells is still a great challenge in current research. (1, 2) In the present study, we have developed multivalent polyglycerol dendron based amphiphiles with well-defined molecular structures that express controlled glycine arrays on their surfaces. The structure-activity relationships with respect to the siRNA complexation, toxicity, and transfection profiles were studied with synthesized amphiphilic polycations. Our findings revealed that a second-generation amphiphilic dendrimer (G2-octaamine, 4) that has eight amine groups on its surface and a hydrophobic C-18 alkyl chain at the core of the dendron, acts as an efficient vector to deliver siRNA and achieve potent gene silencing by investigating the knockdown of luciferase and GAPDH gene activity in HeLa cells. Interestingly, the amphiphilic vector is nontoxic even at higher ratio of N/P 100. To the best of our knowledge this is the first example of successful in vitro siRNA transfection using dendritic amphiphiles. We believe that this supramolecular complex may serve as a new promising alternative for nonviral siRNA delivery systems and will be investigated for in vivo siRNA delivery in the future.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has long been recognized as an important enzyme for energy metabolism and the production of ATP and pyruvate through anaerobic glycolysis in the cytoplasm. Recent studies have shown that GAPDH has multiple functions independent of its role in energy metabolism. Although increased GAPDH gene expression and enzymatic function is associated with cell proliferation and tumourigenesis, conditions such as oxidative stress impair GAPDH catalytic activity and lead to cellular aging and apoptosis. The mechanism(s) underlying the effects of GAPDH on cellular proliferation remains unclear, yet much evidence has been accrued that demonstrates a variety of interacting partners for GAPDH, including proteins, various RNA species and telomeric DNA. The present mini review summarizes recent findings relating to the extraglycolytic functions of GAPDH and highlights the significant role this enzyme plays in regulating both cell survival and apoptotic death.

Dirigent and dirigent-like family proteins contain a number of proteins involved in lignification or in the response to pathogen infection and abiotic stress in plants. In the present study, a full-length cDNA sequence of a dirigent-like gene designated ScDir (GenBank Accession Number JQ622282) was obtained from sugarcane based on the stem full-length cDNA library. The ScDir gene was 819-bp long, including a 564-bp ORF encoding 187 amino acid residues. The protein N-terminus contained signal peptides at amino acid residues of 1-25 and transmembrane regions at 7-26 aa. A his-tagged ScDir protein with an estimated molecular mass of 27.4 kDa was expressed in Escherichia coli system. The expressed ScDir protein had increased the host cell's tolerance to PEG and NaCl. When an endogenous GAPDH gene was used as internal control, results from real-time qPCR demonstrated that the ScDir mRNA amount in sugarcane stems was significantly higher than that in the roots, leaves and buds by 18.64 ± 0.48, 25,635.16 ± 2,966.03 and 721.50 ± 8.17-fold, respectively. The ScDir transcript levels in sugarcane seedling increased under H(2)O(2), PEG or NaCl stress. The expression level of ScDir was significantly upregulated under PEG stress, and the highest level was observed at 12 h after stress. Thus, both the ScDir-hosted cell performance and the enhanced expressions in sugarcane imply that the ScDir gene is involved in the response to abiotic stresses of drought, salts and oxidation. The transcription of the ScDir gene is highly stem-specific, as revealed by real-time qPCR. Key message A novel sugarcane Sc-Dir gene, DIRd subfamily, which is highly stalk-specific expression and involved in the response to artificial stresses of drought, salts, and oxidatives.

Pilobolus crystallinus has three putative glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes (pcgapdh1, pcgapdh2 and pcgapdh3). The results of this study demonstrate that expression of pcgapdh2 was increased by irradiation and that this increased expression was correlated with the formation of asexual reproductive organs (trophocysts). Interestingly, expression of pcgapdh2 was restricted to trophocysts. The formation of trophocysts was likely promoted by light, and the expression of pcgapdh2 was increased as a result of trophocyst formation. This is the first report that shows the regulation of a gapdh gene in an organ-specific manner in fungi.

A major challenge for efficient transgene expression in Dunaliella salina is to find strong endogenous promoters to drive the transgene expression. In the present study, a novel glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter was cloned and used to drive expressions of the bialaphos resistance (bar) gene and of the N-terminal fragment of human canstatin (Can-N). The results showed that the bar gene was transcribed by the GAPDH promoter and integrated into the genome of the transformants of D. salina. Furthermore, the PCR identification, Southern and western blots indicated that Can-N was expressed in transgenic D. salina, demonstrating that the promoter of the D. salina GAPDH gene is suitable for driving expression of heterologous genes in transgenic D. salina.