Search

Analysis of several parameters is required for adequate quality control in umbilical cord blood units (UCBU) when used for therapeutic purposes.To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU.One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the sequence-specific priming technique.CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p0.0001), as well as that of UCB in comparison with that of the segment (p0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY/11 and GP5/GP6+.Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos.Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU.Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming.La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p0.0001); así como la de SCU comparada con la de segmento (p0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+.Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Background Men with non-obstructive azoospermia constitute a challenging subgroup of male infertility patients in whom a genetic cause of defective spermatogenesis may be a contributing factor. The aim of this prospective observational cohort study was to determine whether assessment of meiotic nuclear division 1 (MND1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression (MND1/GAPDH) in testicular tissue could be a prognostic indicator for sperm retrieval and ICSI outcome in patients with non-obstructive azoospermia. The study participants underwent clinical evaluation, conventional semen analysis, serum follicular stimulating hormone (FSH), testosterone assay, scrotal ultrasound examination, microsurgical testicular sperm extraction (mTESE), and assessment of MND1/GAPDH gene expression levels in testicular tissue via quantitative polymerase chain reaction (qPCR) techniques. Results The MND1/GAPDH level was associated with the likelihood of identifying sperm in testicular biopsies (odds ratio (OR) 1.25, 95% confidence intervals (CI) 1.14 to 1.34, p < 0.0001), which was confirmed by the pairwise comparison of high vs. low levels of MND1/GAPDH (OR 5.34, 95% CI 1.97 to 13.16, p = 0.0006). The level of FSH was inversely associated with a lower chance of finding sperm (OR 0.37, 95% CI 0.20 to 0.65, p = 0.001). Compared with small testicular volume, normal volume was inversely associated with the chance of sperm presence (OR 0.16, 95% CI 0.06 to 0.47, p = 0.0002). However, there was no correlation between MND1/GAPDH levels and ICSI outcome. Conclusion Gene expression analysis to predict the likelihood of sperm retrieval following mTESE in patients with non-obstructive azoospermia provides a new avenue for future research, diagnosis and treatment of male factor infertility. Before its wider clinical application, however, this proof-of-concept should be tested in a large multinational, multicenter observational study.

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.

Background and aims: In acute lymphoblastic leukemia (ALL), large numbers of stem cells become lymphoblasts or lymphocytes. Among the genetic factors influencing cancer is the ADAM (a disintegrin and metalloprotease) gene family. Due to the important role of this family in cancer, this study aimed to compare the expression level of ADAM17 gene in patients with ALL and healthy individuals. Material and Methods: In this case-control study, 40 venous blood samples were taken from ALL patients referred to Omid hospital in Isfahan, Iran. Also, 40 venous blood samples were taken from healthy individuals in vitro. Lymphocyte isolation was performed using a ficol and cell RNA was isolated using an RNX-Plus kit. It was then converted to cDNA using the Yekta Tajhiz Azma kit. Finally, reverse transcription-polymerase chain reaction (RT-PCR) technique was used to evaluate the relative expression of ADAM17 gene in blood samples of healthy individuals and patients with leukemia, and the ratio was measured with the reference GAPDH gene. SPSS software version 22 and t test were used to analyze the data. Results: The expression level of ADAM17 gene in patients with ALL compared to the control group showed a significant increase, which was statistically significant (P=0.043). Conclusion: It seems that increasing the expression of ADAM17 gene in people with ALL is a suitable biomarker to diagnose this disease.

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.

OBJECTIVE The aim: This study is designed to investigate the possible association of genetic polymorphism expression of UMOD gene and chronic kidney disease in population. PATIENTS AND METHODS Materials and methods: In the current study, the single-nucleotide polymorphisms (SNPs) were genotyped in the promoter region of the UMOD gene in CKD patients to assess its association with the kidney outcome of CKD. So 50 patients' blood samples suffered from CKD were collected. Among these patients, 21 were men and 29 women, aged 35 - 85 years old. Another group included 50 healthy subjects. DNA was extracted from all blood samples with EDTA using Quick DNA miniprep Kit ZYMO, (Cat№ D3025) according to manufacturer's instructions. Genotyping of 1 common polymorphisms (rs4293393) of the UMOD gene was done and the RNA was extracted and converted to cDNA and a set of primers was used to amplify specific region within the UMOD gene; another set was used to amplify the GAPDH gene to use it in calculation as a reference gene. RESULTS Results and conclusions: After statistical analysis, the results showed that there could be association between having CC mutant polymorphism in UMOD gene and having CKD.

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.

Objective Aim of this study was to define the relationship between RIF (Recurrent Implantation Failure) and endometrial mtDNA copy number. Study Design A total of 50 women of reproductive age including twenty-five patients clinically diagnosed with RIF and twenty-five fertile women as healthy controls were recruited into the study. Endometrial biopsy samples were obtained with a pipelle at the 20–24 days of the menstrual cycle of each participant. Total genomic DNA samples were isolated from endometrial tissues; MT-ND1 (mitochondrially encoded NADH dehydrogenase I) and MT-CO2 (mitochondrially encoded cytochrome C oxidase II) target genes were amplified by droplet digital PCR (ddPCR). Nuclear GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) gene was also used for study normalization. The study has been conducted between February 2019 and June 2019. Result(s) Droplet digital PCR results were analyzed in “QuantaSoft” software. The concentration amount (copies/μl) of each participant’s mitochondrial gene was normalized according to the GAPDH gene concentrations as nuclear reference. mtDNA amounts were compared between RIF patients and healthy controls. Normalized data was statistically evaluated using Mann-Whitney U test and ROC curve analysis. Conclusion(s) It was concluded that the mitochondrial target gene (MT-ND1 and MT-CO2) copy number amount of RIF patients was higher than the one obtained from the healthy group in endometrial tissues. It is thought that higher mtDNA copy number at the RIF group may be related to increased oxidative stress in the endometrium. This stress factors may influence receptivity negatively and cause implantation failure. The receptivity of the endometrium is associated with the number of mtDNA copies and difference can be used as a biomarker for receptivity analysis.

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.

BACKGROUND: Stunting in children increases the risk of degenerative diseases in adulthood, including dyslipidemia, obesity, type 2 diabetes mellitus, and cardiovascular disease. This is based on the result of metabolic changes that may be caused by chronic malnutrition and experienced by stunting children. Stunting in children is associated with metabolic disorders that are based on impaired fat oxidation, a trigger factor for obesity in adulthood. The peroxisome proliferator-activated receptor (PPAR) α gene is a transcriptional factor that regulates fat, carbohydrate, and amino acid metabolism whose genetic variants are linked to the development of dyslipidemia and cardiovascular disease. AIM: The study assessed the effect of metabolic changes in stunting toddler on PPARα gene expression. MATERIALS AND METHODS: An analytical-observational laboratory was done using 41 blood samples, coming from 23 stunting toddlers, and 18 not-stunting toddlers. In all research subjects, anthropometric measurements and examination of PPARα gene mRNA expression were carried out. Analysis of PPARα gene mRNA expression using one-step quantitative reverse transcriptase-polymerase chain reaction using specific primers, as a comparison of gene expression using the GAPDH gene. The relative expression of the PPARα mRNA gene was analyzed using the LIVAK formula. RESULTS: The study obtained a mean of ΔCT in stunting toddlers of 5.81, whereas in stunting toddlers at 5.082. Analysis with LIVAK 2 ^ - formula (ΔCT stunting -ΔCT not stunting) obtained PPARα mRNA gene expression of 0.6. CONCLUSION: We conclude that there is a decrease in PPARα gene expression in stunting toddlers.

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.

Gastric cancer is one of the most serious and lethal kinds of cancer in the world. It is a multi-step, multi-factor, and elaborated process that is associated to gene abnormal expression. This study intended to investigate the WNT16 gene’s expression in human gastric tumor and the margin tissues of the stomach (normal tissues). Correspondingly, 40 samples (20 tumoral tissues and 20 non tumoral or margins tissues) were investigated in Imam Khomeini Hospital in Sari City, Mazandaran Province, Iran. In this way, real-time PCR, Taqman assay was employed to evaluate the upregulation and downregulation of this gene in both tissues in triplicate form. The GAPDH gene was selected as housekeeping gene. Conspicuously, the results have shown a remarkable modification in tumoral tissues, and the gene expression increased significantly in tumoral tissue. Conclusively, the upregulation of WNTt16 gene expression in tumoral tissues was impressive and the P value was 0.005 and the SE range was 0.064–142.154.

Digital PCR enabled high-sensitivity and quantitative measurements of rare biological variants. A new digital droplet-enabled PCR technology was introduced in this paper, which partitioned genetic targets into a planar nanoliter droplet array by using a microfluidic impact printer (MIP) with a disposable microfluidic chip. The accuracy of this MIP-enabled PCR technology was verified by detecting a series of concentration gradients of GAPDH gene across spanning four orders of magnitude (from 0.464 copies/μL to 464 copies/μL). Furthermore, this technology was applied to detect the expressions of p53 gene in colon cancer tissues and adjacent nontumorous tissues, from which the copies of the nucleic acids could be absolute-quantitatively determined. The outcomes were consistent with the results of using the conventional real-time PCR, demonstrating a great potential of the MIP-enabled digital PCR in detecting gene expression in clinical samples.

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.

BACKGROUND: Methods of diagnostic molecular biology are routinely applied on formalin-fixed, paraffin-embedded tissues processed via conventional method. Recently, there has been a growing interest to use microwave technology in histopathology laboratories to overcome the deficiencies of the conventional processing method. Thefore, this study was aimed to compare and analyze the quality and quantity of DNA obtained from tissues processed by conventional and microwave tissue processing techniques and to further ascertain the applicability of the latter for PCR (polymerase chain reaction based research).METHODS: Thirty fresh tissues of oral squamous cell carcinoma (OSCC) were included, and each sample was cut into two equivalent halves. One tissue half was processed by conventional manual method whereas the other half was processed using a domestic microwave oven. DNA was obtained from all the tissues which were then subjected to Polymerase chain reaction (PCR) to evaluate GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) gene expression.RESULTS: The results revealed better DNA yield from microwave processed tissue while the quality of the DNA was alike from both the techniques.CONCLUSION: On the basis of the results obtained, it can be concluded that DNA produced by microwave processed tissues was similar to that obtained by conventional processing technique in terms of quantity and quality. Thus, microwave processed tissue samples can be successfully used for further molecular studies and researches.

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

Objective To evaluate the effect of imatinib mesylate on KAI1 gene expression and apoptosis properties in human gastric carcinoma AGS cell line. Methods Cell viability was assessed by MTT assay and quantitative real time PCR method was applied for investigation of Bax, Bcl-2 , and KAI1 gene expression in AGS cells. The quantity of KAI1, Bax , and Bcl-2 compared to GAPDH gene expressions were examined using the formula 2 -ΔΔCt . Furthermore, cell apoptosis/necrosis was carried out by annexin V/PI staining and quantified with flow cytometry after treatment with imatinib. Results Imatinib mesylate was showed to have a dose-dependent toxicity effect against AGS cells. KAI1 / GAPDH gene expression ratios were 1.07 ± 0.02 ( P > 0.05), 1.68 ± 0.19 ( P > 0.05), 3.60 ± 0.55 ( P P Bax detected by real-time PCR after treatment with imatinib mesylate were significantly increased. Also, the number of apoptotic cells was increased from 3.72% (statistically significant; P Conclusions The results suggest that imatinib mesylate can induce apoptosis pathway in a dose-dependent mode and might modulate metastasis by up regulating KAI1 gene expression in human gastric carcinoma AGS cell line.

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.