Phosphoglycerate Kinase Gene Research Articles

The Effects of a Saccharomyces cerevisiae Strain Overexpressing the Endopolygalacturonase PGU1 Gene on the Aminoacidic, Volatile, and Phenolic Compositions of Cabernet Sauvignon Wines

The addition of pectinase enzymes during the maceration stage of grape skins in order to improve the extraction yields and color of red wines is a common practice in many wineries. The objective of this work was to study in depth the changes that occurred in the aminoacidic, volatile, and phenolic compositions of Cabernet Sauvignon wines fermented with a Saccharomyces cerevisiae strain genetically modified with the gene encoding for endopolygalacturonase (PGU1) in transcriptional fusion with the promoter of the phosphoglycerate kinase (PGK1) gene, both from S. cerevisiae origin. A higher yield extraction of wine was obtained in wines fermented with the modified strain (PW), increasing by around 6.1% compared to the control wine (CW). Moreover, there was a 40% decrease in the malic acid content in the PW, thus suggesting that this modified yeast could be investigated as a malic acid-reducing agent. There were slight differences in other aroma volatile compounds studied as well as in the phenolic content. However, there was a considerable increase in the amino acid content in the PW.

Morphological and proteomic study of waterlogging tolerance in cotton

Floating seedling cultivation technique is a novel seedling method in cotton and it provides an ideal model to study cotton growing under waterlogging stress. Morphological character and proteomic profile of the primary root from the seedling cultured by the new technology were evaluated in this study. Compared to seedlings cultured by the traditional method, the diameter of the taproot from floating technology is small at all five seedling stages from one-leaf stage to five-leaf stage. There are similar changes between the thickness of cortex and diameter of stele, which increased from the one- to the two-leaf stage but decreased from the two- to the five-leaf stage. At the one-leaf stage, the number and volume of mitochondria in the primary root-tip cells were less than those in the control. At the two-leaf stage, there was significantly less electron-dense material in the primary root-tip cells than those in the control group. From the one- to the two-leaf stage, the vacuole volume was significantly smaller than that in the control. Total 28 differentially expressed proteins were revealed from aquatic and control group roots of cotton seedlings at the three-leaf stage by two-dimensional electrophoresis, which included 24 up-regulated and four down-regulated proteins. The relative expression of the phosphoglycerate kinase (PGK) gene in aquatic roots increased from the one- to the four-leaf stage but declined rapidly from the four- to the five-leaf stage. The relative expression of the 14–3-3b gene tended to decrease from the one- to the five-leaf stage. The PGK and 14–3-3b genes were specifically expressed in the aquatic roots at the three-leaf stage. In brief, these changes induced waterlogging resistance in the aquatic roots of cotton seedlings in the floating nursery, thereby causing the roots to adapt to the aquatic environment, promoting the growth and development of cotton seedlings.

Transcriptomic analysis of the response of Avena sativa to Bacillus amyloliquefaciens DGL1.

Bacillus amyloliquefaciens DGL1, isolated from the arid sandy areas in Dagler, Qinghai Province, China, promotes the growth of Avena sativa variety "Qing Yan 1". To elucidate the transcriptomic changes in the oat root system following interaction with DGL1 and to reveal the molecular mechanism by which DGL1 promotes oat growth, treatment and control groups of oat roots at 2, 4, 8, and 12 h after inoculation with a suspension of strain DGL1 were analyzed using Illumina high-throughput transcriptome sequencing technology. The differentially expressed genes were determined through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, and the metabolic pathways and key genes were analyzed. The results showed that 7874, 13,392, 13,169, and 19,026 differentially expressed genes were significantly enriched in the glycolysis/gluconeogenesis pathway, amino acid metabolism, nitrogen metabolism, plant hormone signal transduction, and other related metabolic pathways in the oat roots at 2, 4, 8, and 12 h after inoculation with a DGL1 suspension. The GO and KEGG enrichment analyses revealed that the genes encoding plasma membrane ATPase, phosphoglycerate kinase gene PGK, ammonium transporter protein gene AMT, cellulose synthase gene CSLF6, and growth hormone response family gene IAA18 were significantly upregulated. It is hypothesized that the pro-growth mechanism of strain DGL1 in oats is the result of the coordination of multiple pathways through the promotion of oat energy metabolism, phytohormone signaling, secondary metabolite synthesis, and amino acid metabolism.

Changes in terpene biosynthesis and submergence tolerance in cotton

BackgroundFlooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is unknown.ResultsThe transcriptome results showed that a total of 6,893 differentially expressed genes (DEGs) were discovered under submergence stress. Gene Ontology (GO) enrichment analysis showed that DEGs were involved in various stress or stimulus responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs related to plant hormone signal transduction, starch and sucrose metabolism, glycolysis and the biosynthesis of secondary metabolites were regulated by submergence stress. Eight DEGs related to ethylene signaling and 3 ethylene synthesis genes were identified in the hormone signal transduction. For respiratory metabolism, alcohol dehydrogenase (ADH, GH_A02G0728) and pyruvate decarboxylase (PDC, GH_D09G1778) were significantly upregulated but 6-phosphofructokinase (PFK, GH_D05G0280), phosphoglycerate kinase (PGK, GH_A01G0945 and GH_D01G0967) and sucrose synthase genes (SUS, GH_A06G0873 and GH_D06G0851) were significantly downregulated in the submergence treatment. Terpene biosynthetic pathway-related genes in the secondary metabolites were regulated in submergence stress.ConclusionsRegulation of terpene biosynthesis by respiratory metabolism may play a role in enhancing the tolerance of cotton to submergence under flooding. Our findings showed that the mevalonate pathway, which occurs in the cytoplasm of the terpenoid backbone biosynthesis pathway (ko00900), may be the main response to submergence stress.

E2F regulation of the Phosphoglycerate kinase gene is functionally important in Drosophila development

The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster. We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase (Pgk), being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acidcycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in PgkΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in PgkΔE2F mutants. Moreover, PgkΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the PgkΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk.

Integrated Multiomics Analysis of Salivary Exosomes to Identify Biomarkers Associated with Changes in Mood States and Fatigue.

Fatigue and other deleterious mood alterations resulting from prolonged efforts such as a long work shift can lead to a decrease in vigilance and cognitive performance, increasing the likelihood of errors during the execution of attention-demanding activities such as piloting an aircraft or performing medical procedures. Thus, a method to rapidly and objectively assess the risk for such cognitive fatigue would be of value. The objective of the study was the identification in saliva-borne exosomes of molecular signals associated with changes in mood and fatigue that may increase the risk of reduced cognitive performance. Using integrated multiomics analysis of exosomes from the saliva of medical residents before and after a 12 h work shift, we observed changes in the abundances of several proteins and miRNAs that were associated with various mood states, and specifically fatigue, as determined by a Profile of Mood States questionnaire. The findings herein point to a promising protein biomarker, phosphoglycerate kinase 1 (PGK1), that was associated with fatigue and displayed changes in abundance in saliva, and we suggest a possible biological mechanism whereby the expression of the PGK1 gene is regulated by miR3185 in response to fatigue. Overall, these data suggest that multiomics analysis of salivary exosomes has merit for identifying novel biomarkers associated with changes in mood states and fatigue. The promising biomarker protein presents an opportunity for the development of a rapid saliva-based test for the assessment of these changes.

Extensive Variation in Gene Expression is Revealed in 13 Fertility-Related Genes Using RNA-Seq, ISO-Seq, and CAGE-Seq From Brahman Cattle.

Fertility is a key driver of economic profitability in cattle production. A number of studies have identified genes associated with fertility using genome wide association studies and differential gene expression analysis; however, the genes themselves are poorly characterized in cattle. Here, we selected 13 genes from the literature which have previously been shown to have strong evidence for an association with fertility in Brahman cattle (Bos taurus indicus) or closely related breeds. We examine the expression variation of the 13 genes that are associated with cattle fertility using RNA-seq, CAGE-seq, and ISO-seq data from 11 different tissue samples from an adult Brahman cow and a Brahman fetus. Tissues examined include blood, liver, lung, kidney, muscle, spleen, ovary, and uterus from the cow and liver and lung from the fetus. The analysis revealed several novel isoforms, including seven from SERPINA7. The use of three expression characterization methodologies (5′ cap selected ISO-seq, CAGE-seq, and RNA-seq) allowed the identification of isoforms that varied in their length of 5′ and 3′ untranslated regions, variation otherwise undetectable (collapsed as degraded RNA) in generic isoform identification pipelines. The combinations of different sequencing technologies allowed us to overcome the limitations of relatively low sequence depth in the ISO-seq data. The lower sequence depth of the ISO-seq data was also reflected in the lack of observed expression of some genes that were observed in the CAGE-seq and RNA-seq data from the same tissue. We identified allele specific expression that was tissue-specific in AR, IGF1, SOX9, STAT3, and TAF9B. Finally, we characterized an exon of TAF9B as partially nested within the neighboring gene phosphoglycerate kinase 1. As this study only examined two animals, even more transcriptional variation may be present in a genetically diverse population. This analysis reveals the large amount of transcriptional variation within mammalian fertility genes and illuminates the fact that the transcriptional landscape cannot be fully characterized using a single technology alone.

Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in Tilletia indica Isolates Inciting Karnal Bunt of Wheat.

Karnal bunt of wheat is an internationally quarantined disease affecting trade, quality, and production of wheat. During 2015–2016, a severe outbreak of Karnal bunt disease occurred in north-western plain zone of India. The present study was undertaken to decipher genetic variations in Indian isolates of Tilletia indica collected from different locations. Seven multilocus sequence fragments were selected to differentiate and characterize these T. indica isolates. A phylogenetic tree constructed based on pooled sequences of actin-related protein 2 (ARP2), β-tubulin (TUB), eukaryotic translation initiation factor 3 subunit A (EIF3A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), histone 2B (H2B), phosphoglycerate kinase (PGK), and serine/threonine-protein kinase (STPK) showed that isolate KB-11 (Kaithal, Haryana) was highly conserved as it was located in cluster 1 and has the maximum sequence similarity with the reference strain. Other isolates in cluster 1 included KB-16 and KB-17, both from Uttar Pradesh, and KB-19 from Haryana. Isolates KB-07 (Jind, Haryana) and KB-18 (Mujaffar Nagar, Uttar Pradesh) were the most diverse and grouped in a subgroup of cluster 2. Maximum numbers of single nucleotide polymorphisms (SNPs) (675) were in the PGK gene across the T. indica isolates. The minimum numbers of SNPs (67) were in KB-11 (Kaithal, Haryana), while the maximum number of SNPs (165) was identified in KB-18, followed by 164 SNPs in KB-14. KB-18 isolate was found to be the most diverse amongst all T. indica isolates. This first study on multilocus sequence typing (MLST) revealed that the population of T. indica was highly diverse.

The genome of Shanputao (Vitis amurensis) provides a new insight into cold tolerance of grapevine.

Vitis amurensis (Shanputao) is the most cold tolerant Vitis species and so is of great interest to grape breeders and producers in areas with low winter temperatures. Here, we report its high-quality, chromosome-level genome assembly based on a combination of sequence data from Illumina and PacBio platforms, BioNano optical mapping and high-throughput chromosome conformation Capture (Hi-C) mapping. The 604.56-Mb genome contains 32885 protein-coding genes. Shanputao was found to share a common ancestor with PN40024 (V. vinifera) approximately 2.17-2.91million years ago, and gene expansion observed in Shanputao might contribute to the enhancement of cold tolerance. Transcriptome analysis revealed 17 genes involved in cold signal transduction, suggesting that there was a different response mechanism to chilling temperature and freezing conditions. Furthermore, a genome-wide association study uncovered a phosphoglycerate kinase gene that may contribute to the freezing resistance of buds in the winter. The Shanputao genome sequence not only represents a valuable resource for grape breeders, but also is important for clarifying the molecular mechanisms involved in cold tolerance.

Real-Time-PCR Assay Based on Phosphoglycerate Kinase Gene for Detection of Entamoeba histolytica Trophozoites in Stool Samples in Holy Karbala, Iraq

Amebiasis is an important cause of diarrheal disease worldwide and has been associated with childhoodmalnutrition. E.histolytica diagnosed usually by microscope, which consider as traditional diagnosis andare neither sensitive nor specific detection of Entamoeba histolytica(1). Real-time PCR assay developed forsensitive and specific detection of the intestinal Protozoan parasites Entamoeba histolytica directly fromhuman feces(2). The RT-PCR assay was able to detect as little as 0.1 parasite per g of feces(3). The currentstudy based on Phosphoglycerate kinase gene (PGK) is a major enzyme used in glycolysis, in the first ATPgenerating step of the glycolytic pathway so the PGK is an enzyme that catalyzes the reversible transfer ofa phosphate group from 1,3-bisphosphoglycerate (1,3-BPG) to ADP producing 3-phosphoglycerate (3-PG)and ATP(4). In our study, depend on PGK as target for Real time PCR assay for detection the trophozoitestage of E.histolytica in stool samples of infected persons.In the current study, a total of 300 human fecal samples were collected from children (less than one year-15year) that suspected to infection with amoebiasis which admitted to hospitals and primary care centers in thecity center, Al-Hindiya district and Nahiat Al -Hurr (100 samples from each region) during the period fromFebruary 2019 to January 2020. Fecal samples processed by direct wet smear and formalin ethyl acetateconcentration method followed by iodine staining and was microscopically examined for E.histolytica.Microscopically positive samples were then subject to Real-time PCR. This is the first study in Iraq using thePhosphoglyceratekinase gene as target for molecular techniques to determine the presence of E. histolyticatrophozoites.in stool samples.

Phosphoglycerate kinase: structural aspects and functions, with special emphasis on the enzyme from Kinetoplastea.

Phosphoglycerate kinase (PGK) is a glycolytic enzyme that is well conserved among the three domains of life. PGK is usually a monomeric enzyme of about 45 kDa that catalyses one of the two ATP-producing reactions in the glycolytic pathway, through the conversion of 1,3-bisphosphoglycerate (1,3BPGA) to 3-phosphoglycerate (3PGA). It also participates in gluconeogenesis, catalysing the opposite reaction to produce 1,3BPGA and ADP. Like most other glycolytic enzymes, PGK has also been catalogued as a moonlighting protein, due to its involvement in different functions not associated with energy metabolism, which include pathogenesis, interaction with nucleic acids, tumorigenesis progression, cell death and viral replication. In this review, we have highlighted the overall aspects of this enzyme, such as its structure, reaction kinetics, activity regulation and possible moonlighting functions in different protistan organisms, especially both free-living and parasitic Kinetoplastea. Our analysis of the genomes of different kinetoplastids revealed the presence of open-reading frames (ORFs) for multiple PGK isoforms in several species. Some of these ORFs code for unusually large PGKs. The products appear to contain additional structural domains fused to the PGK domain. A striking aspect is that some of these PGK isoforms are predicted to be catalytically inactive enzymes or ‘dead’ enzymes. The roles of PGKs in kinetoplastid parasites are analysed, and the apparent significance of the PGK gene duplication that gave rise to the different isoforms and their expression in Trypanosoma cruzi is discussed.

Varying Expression of Mu and Kappa Opioid Receptors in Cockatiels (Nymphicus hollandicus) and Domestic Pigeons (Columba livia domestica).

Avian species have varying analgesic responses to opioid drugs. Some of this variability could be due to extrinsic factors such as administration route or dose. However, intrinsic factors such as gene expression or polymorphic differences in opioid receptors may be important components.ObjectivesThe objectives of this study were to determine the relative gene expression and polymorphisms present for mu and kappa opioid receptors (OPRM1 and OPRK1) in the cerebrum, brainstem, spinal cord, and footpad of cockatiels and pigeons.MethodsTissue biopsies were obtained from 11 adult cockatiels (6 male and 5 female) and 11 adult pigeons (6 male and 5 female). RNA was extracted and qPCR was performed to determine the level of gene expression for OPRM1 and OPRK1 relative to a reference gene phosphoglycerate kinase 1 (PGK1) using the ΔΔCt method. Sanger sequencing was performed to identify polymorphisms, if present.ResultsThere were higher expression levels of OPRM1 compared to OPRK1 in all tissues examined regardless of species (p < 0.001, FDR p < 0.001) Cockatiels had less OPRK1 expression in the cerebrum compared to pigeons (p = 0.005, FDR p = 0.004). Cockatiels had more OPRM1 expression in the brainstem (p = 0.045, FDR p = 0.029), but less OPRM1 expression in the footpad compared to pigeons (p = 0.029, FDR p = 0.021). No other significant differences in OPRM1 or OPRK1 expression were identified across species. Two missense polymorphisms were identified in OPRK1; none were found in OPRM1.ConclusionThe differential expression of opioid receptors between cockatiels and pigeons could have implications for variability in analgesic response between these two species.

RNA‐seq‐based selection of reference genes for RT‐qPCR analysis of pitaya

Reverse‐transcription quantitative real‐time PCR (RT‐qPCR) is a primary tool for measuring gene expression levels, and selection of appropriate reference genes is crucial for accurate and reproducible results of gene expression under various experimental conditions. However, no systematic evaluation of reference genes in pitaya (Hylocereus undatus Britt.) has been performed. Here, we examined the expression of five candidate reference genes, namely elongation factor 1‐alpha (HuEF1‐α), 18S ribosomal RNA (Hu18S rRNA), ubiquitin (HuUBQ), actin (HuACT), and ubiquitin‐conjugating enzyme (HuUQT), under different conditions in pitaya. The expression stabilities of these five genes were evaluated using two computation programs: geNorm and NormFinder. The results were further validated by normalizing the expression of the phosphoglycerate kinase (HuPGK) and ethylene‐responsive transcription factor (HuERF) genes. Our results indicate that combined use of HuUBQ and HuUQT is the most stable reference under all of the experimental conditions examined. HuEF1‐α, HuUBQ, and HuUQT are the top three most stable reference genes under salt stress, drought stress, and heat stress, and across different cultivars. HuEF1‐α, HuACT, and HuUQT exhibited the most stable expression patterns across different tissues. Our results will allow researchers to select the most appropriate reference genes for gene expression studies of pitaya under different conditions.

Phylogenetic relationships in the genus Avena based on the nuclear Pgk1 gene.

The phylogenetic relationships among 76 Avena taxa, representing 14 diploids, eight tetraploids, and four hexaploids were investigated by using the nuclear plastid 3-phosphoglycerate kinase gene (Pgk1). A significant deletion (131 bp) was detected in all the C genome homoeologues which reconfirmed a major structural divergence between the A and C genomes. Phylogenetic analysis indicated the Cp genome is more closely related to the polyploid species than is the Cv genome. Two haplotypes of Pgk1 gene were obtained from most of the AB genome tetraploids. Both types of the barbata group showed a close relationship with the As genome diploid species, supporting the hypothesis that both the A and B genomes are derived from an As genome. Two haplotypes were also detected in A. agadiriana, which showed close relationships with the As genome diploid and the Ac genome diploid, respectively, emphasizing the important role of the Ac genome in the evolution of A. agadiriana. Three homoeologues of the Pgk1 gene were detected in five hexaploid accessions. The homoeologues that might represent the D genome were tightly clustered with the tetraploids A. maroccana and A. murphyi, but did not show a close relationship with any extant diploid species.

Molecular and biochemical characterization of natural and recombinant phosphoglycerate kinase B from Trypanosoma rangeli

T. rangeli epimastigotes contain only a single detectable phosphoglycerate kinase (PGK) enzyme in their cytosol. Analysis of this parasite's recently sequenced genome showed a gene predicted to code for a PGK with the same molecular mass as the natural enzyme, and with a cytosolic localization as well. In this work, we have partially purified the natural PGK from T. rangeli epimastigotes. Furthermore, we cloned the predicted PGK gene and expressed it as a recombinant active enzyme. Both purified enzymes were kinetically characterized and displayed similar substrate affinities, with KmATP values of 0.13 mM and 0.5 mM, and Km3PGA values of 0.28 mM and 0.71 mM, for the natural and recombinant enzyme, respectively. The optimal pH for activity of both enzymes was in the range of 8–10. Like other PGKs, TrPGK is monomeric with a molecular mass of approximately 44 kDa. The enzyme's kinetic characteristics are comparable with those of cytosolic PGK isoforms from related trypanosomatid species, indicating that, most likely, this enzyme is equivalent with the PGKB that is responsible for generating ATP in the cytosol of other trypanosomatids. This is the first report of a glycolytic enzyme characterization from T. rangeli.

A Hemizygous Deletion Within the PGK1 Gene in Males with PGK1 Deficiency.

Phosphoglycerate kinase-1 (PGK1) deficiency is a rare X-linked disorder caused by pathogenic variants in the PGK1 gene. Complete loss-of-function variants have not been reported in this gene, indicating that residual enzyme function is critical for viability in males. Therefore, copy number variants (CNVs) that include single exon or multiple exon deletions or duplications are generally not expected in individuals with PGK1 deficiency. Here we describe a 64-year-old male presenting with a family history (three additional affected males) and a personal history of childhood-onset metabolic myopathy that involves episodes of muscle pain, stiffness after activity, exercise intolerance, and myoglobinuria after exertion. Biochemical analysis on a muscle biopsy indicated significantly reduced activity (15% compared to normal) for phosphoglycerate kinase (PGK1), a glycolytic enzyme encoded by PGK1. A diagnosis of PGK1 deficiency was established by molecular analysis which detected an approximately 886kb deletion involving the polyadenylation site in the 3'UTR of the PGK1 gene. RNA analysis showed significantly reduced PGK1 transcript levels (30% compared to normal). This is the first deletion reported in the PGK1 gene and is the first pathogenic variant involving the 3'UTR polyadenylation site of this gene. Our report emphasizes the role of 3'UTR variants in human disorders and underscores the need for exploring noncoding regions of disease-associated genes when seeking a molecular diagnosis.

Polyclonal origin of parathyroid tumors is common and is associated with multiple gland disease in primary hyperparathyroidism

Parathyroid tumors are mostly considered monoclonal neoplasms, the rationale for focused parathyroidectomy in primary hyperparathyroidism. We reported that flow sorting parathyroid tumor cells and methylation-sensitive polymerase chain reaction (me-PCR) of polymorphic human androgen receptor gene and phosphoglycerate kinase gene alleles in deoxyribonucleic acid reveals that ≤35% of parathyroid tumors are polyclonal. We sought to confirm these findings and assess for clinical relevance. Parathyroid tumors from 286 female primary hyperparathyroidism patients were analyzed for clonal status. Tumor clonal status was compared with clinical variables and operative findings. Statistical analysis was performed and significance was established at P < .05. In the study, 176 (62%) patients were informative for human androgen receptor gene and/or phosphoglycerate kinase gene. Assignment of clonal status was made in 119 (68%) tumors, of which 64 (54%) were monoclonal and 55 (46%) were polyclonal. Comparison of tumor clonal status to clinical variables in patients with complete operative data (N = 82) showed that while clinical features were the same between tumor types, patients with polyclonal tumors more often had multiple gland disease (risk ratio 4.066, confidence interval, 1.016-16.26; P = .039) potentially missed at unilateral neck exploration. This work confirms that primary hyperparathyroidism is often the result of polyclonal tumors and that parathyroid tumor clonal status may be associated with multiple gland disease.

Retraction: Hypoxia-Inducible Factor (HIF) 1α Accumulation and HIF Target Gene Expression Are Impaired after Induction of Endotoxin Tolerance.

The oxygen-sensitive transcription factor hypoxia-inducible factor 1 (HIF-1) is known as the key regulator of hypoxia-induced gene expression. In addition to hypoxia, endotoxins such as bacterial LPS as well as proinflammatory cytokines have been shown to induce HIF-1, suggesting an integrative role for HIF-1 in conditions of hypoxia and inflammation. Cells can become tolerant to endotoxins by repetitive exposure to LPS. Herein, we studied the effect of endotoxin tolerance on HIF-1alpha accumulation and expression of HIF target genes in human monocytic cells and primary mouse peritoneal macrophages. Tolerant cells had reduced levels of HIF-1alpha under hypoxia, which was due to lowered levels of HIF-1alpha mRNA. HIF-1alpha expression is under control of NF-kappaB and increased DNA binding of the p52 subunit of NF-kappaB was found in tolerant cells. Knock down of p52 abolished the effects of endotoxin tolerance on HIF-1alpha expression, which suggest a negative regulatory role of p52 on HIF-1alpha transcription during endotoxin tolerance. Endotoxin tolerant cells showed diminished expression of the HIF target genes phosphoglycerate kinase 1 and adrenomedullin and reduced viability under hypoxic conditions, as well as a significantly reduced invasion. Peritoneal macrophages from endotoxin-tolerant mice made showed significantly reduced HIF-1alpha protein accumulation and subsequent HIF target gene expression. We conclude that endotoxin tolerance impairs HIF-1alpha induction which reduces the ability of monocytic cells to survive and function under hypoxic conditions.

Lentiviral Vectors Incorporating Ubiquitous Chromatin Opening Element Driving Canine CD18 Expression

Leukocyte adhesion deficiency type 1 (LAD1) in humans is caused due to mutations in the ITGB2 gene encoding the leukocyte CD18 subunit (b2 integrin). This results in defective leukocyte adhesion and migration leading to recurrent episodes of life-threatening bacterial infection. Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific large animal model of LAD1 in which new therapeutic approaches could be tested. Our previous studies have demonstrated variable efficiency of CD18 expression under the control of several promoters. These include cellular promoters such as those of human elongation factor 1a (hEF1a): long (1169bp) and short (248bp) fragments, human phosphoglycerate kinase (hPGK), human CD11b and human CD18 genes. In addition, murine stem cell virus (MSCV) promoter has also been demonstrated to lead to very high levels of CD18 expression in CLAD CD34+ cells thereby reversing the CLAD phenotype in dogs previously treated with both foamy and lentiviral vectors. But, due to potential genotoxicity associated with the use of viral promoters, we continued our efforts in search of novel cellular promoters. One such promoter is the ubiquitous chromatin opening element (UCOE) from the human heterogeneous ribonucleoprotein A2/B1 and chromobox homolog 3 (HNRPA2B1-CBX3) loci. UCOE has been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., Blood 2007).It has also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., Mol Ther. 2010). Since the full-length element is about 2.6 kb, we cloned and tested different fragment lengths of the UCOE promoter in a SIN lentiviral vector (pCL20) in CLAD CD34+ cells in vitro. Efficiency of expression of CD18 obtained with the six promoter fragments of UCOE (in bp), namely U3'631, U3'1262, U3'652, U5'1357, U5'723 and U5'655 were compared to those obtained with an MSCV promoter. Functional viral titers were first determined using a human LAD EBV-transformed B-cell line that lacks endogenous human CD18. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells after a 24h cytokine prestimulation in vitro, the percentage of CD18+ cells 5 days after transduction were as follows: U3'631 - 8.49%, U3'1262 - 15.9%, U3'652 - 21.3% (tested at MOI 100), U5'1357 - 2.05% (tested at MOI 30), U5'723 - 2.44% (tested at MOI 20), U5'655 - 3.01% (tested at MOI 50) and MSCV - 35.3% (tested at MOI 100). The CD18 expression levels driven by some of these promoter fragments were comparable to those driven by cellular promoters mentioned previously. The UCOE is promising in that it could overcome possible gene silencing effects when used in vivo, unlike promoters such as EF1a and PGK which were largely subjected to post-transcriptional gene silencing with sub-therapeutic levels of CD18 as previously tested in the dog model. Hence, functional correction of the CD18 defect could be achieved with candidate UCOE-incorporating SIN lentiviral vector(s) when used in the treatment of CLAD dogs. DisclosuresNo relevant conflicts of interest to declare.

Enhancement of wildlife disease surveillance using multiplex quantitative PCR: development of qPCR assays for major pathogens in UK squirrel populations.

Rapid development in polymerase chain reaction (PCR) technology has revolutionised the speed and accuracy of many diagnostic assays. However, comparatively few wildlife epidemiological studies use quantitative PCR (qPCR) for pathogen detection, even fewer employ an internal control, to ensure confidence in negative results, and PCR’s ability to multiplex and therefore detect several targets in a single reaction is underutilised. Here, we describe the development of two multiplex qPCR assays for the red and grey squirrel that detect the pathogens squirrelpox virus (SQPV) and adenovirus in squirrels (SADV), both of which cause mortality in the red squirrel. Both assays use a section of the squirrel phosphoglycerate kinase gene as an endogenous internal control that identifies and compensates for both, inadequate sampling or PCR inhibition. Tests on infected squirrel tissue demonstrate that simple swab samples (particularly from distal antebrachial skin) are sufficient to detect and identify the relative quantity of SQPV DNA in both squirrel species, while rectal swabs and blood cell pellets can be used to reliably indicate SADV infection. These assays are sensitive and specific with an endogenous internal control providing confidence in negative results and allowing comparison across laboratories. Using such assays should prove advantageous in wildlife studies with limited resources while allowing the maximum data yield.Electronic supplementary materialThe online version of this article (doi:10.1007/s10344-016-1031-z) contains supplementary material, which is available to authorized users.